Paclitaxel (Taxol®) is a widely used chemotherapeutic agent that has a major dose limiting side-effect of painful peripheral neuropathy. Currently there is no effective therapy for the prevention or treatment of chemotherapy-induced painful peripheral neuropathies. Evidence for mitochondrial dysfunction during paclitaxel-induced pain was previously indicated with the presence of swollen and vacuolated neuronal mitochondria. As mitochondria are a major source of reactive oxygen species (ROS), the aim of this study was to examine whether pharmacological inhibition of ROS could reverse established paclitaxel-induced pain or prevent the development of paclitaxel-induced pain. Using a rat model of paclitaxel-induced pain , the effects of a non-specific ROS scavenger, N-tert-Butyl-α-phenylnitrone (PBN) and a superoxide selective scavenger, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL) were compared. Systemic 100 mg/kg PBN administration markedly inhibited established paclitaxel-induced mechanical hypersensitivity to von Frey 8 g and 15 g stimulation and cold hypersensitivity to plantar acetone application. Daily systemic administration of 50 mg/kg PBN (days −1 to 13) completely prevented mechanical hypersensitivity to von Frey 4 g and 8 g stimulation and significantly attenuated mechanical hypersensitivity to von Frey 15 g. Systemic 100 mg/kg TEMPOL had no effect on established paclitaxel-induced mechanical or cold hypersensitivity. High dose (250 mg/kg) systemic TEMPOL significantly inhibited mechanical hypersensitivity to von Frey 8 g & 15 g, but to a lesser extent than PBN. Daily systemic administration of 100 mg/kg TEMPOL (day −1 to 12) did not affect the development of paclitaxel-induced mechanical hypersensitivity. These data suggest that ROS play a causal role in the development and maintenance of paclitaxel-induced pain, but such effects cannot be attributed to superoxide radicals alone. Paclitaxel is a taxane-derived chemotherapeutic used alone, or in combination therapy, for the treatment of ovarian, breast and advanced non-small cell lung cancers, and AIDS-related Kaposi's sarcoma. Paclitaxel binds to β-tubulin of microtubules 2−, hydroxyl radical OH., are by-products of oxidative phosphorylation and usually decomposed by specialised cellular enzymes e.g.superoxide dismutases, peroxidases. In the 1990s, the role of ROS in neuropathic pain was demonstrated with the inhibition of CCI-evoked heat hyperalgesia by novel antioxidants 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL) Reactive oxygen species (ROS) e.g. superoxide radical OOur interest in the role of ROS in chemotherapy-induced pain developed after finding swollen/vacuolated mitochondria in peripheral sensory nerves of paclitaxel-treated rats, in the absence of axonal degeneration ad libitum. Bedding/cages were changed twice a week and only rats were housed in the same room. Prior to any behavioural testing, rats were habituated to the testing environment for 30 minutes on two or three separate days.All experiments were carried out in strict accordance with the UK Animals (Scientific Procedures) Act 1986 and the ethical guidelines issued by the International Association for the Study of Pain Following habituation to the behavioural testing environment and baseline measurements of mechanical sensitivity (see Section 2.2), rats were injected intraperitoneally (i.p.) with 2 mg/kg paclitaxel on four alternate days  as previously described Animals were placed on an elevated platform of small metal rods (spaced 8 mm apart) in individual Perspex boxes (dimensions 15 cm×16 cm×21 cm). Animals were allowed to acclimatise for 5–10 minutes before testing. Mechanical hypersensitivity was assessed using three von Frey filaments with bending forces of 4 g, 8 g and 15 g, in ascending order of force, as previously described N-tert-Butyl-α-phenylnitrone  was dissolved in sterile 0.9% saline for injection  resulting in a clear, colourless solution. PBN was administered intraperitoneally (i.p.) in either a treatment or prophylactic dosing paradigm, to test if PBN could treat established paclitaxel-induced pain or prevent the development of paclitaxel-induced pain.Following habituation and baseline testing, all rats received paclitaxel as described above and the emergence of mechanical hypersensitivity was monitored. On day 26 post paclitaxel initiation, von Frey testing was performed on all rats and rats were then divided into two groups displaying similar levels of mechanical hypersensitivity. Rats then received an i.p. injection of 100 mg/kg PBN (n = 9) or an equivalent volume of vehicle . Rats were tested again for mechanical hypersensitivity at one hour, three hours and 24 hours following PBN/vehicle administration. This process was repeated for the next two consecutive days i.e. Day 27 & Day 28 post paclitaxel treatment. Thus rats with established paclitaxel-induced mechanical hypersensitivity received either three daily injections of 100 mg/kg PBN or saline, with each injection followed 1, 3 and 24 hours later by von Frey testing. Throughout the experiment, behavioural testing was performed under blind conditions by a single experimenter (MF). Injections were performed by another scientist. PBN/vehicle treatments were randomised within the equal groups of 6 animals being tested in a given session. These methods provided a concurrent vehicle-treated group throughout the experiments to control for potential variety in behavioural response due to the time of day. Following completion of the experiment, the identity of the treatment received by each rat was revealed and the data analysed.Following habituation and four baseline measurements, rats were divided into two groups based on their responses to von Frey stimulation providing two groups with similar average baseline mechanical sensitivity. Rats received daily i.p. doses of either 50 mg/kg PBN (n = 8) or an equivalent volume of vehicle  for 15 consecutive days (day -1 through to day 13). On days 0, 2, 4 & 6 when paclitaxel was also administered, rats received PBN/vehicle injection before the paclitaxel injection. 50 mg/kg PBN was used in this experiment as opposed to 100 mg/kg due to concerns over tolerability to large injection volumes during an extended period. Mechanical sensitivity was then assessed in the mornings on days 7, 10, 14, 17, 19, 21, 25, 28, 31, 34, 38, 41 and 45 following the initiation of paclitaxel treatment (day 0). Throughout the experiment, behavioural testing and PBN/vehicle administration was performed under blind conditions by a single experimenter (MF). Following completion of the experiment, the identity of the treatment received by each rat was revealed and the data analysed.4-Hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl  dissolved in sterile 0.9% saline for injection  resulting in an orange solution. TEMPOL was administered intraperitoneally (i.p.) in either a prophylactic or treatment dosing paradigm to test if TEMPOL could treat established paclitaxel-induced pain or prevent the development of paclitaxel-induced pain.Following habituation and baseline testing, all rats received paclitaxel as described above and the emergence of mechanical hypersensitivity was monitored. On day 27 post paclitaxel initiation, von Frey testing was performed on all rats and rats were then divided into three groups displaying similar levels of mechanical hypersensitivity. Rats then received an i.p. injection of either 100 mg/kg TEMPOL (n = 8), 250 mg/kg TEMPOL (n = 8) or an equivalent volume of vehicle . Rats were tested again for mechanical hypersensitivity at one hour, three hours and 24 hours following TEMPOL/vehicle administration. Throughout the experiment, behavioural testing was performed under blind conditions by a single experimenter (SJLF). Injections were performed by another scientist. TEMPOL/vehicle treatments were randomised within the equal groups of 8 animals being tested in a given session. These methods provided a concurrent vehicle-treated group throughout the experiments to control for potential variety in behavioural response due to the time of day. Following completion of the experiment, the identity of the treatment received by each rat was revealed and the data analysed. Initially this TEMPOL treatment dosing paradigm was intended to run over three consecutive days (as performed for PBN). However due to significant side-effects  observed immediately following 250 mg/kg administration the experiment was curtailed. These side-effects were not evident at the one hour testing time point.In the prophylactic paradigm, following habituation and baseline testing, rats were divided into two groups based on their responses to von Frey stimulation providing two groups with similar baseline mechanical sensitivity. Rats received daily i.p. doses of either 100 mg/kg TEMPOL (n = 9) or an equivalent volume of vehicle  for 14 consecutive days (day −1 through to day 12). On days 0, 2, 4 & 6 when paclitaxel was also administered, rats received TEMPOL/vehicle injection before the paclitaxel injection. Mechanical sensitivity was then assessed in the mornings on days 7, 13, 17, 20, 24, 27, 33, 39 and 45 following the initiation of paclitaxel treatment (day 0). Throughout the experiment, behavioural testing and TEMPOL/vehicle administration was performed under blind conditions by a single experimenter (SJLF). Following completion of the experiment, the identity of the treatment received by each rat was revealed and the data analysed. As TEMPOL dissolved to give an orange solution, blinding procedures were more challenging than in PBN experiments. KCL Biological Services Unit staff kept SJLF blind to treatment received by randomisation of rat order within the elevated testing environment (at day 7) and by renumbering rats from day 12 onwards.0 = no response, 1 = quick withdrawal, flick or stamp of the paw, 2 = prolonged withdrawal or repeated flicking (≥3) of the paw, 3 = repeated flicking of the paw with licking directed at the ventral side of the paw. Acetone was applied alternately three times to each paw and the responses scored categorically. At least 12 minutes had elapsed before the next application of acetone was applied to the same hind paw. Cumulative scores were then generated by adding the 6 scores for each rat together, the minimum score being 0  and the maximum possible score being 18 . All testing was performed on rats when they were alert, not grooming and with all four paws in contact with the platform.Animals were placed on an elevated platform of small metal rods (spaced 8 mm apart) in individual Perspex boxes (dimensions 15 cm×16 cm×21 cm). Animals were allowed to acclimatise for 5–10 minutes before testing. Cold hypersensitivity was assessed using acetone, as previously described Following habituation and baseline testing, 24 rats received paclitaxel as described above and the emergence of cold hypersensitivity was monitored. Due to time constraints, this experiment using 24 rats was conducted on two separate days. On day 27 post paclitaxel initiation, acetone testing was performed on 15 rats and rats were then divided into three groups (n = 5) displaying similar levels of cold hypersensitivity. Rats then received an i.p. injection of either 100 mg/kg PBN, 100 mg/kg TEMPOL or an equivalent volume of vehicle . Rats were tested again for cold hypersensitivity at one hour, three hours and 24 hours following PBN/TEMPOL/vehicle administration. On day 30 post paclitaxel initiation, acetone testing was performed on the remaining 9 rats and rats were then divided into three groups (n = 3) displaying similar levels of cold hypersensitivity. Similarly, rats then received an i.p. injection of either 100 mg/kg PBN, 100 mg/kg TEMPOL or an equivalent volume of vehicle . Rats were tested again for cold hypersensitivity at one hour, three hours and 24 hours following PBN/TEMPOL/vehicle administration. Throughout the experiment, behavioural testing was performed under blind conditions by a single experimenter (LAG). Injections were performed by another scientist. PBN/TEMPOL/vehicle treatments were randomised within the groups of animals (7–9) being tested in a given session. These methods provided a concurrent vehicle-treated group throughout the experiments to control for potential variety in behavioural response due to the time of day for example. Following completion of the experiment, the identity of the treatment received by each rat was revealed and the data pooled from each part of the experiment resulting in n = 8 for each treatment group.One tailed unpaired t-tests with Bonferroni correction were used to compare the effects of repeated PBN treatment to vehicle treatment on established paclitaxel-induced mechanical hypersensitivity . One wayA cumulative dose of 8 mg/kg paclitaxel administered in four i.p. injections resulted in significant mechanical and cold hypersensitivity, assessed by responses to von Frey 4 g, 8 g and 15 g stimulation and acetone application, respectively. Maximal mechanical hypersensitivity was observed around day 27 post paclitaxel initiation as previously described Prophylactic PBN administration before, during and after paclitaxel administration  had a marked preventative effect on the development of paclitaxel-induced mechanical hypersensitivity . No sign2−) using TEMPOL, a superoxide dismutase mimetic. Given these inhibitory effects on paclitaxel-induced pain of global ROS inhibition by PBN, we investigated whether these inhibitory effects could be replicated with selective inhibition of superoxide radicals  are illustrated in Paclitaxel is also known to induce cold hypersensitivity in humans in vivo. We have observed the effects of systemic administration of a non-specific ROS scavenger, PBN, and a superoxide-specific scavenger, TEMPOL, in both treatment and prophylactic dosing paradigms. The rationale for these experiments was to test if PBN and/or TEMPOL could reverse established paclitaxel-induced pain and/or prevent the development of paclitaxel-induced pain, as both scenarios have significant clinical relevance.In this study, we have examined the role of ROS in the maintenance and development of paclitaxel-induced painful peripheral neuropathy The first administration of PBN markedly inhibited established paclitaxel-induced mechanical hypersensitivity to von Frey 8 g and 15 g stimulation for over three hours. Similarly, repeated bolus PBN treatment on the following two days also significantly inhibited this mechanical hypersensitivity, but to a lesser extent, perhaps indicating tolerance to repeated PBN administration. Mechanical hypersensitivity to von Frey 4 g was unaffected by PBN treatment, which could suggest a lack of anti-allodynic effect by PBN or the relatively small window of hypersensitivity to von Frey 4 g in this cohort of rats to elicit a statistically significant inhibition. PBN also significantly inhibited paclitaxel-induced cold hypersensitivity. In comparison, prophylactic PBN dosing completely prevented the development of mechanical hypersensitivity to von Frey 4 g & 8 g stimulation through to day 45 post paclitaxel initiation. Prophylactic PBN dosing also delayed the appearance of, and reduced the magnitude of mechanical hypersensitivity to von Frey 15 g. These effects demonstrate that ROS play a role in both the maintenance and development of paclitaxel-induced pain.The effects of TEMPOL on paclitaxel-induced mechanical hypersensitivity we observed were quite different to the inhibitory effects of PBN. The same dose of TEMPOL (100 mg/kg) did not inhibit established paclitaxel-induced mechanical hypersensitivity. High dose (250 mg/kg) TEMPOL inhibited mechanical hypersensitivity to von Frey 8 g and 15 g at one hour, but to a lesser extent than observed with PBN and these effects were not present at three hours post administration. Similar to PBN, TEMPOL had no effect on established mechanical hypersensitivity to von Frey 4 g. However, in marked contrast to PBN, prophylactic TEMPOL showed no inhibitory effects on the development of paclitaxel-induced mechanical hypersensitivity through to day 45 post paclitaxel initiation. Prophylactic TEMPOL was administered at twice the prophylactic PBN dose that prevented development of paclitaxel-induced mechanical hypersensitivity. It is possible that higher doses of TEMPOL may have an inhibitory effect on the development of paclitaxel-induced pain. However, given the side-effects observed in the minutes following 250 mg/kg TEMPOL administration in the established pain study, we decided that 100 mg/kg was the maximally tolerated dose for repeated prophylactic dosing. The overall lack of effect of TEMPOL in this study suggests that superoxide radicals do not play a role in the maintenance or development of paclitaxel-induced pain. The lack of parallel effects of PBN and TEMPOL in paclitaxel-induced pain provides further evidence that chemotherapy-induced painful peripheral neuropathies have different causal mechanisms to other pain states. Previously, both PBN and TEMPOL at similar doses have been shown to inhibit SNL-evoked heat and mechanical hypersensitivity in a similar manner A recent study has examined the effects of PBN on paclitaxel-induced mechanical hypersensitivity The causal mechanism(s) for chemotherapy-induced painful peripheral neuropathy are unclear. Various rodent models of paclitaxel-induced painful peripheral neuropathy have been reported using different systemic dosing schedules and cumulative doses of paclitaxel The inhibitory effects of a non-specific ROS scavenger reported here indicate that ROS has a causal role in paclitaxel-induced pain. While we have provided evidence to suggest superoxide radicals do not play a major role in paclitaxel-induced painful peripheral neuropathy, further study could address the contribution of other free radicals such as hydrogen peroxide, peroxynitrite and hydroxyl radicals. Considering that mitochondria are a major source of ROS, this perhaps demonstrates a consequential mechanism of how the atypical mitochondria in peripheral sensory axons lead to paclitaxel-induced pain. Alternatively, as superoxide radicals are predominantly derived from mitochondria, it is possible that the ROS responsible for paclitaxel-induced pain are generated from other sites in the cell. In this model of paclitaxel-induced painful peripheral neuropathy, atypical mitochondria were observed at day 7 (where no pain behaviour is observed) and day 27 (peak pain severity) In conclusion, this study demonstrates that global inhibition of ROS can inhibit established paclitaxel-induced pain and prevent the development of paclitaxel-induced pain, whereas selective inhibition of superoxide radicals was mostly ineffective. The causal role of ROS in paclitaxel-induced painful peripheral neuropathy highlights a potential novel therapeutic strategy for the prevention and treatment of this major dose-limiting side-effect.


EGFR overexpression in head and neck squamous cell carcinoma (HNSCC). For this reason, expression/mutation of EGFR were analyzed in 30 dysplastic head and neck lesions and 148 HNSCC samples of Indian patients along with 3 HNSCC cell lines. In addition, deletion/methylation/mutation/expression of SH3GL2 (associated with EGFR degradation) and CDC25A (associated with dephosphorylation of EGFR) were analyzed in the same set of samples. Our study revealed high frequency of EGFR overexpression (66–84%), low frequency of gene amplification (10–32.5%) and absence of functional mutation in the dysplastic lesions and HNSCC samples. No correlation was found between protein overexpression and mRNA expression/gene amplification status of EGFR. On the other hand, frequent alterations (deletion/methylation) of SH3GL2 (63–77%) and CDC25A (37–64%) were seen in the dysplastic and HNSCC samples. Two novel single nucleotide polymorphism (SNPs) were found in the promoter region of SH3GL2. Reduced expression of these genes showed concordance with their alterations. Overexpression of EGFR and p-EGFR were significantly associated with reduced expression and alterations of SH3GL2 and CDC25A respectively. In-vitro demethylation experiment by 5-aza-2′-deoxycytidine (5-aza-dC) showed upregulation of SH3GL2 and CDC25A and downregulation of EGFR expression in Hep2 cell line. Poor patient outcome was predicted in the cases with alterations of SH3GL2 and CDC25A in presence of human papilloma virus (HPV) infection. Also, low SH3GL2 and high EGFR expression was a predictor of poor patient survival. Thus, our data suggests that overexpression of EGFR due to its reduced degradation and dephosphorylation is needed for development of HNSCC.The aim of this study is to understand the mechanism of  EGFR) protein, deletion in chromosome 9p21, p16/p14 inactivation, trisomy of chromosome 7 and telomerase activation were suggested to be associated with the development of hyperplastic lesions of head and neck EGFR is quite important due to its regulation of multiple cell signaling cascades. Nowadays, multiple therapeutic targets have been made against EGFR to treat this tumor, but success is still far behind Head and neck squamous cell carcinoma is the sixth most common cancer worldwide and it accounts for 30–40% of all cancer types in the Indian subcontinent EGFR protein was seen in HNSCC yet amplification of this locus was not prevalent (10–30%) EGFR in HNSCC. It was evident that binding of EGF to EGFR triggered a series of biochemical events including autophosphorylation of specific tyrosine residues  in its kinase domain SH3GL2 interaction SH3GL2 (located at chromosome 9p22.2) have been reported in head and lesions SH3GL2 interacts with CIN85, whereas the LPAAT domain of SH3GL2 is associated with lysophophatydic acid acyl tyransferase activity which converts lysophosphotidic acid (LPA) into phosphatidic acid (PA) needed for membrane curvature for encapsulation of EGFRSH3GL2 and EGFR in HNSCC has not yet been studied. On the other hand, Shang, et al CDC25A, a dual phosphatase, could regulate the activity of EGFR through dephosphorylation of the tyrosine residues. Its frequent deletion (53%) and reduced expression (64%) have been reported in HNSCC SH3GL2, association of CDC25A with EGFR in HNSCC has not yet been studied.Though, frequent (80–90%) overexpression of EGFR protein overexpression, we analyze the alterations of EGFR, SH3GL2 and CDC25A in the same set of HNSCC samples. At first, alterations  of EGFR were analyzed in primary head and neck lesions of Indian patients and some HNSCC cell lines. Then, alterations (deletion/promoter methylation/mutation/expression) of SH3GL2 and CDC25A were analyzed in these samples. Our data suggests that overexpression of EGFR protein might be due to the impairment of the SH3GL2 associated endocytosis mechanism, whereas down regulation of CDC25A in this tumor might lead to the EGFR protein in its active state.Thus, to understand the molecular mechanism of The Institutional Ethical board of Chittaranjan National Cancer Institute, Kolkata approved the usage of Human specimens in this study. The above board approved usage of these human clinical samples specifically in this study, related to the involvement of the study of EGFR homeostasis in HNSCC. The tumor specimens were collected from the hospital section of Chittaranjan National Cancer Institute, Kolkata, after obtaining written, informed consent of the concerned patients, in stipulated format, approved by the above mentioned Institutional Ethical board of Chittaranjan National Cancer Institute, Kolkata, India. Blood samples were collected from healthy control with written informed consent as approved by the Institutional ethical Board.Total 178 tumor tissue samples of head and neck lesions as well as matched adjacent normal tissues were collected from Chittaranjan National Cancer Institute, Kolkata, India. The primary tumors were collected from patients after surgical resection having no previous treatment record. All these tumors were graded and staged according to UICC TNM classification. Samples were frozen immediately after collection at -80°C until use. Part of the freshly operated tissues was directly collected in TRIzol reagent  for RNA isolation and another part was fixed in formalin and embedded in paraffin for immunohistochemical analysis. Clinicopathological information of the patients (n = 178) were presented in The contaminant normal cells in the head and neck lesions were removed by microdissection procedure from cryosections (5 µm) using surgical knives under a dissecting microscope . The representative sections from different regions of the specimens were stained with hematoxylin and eosin for diagnosis as well as for marking of the dysplastic epithelium/tumor rich regions. The samples containing >60% dysplastic epithelium/tumor cells were taken for isolation of DNA according to the standard procedure EGFR, SH3GL2 and CDC25A was done by immunohistochemical analysis in 50 primary tumor samples and by immunoflorescence analysis in Hep2, KB, SCC084 according to the standard procedure as describe earlier CDC25A (sc-6947), SH3GL2 (sc-10874), EGFR (sc-03) and p-EGFR (sc-57541) at a dilution of 1∶80 at 4°C. HRP conjugated rabbit anti-goat (sc-2020), goat anti rabbit (sc-2004) and goat anti mouse (sc-2005) secondary antibody was added 1∶500 dilutions. The slides were developed using 3–3′ diaminobenzidine (DAB) as the chromogen and counterstained with hematoxylin and photographed in Bright Field microscope . The staining intensity  and the percentage of positive cells  were detected by 2 observers independently and by combining the two scores, final evaluation of expression was done  The protein expression of For immunofluroscence analysis, cover slip culture of Hep2, KB and SCC 084 cell lines were reacted with the same dilution of primary antibody of these genes after permeabilisation with 0.5% Triton X-100 and blocking with 5% BSA. After washing, the coverslips were incubated with FITC conjugated corresponding secondary antibody goat anti mouse (sc-2010), goat anti rabbit (sc-2012) and rabbit anti goat (sc-2777) at 1∶500 dilution and mounted with glycerol after thorough washing. Imaging of the cover slip was performed in florescence microscope .EGFR amplification was carried out using differential polymerase chain reaction (DPCR) method EGFR gene was selected for amplification analysis. The dopamine D2 receptor gene (DRD2) gene was used as internal control due to low frequency of alterations reported in HNSCC A quantitative measurement of EGFR and SH3GL2 was screened in 30 dysplastic lesions, 148 invasive samples and 3 oral cancer cell lines by single strand conformation polymorphism (SSCP) analysis using [α-P32] dCTP as described by Tripathi el al EGFR. For mutation analysis of SH3GL2, important enzymatic domain and alternating splice site of the gene were selected. Exon 9 & exon-10 encoded the SH3domain and exon-1 & exon-2 encoded the lysophosphatydic acid acyl transferase enzymatic domain of the gene. The alternating splice sites were located in exons 3, 4, &5. All these exons were selected for mutation analysis of SH3GL2. Primer sequences and locations were presented in The mutation of http://www.ensembl.org, Release-49). An intragenic microsatellite marker D9S157 located 17.61 Mb from p-ter of chr. 9 was taken for analysis of SH3GL2 and D3S3560 locus located 48.16 Mb from p-ter of chr. 3 and 4.8 Kb telomeric of the gene was taken for deletion analysis of CDC25A. Details of the markers was shown in In microsatellite based deletion mapping, a standard polymerase chain reaction (PCR) containing [γ-P32] ATP end labeled forward primer was done in a 20 µl reaction volume according standard procedure as describe earlier SH3GL2 and CDC25A was analyzed by PCR-based methylation sensitive restriction analysis (MSRA) method using MspI/HpaII restriction enzymes  using standard procedure as describe previously The promoter methylation of the candidate genes SH3GL2, CDC25A and EGFR were analyzed in 22 primary HNSCC samples and their corresponding normal head and neck tissues and 3 cell lines  using primers as mentioned in mRNA expression of In our previous analysis of 5-aza-dc experiment at different concentrations (5–50 µM) it was evident that the cell viability was unaltered up to 5 days of 5-aza-dc treatment at 20 µM concentration. For this reason, the 5-aza-dc concentration was taken upto 20 µM in Hep2 cell line. Subconfluent cultures of Hep2 was treated with 5 M, 10 M and 20 M demethylating agent, 5-aza-dC  for 5 days. Controls without 5-aza-dC were cultured concomitantly in the same manner. After completion of treatment, cells are harvested and proceeded for next experiment.For kinetic study, subconfluent cultures of Hep2 cell line were treated with 20 µm 5-aza-dc in six Petri dish and the cells were harvested at different time point like 0 hour, 24 hours, 48 hours, 72 hours, 96 hours and 120 hours after aza treatment and protein was isolated from the cells. For immunoflorescence analysis after aza treatment, Hep2 cells were grown over night on cover slip and treated with 20 µm 5-aza-dc for 72 hour. Then the cells were fixed with chilled methanol and used for immunoflorescence analysis.5 cells were plated in 35 mm petri dish. After 24 hour, media was replaced with serum and antibiotic free media and siRNAs were transfected using Lipofectamin 2000 (Invitrogen) according to the manufacturer protocol. The siRNA of SH3GL2 (SC-35304), CDC25A (SC-29254) and scrambled control (SC-37007) were purchased from Santa Cruz Biotechnology, CA, USA and were used at a final concentration of 80 pmole. After siRNA transfection, cells were harvested at different time point like 24 hour, 48 hour, 72 hour and 96 hour for each gene and RNAs were isolated to find out the time point for maximum gene knock down by real time quantitation. After characterizing the time for optimum gene knock down, cells were again treated with siRNA and harvested after particular time to isolate the protein.The siRNA mediated knock down of SH3GL2 and CDC25A experiment was done in oral cancer cell line SCC084 according to standard procedure EGFR, p-EGFR(sc-12351), SH3GL2, CDC25A, Actin (sc-8432) and alpha-tubulin (sc-5286) and HRP-tagged secondary antibodies and developed with luminol . The signal intensities were scanned by densitometric scanning (Bio-Rad GS-800). The same membrane was used for incubation with different antibodies after stripping with 0.2M NaOH (See Supplementary method for details of the protocol Proteins were extracted from cells by using standard protocol Presence of HPV in the head and neck lesions was detected by PCR using primers (MY09 and MY11) from the consensus L1 region followed by typing of HPV 16/18 in the L1 positive samples as described previously Fisher’s exact test was used to determine the association between tumors genetic profile and different clinicopathological features. All statistical tests were 2-sided and considered significant at probability value, p<0.05. Survival curves were calculated according to Kaplan–Meier method in 148 HNSCC samples. For this method, p values were evaluated by the log rank test for censored survival data. A Cox-proportional hazards regression model was used to test the statistical significance of several potential prognostic factors like clinical stage, tumor site, tobacco exposure, HPV infection and alterations of the candidate TSGs that were jointly predictive of overall survival of the patients. From this model we estimated the hazard ratio (HR) for each potential prognostic factor with a 95% confidence interval (CI) in univariate and a multivariate fashion. All the statistical analysis was performed using statistical programs Epi Info 6.04 b, SPSS 9.0 .20. Transcription factor binding site of SH3GL2 promoter was analyzed uising on line server Alibaba 2.1 TF Binding Prediction software 21.All the oligonucleotides primers used in different experiment were designed using primer-3 software EGFR was seen in basal layer followed by gradual decrease in parabasal and spinous layers  of dysplastic lesions. In invasive HNSCC tumor, overexpression of EGFR was observed in 84% (37/44) samples irrespective of tumor stages  of the head and neck lesions showed fication . Gradualfication . No amplfication . Thus, EEGFR is a rare event in HNSCC.In SSCP analysis, about 13% (24/178) and 20% (35/178) of the HNSCC samples showed altered bands in exon-18 and exon-20 respectively , c, d. NEGFR mRNA with mean fold expression of 1.46 (±10.58)  of the HNSCC samples showed overexpression of (±10.58) . The mRN(±10.58) . No overSH3GL2 and 60 of 178 at CDC25A locus), high frequency of deletion was observed in SH3GL2  and CDC25A  loci (SH3GL2 and CDC25A were observed in 27% (7/26) and 37% (6/16) dysplastic lesions respectively. Comparable frequency of SH3GL2 deletion (30–46%) was observed in subsequent stages, while deletion frequencies of CDC25A (37–41%) were comparable upto stage-I/II followed by significant increase (61–64%) in stage-III/IV of the HNSCC samples (SH3GL2 and CDC25A loci in varying frequencies (4–16%)  in tumor samples (After excluding non-informative tumor samples (21 of 178 at 08) loci . The del samples . In addi (4–16%) . This in samples .SH3GL2 gene in HNSCC lesions  of methylation in subsequent stages of HNSCC  in dysplastic lesions and became comparable (64%–77%) in subsequent stages of tumor progression  of promoter methylation was seen in the  lesions . No proms report  [14]. Inn of DNA . Concordn of DNA . In dyspof HNSCC . Thus itgression .In SSCP analysis, altered bands in exon-1 were seen in 18% (32/178) HNSCC samples and also in their respective normal samples . No otheSH3GL2 is a rare event in HNSCC.To identify the nature of observed sequence variations, sequencing analysis of these variants was done in 52 control DNA samples. The −31 heterozygous allele variant (C>T) was observed in 4% (2/52) control samples , c. On tSH3GL2 mRNA expression with 6.4 (±13.5632) mean fold reduction (CDC25A reduced expression was seen in 60% (15/25) of the samples with 2.4 (±14.41543) mean fold reduction   (It was evident that 80% (20/25) primary HNSCC samples showed ≥2 fold reduction of eduction . In caseeduction . No chan0.00278) .SH3GL2, CDC25A and p-EGFR in the basal/parabasal/spinous cells of normal oral epithelium  of the samples  (SH3GL2 showed significant association (p = 0.0011) with overexpression of EGFR in the samples (CDC25A was observed in 70% (35/50) of the samples. Cytoplasmic and perinuclear expression of CDC25A was seen in the cell lines with low expression in Hep2 (CDC25A was concordant with its deletion pattern of the samples (p = 0.011)  of the samples (p-EGFR was seen in the cell lines (p-EGFR in the tumors showed correlation with reduced expression of CDC25A (p = 0.0117 )  . In addi samples . In the  in Hep2 , b, c. T= 0.011) . In case samples . Cytoplall lines , b, c. I0.0117 ) .SH3GL2 and CDC25A mediated regulation of EGFR/p-EGFR demethylation experiment in presence of 5-aza-dc was done in Hep2 cell line. It was evident that the mRNA expression of SH3GL2 and CDC25A was gradually increased with increasing concentration of 5-aza-dc with significant increase of SH3GL2 at higher concentration . In concordance with mRNA expression, protein expression of SH3GL2 and CDC25A was significantly reduced after 72 hour of siRNA treatment  c &d. InInfection by HPV is considered as one of the important etiological factors for HNSCC development. HPV typing was done in this study to analyze the frequency of high-risk HPVs in our samples. HPV DNA was detected in 52.2% (93/178) of the tumors. Among the HPV positive samples, 92.5% (86/93) were HPV-16-positive and 7.5% (7/93) were HPV-18 positive. HPV infection was found to be significantly associated with tobacco consumption (p = 0.0341) .CDC25A and SH3GL2 (p = 0.02). The patients having alterations in both SH3GL2 and CDC25A had the worse overall survival indicating prognostic significance of these genes among the HNSCC patients    whereas, 0.0461) . Interesnfection  and ii) n tumors . On the d p-EGFR .SH3GL2, EGFR amplification and absence of HPV infection showed hazardous to survival of the patient  and EGFR amplification  in absence of HPV infection  were significant predictors for hazardous life and poor survival of patients  of dysplastic and HNSCC samples (EGFR locus amplification (10–30%) has also been reported in HNSCC EGFR related to lung cancer, we did not find any such mutations. Like our data, infrequent mutation of EGFR has been reported in HNSCC EGFR overexpression in this tumor having absence of genetic alterations in EGFR has not yet been elucidated. Although, a significant correlation was seen between gene amplification and mRNA expression, protein overexpression did not correlate with mRNA expression status of EGFR  of SH3GL2 and CDC25A genes associated with EGFR homeostasis. Frequent deletion/methylation of SH3GL2  was evident in the head and neck lesions with comparable frequencies (63% to 77%) in dysplastic lesions and HNSCC samples  in the promoter region of this gene  of CDC25A and expression of p-EGFR were done in same set of the head and neck lesions. Frequent deletion of CDC25A was seen in dysplastic lesions and subsequent stages as seen our previous report  in varying frequencies in numerous human cancer CDC25A was seen in HNSCC samples as seen in our earlier study CDC25A (30–80%) has been reported in different carcinomas in liver, esophagus, colon, breast including head and neck CDC25A was observed in our samples, the upregulation of this gene by 5-aza-dc in Hep2 cell line suggests the presence of methylation in the some other regulatory regions Click here for additional data file.Figure S2Molecular alterations of SH3GL2.(TIF)Click here for additional data file.Figure S3ICC analysis of EGFR, p-EGFR, SH3GL2 and CDC25A in presence and absence of 5-aza-dc.(TIF)Click here for additional data file.Figure S4Alterations(deletion/methylation) pattern of SH3GL2 and CDC25A in dysplasia and HNSCC samples.(TIF)Click here for additional data file.Figure S5Demethylation experiment of SH3GL2 and CDC25A in Hep2 cell line.(TIF)Click here for additional data file.Table S1Clinical information of control samples.(DOC)Click here for additional data file.Table S2Primer profile used in different experiment.(DOC)Click here for additional data file.Table S3Information of microsatellite markers.(DOC)Click here for additional data file.Table S4Correlation between DPCR and QPCR.(DOC)Click here for additional data file.Table S5Molecular alterations of EGFR.(DOC)Click here for additional data file.Table S6Association of alterations of genes with different clinicopathological parametres.(DOC)Click here for additional data file.Table S7Compilation of mutation analysis of SH3GL2.(DOC)Click here for additional data file.Table S8Effect of 5-aza-dc treatment on expression of EGFR, SH3GL2 and CDC25A.(DOC)Click here for additional data file.Doc S1Supplementary methods.(DOC)Click here for additional data file.


Fusarium oxysporum G12 was cloned together with its native preprosequence and a C-terminal His-tag, and successfully expressed both in Escherichia coli and Pichia pastoris. The enzyme was subsequently purified and characterized. Among all tested substrates, the highest catalytic efficiency (kcat/Km) was found with 1-methyl-β-D-galactopyranoside (2.2 mM−1 s−1). The Michaelis constant (Km) for D-galactose was determined to be 47 mM. Optimal pH and temperature for the enzyme activity were 7.0 and 40°C, respectively, and the enzyme was thermoinactivated at temperatures above 50°C. GalOx contains a unique metalloradical complex consisting of a copper atom and a tyrosine residue covalently attached to the sulphur of a cysteine. The correct formation of this thioether bond during the heterologous expression in E. coli and P. pastoris could be unequivocally confirmed by MALDI mass spectrometry, which offers a convenient alternative to prove this Tyr-Cys crosslink, which is essential for the catalytic activity of GalOx.A gene coding for galactose 6-oxidase from  Fusarium graminearum (formerly classified as Dactylium dendroides) being perhaps the best studied representative. GalOx catalyzes the oxidation of primary alcohols  to aldehydes, accompanied by the reduction of molecular oxygen to hydrogen peroxide Galactose oxidase  is a member of the radical copper oxidase family GalOx is characterized by a broad substrate tolerance, yet strict stereospecificity, for various alcohol substrates F. graminearum GalOx, Tyr272, one of the copper ligands, is covalently linked at Cε to the sulphur atom of Cys228. This bond seems to have partial double-bond character with Cβ of Cys228 lying in the same plane as the ring of Tyr272 •-Cys redox cofactor in GalOx is a self-processing reaction requiring only the apoprotein, copper, and dioxygen; no other proteins or enzymes are required for the processing and assembly of the catalytically active enzyme II, Tyr•) and the reduced form  are involved in the catalytic cycle. The semi-reduced form  is catalytically inactive but can be oxidized to the catalytically active oxidized form Typically, wild-type fungal GalOx is produced as a prepro form carrying a N-terminal signal sequence, which is removed upon secretion, yielding the immature proform. The prosequence in this form was suggested to function as an intramolecular chaperone supporting copper binding and cofactor formation F. graminearum has been cloned and expressed in E. coli with low yields P. pastorisA. nidulansWild type GalOx of F. oxysporum both in E. coli and P. pastoris. We investigated the biochemical properties of the enzyme, and for the first time confirmed the formation of the unique thioether bond in both expression systems directly using mass spectrometry.In this study, we report the successful heterologous expression of a novel, not-yet-characterized GalOx from E. coli strain BL21 (DE3) and P. pastoris strain X-33 were purchased from Invitrogen . E. coli NEB 5-alpha was from New England BioLabs. The HisPrep FF 16/10 column was from GE Healthcare Bioscience AB . F. oxysporum strain G12 was kindly provided by Gerhard Adam .All chemicals were reagent-grade or better and purchased from Sigma-Aldrich  unless otherwise stated. 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) was obtained from Amresco . Restriction enzymes, ligase and standards for Agarose gel electrophoresis (GeneRuler DNA Ladder Mix) were obtained from Fermentas  while the Phusion polymerase was from New England BioLabs . SDS-PAGE protein standard (Precision Plus Protein prestained standard) was from BioRad . The cloning vector pJET 1.2 was purchased from Fermentas and the expression vector pET21a from Novagen . The expression vectors pPICZB and pPICZαA, Fusarium oxysporum strain G12 was cultivated in shaken flasks for 2 days at 25°C and 110 rpm in Sabouraud medium . Fungal mycelium was collected by centrifugation  and washed. Subsequently, genomic DNA was isolated from 100 mg of frozen mycelia ground in liquid nitrogen using the Wizard SV Genomic DNA Purification System . The GalOx gene including its prepro sequence was amplified by PCR using primers based on the published genome of F. oxysporum5′-ATGAAGCCCCTTTGGACACTTGC-3′ and 5′-CTACTGAGTAACGAGAAGAGTACTCGC-3′. The resulting PCR product of approximately 2 kb was purified from the agarose gel using the Wizard SV Gel & PCR CleanUp System (Promega) and ligated into the pJET 1.2 vector using the CloneJET PCR Cloning Kit (Fermentas).E. coli NdeI and SalI restriction sites were introduced by PCR using the following Primers (restriction sites underlined): CATATGAAGCCCCTTTGGACAC-3′5′-CAGTG and GTCGACCTGAGTAACGAGAAGAG-3′5′-CGAA. After digestion with NdeI and SalI the PCR product was ligated into the multiple cloning site of the expression vector pET21a in frame with the C-terminal His6-tag and transformed into E. coli BL21 (DE3). To confirm the correct sequence, the recombinant plasmid was analyzed by restriction digestion and sequenced by VBC Biotech . Two different vectors were constructed for expression in P. pastoris. The first vector contained the full-length GalOx cDNA including the native prepro signal. In the second vector this sequence was replaced by the α-factor of S. cerevisiae, which serves as secretion signal for the mature protein. For the first construct the full-length GalOx cDNA was cloned into the expression vector pPICZB under control of the methanol-inducible AOX promoter and in frame with a C-terminal His6-tag. To this end, the restriction sites EcoRI and NotI were introduced by PCR , and after digestion the PCR product was ligated into the vector using the Quick Ligation Kit (New England BioLabs). The ligation product was transformed into the electrocompetent E. coli strain NEB 5-alpha, and positive clones were selected on Low Salt LB agar plates containing 25 µg mL−1 Zeocin. Plasmid DNA was isolated using the Pure Yield Plasmid Miniprep System (Promega) and linearized with SacI within the 5′ AOX1 region to promote integration. The restriction enzyme was heat inactivated and the plasmid purified with the Wizard SV Gel and PCR Clean-Up System (Promega). Electrocompetent P. pastoris X-33 cells were prepared and transformed with the linearized plasmid according to the operating instructions and applications guide of the MicroPulser electroporation apparatus (BioRad). Transformants were selected for growth on YPD plates containing 100 µg mL−1 Zeocin. In the second construct the prepro sequence of the mature GalOx cDNA was replaced with the α-mating factor of S. cerevisiae. To produce the truncated cDNA a different forward primer was used for PCR (5′-TCGCAGAATTCATGGCCTCAGCTCCCATCG-3′) and the resulting PCR product was ligated into the pPICZαA vector as described above.For expression in E. coli BL21 (DE3) for production of the recombinant enzyme was performed in double concentrated LB medium  containing 50 mg L−1 ampicillin to maintain the expression plasmid. E. coli was grown in baffled shaking flasks at 37°C until an OD600 of 0.4–0.6 was reached. Recombinant protein production was induced by the addition of 5% lactose and cultivation was continued at 25°C for 16 h Cultivation of P. pastoris was also carried out in baffled shaken flasks. Precultures (20 mL) were grown at 30°C in YPD medium containing 25 µg mL−1 Zeocin. After 16 h the precultures were used to inoculate 1-L baffled shaken flasks containing 200 mL of BMMY medium (buffered methanol complex medium) and further incubated at 30°C. After 3 days a linear methanol feed (2% per day) was started. Samples were taken every day, clarified by centrifugation, and protein concentrations as well as GalOx activities were assayed in the supernatant.Production of recombinant GalOx in E. coli biomass and the P. pastoris supernatant, respectively. E. coli biomass was resuspended in phosphate buffer  after harvesting the cells by centrifugation . Cell disruption was performed by addition of lysozyme (1 mg mL−1) and incubation for 30 min at 37°C, followed by sonication, as previously described Pichia cultivations was purified from the culture supernatant as described above after cells had been removed by centrifugation .Recombinant GalOx was purified from both, the SDS-PAGE was performed as described by Laemmli F. oxysporum were recorded using a Chirascan CD Spectrophotometer  in the wavelength range of 180 to 260 nm. The instrument was flushed with nitrogen and the conditions were set as follows: path length was 1 mm, spectral bandwidth 3 nm and the scan time per point was set to 10 s. CD measurements were performed with 0.5 mg mL−1 GalOx in 5 mM potassium phosphate buffer (pH 7.0).Far UV electronic circular dichroism (CD) spectra of GalOx from Coomassie brilliant blue stained protein bands were excised from the gel, cut into small pieces and washed with 50 mM 4-ethylmorpholine acetate (pH 8.1) in 50% acetonitrile (MeCN). After complete destaining, the supernatant was removed and the gel was partly dried in a SpeedVac concentrator. The protein digestion using 100 ng of sequencing-grade trypsin  was performed overnight at 37°C in a cleavage buffer containing 25 mM 4-ethylmorpholine acetate (pH 8.1) and 5% MeCN. Alternatively, the proteins were digested sequentially by trypsin (as described above) and Asp-N protease  as well as using the sequence Asp-N and trypsin, always in the same cleavage buffer. The digestions were stopped by addition of 5 µL of 5% trifluoroacetic acid in MeCN. An aliquot of the peptide mixture (0.5 µL) was deposited on the MALDI target and allowed to air-dry at room temperature. After complete evaporation, 0.5 µL of the matrix solution  was added.+ ions of trypsin autoproteolytic fragments (842.5 and 2211.1 Da).MALDI mass spectra were measured on an Ultraflex III MALDI-TOF/TOF instrument  equipped with LIFT technology for MS/MS analysis. MALDI mass spectra were also obtained on a high accuracy APEX-Qe FT-ICR instrument equipped with a 9.4 T superconducting magnet and a Combi ESI/MALDI ion source . The spectra were acquired in the mass range of 700–5000 Da and calibrated internally using the monoisotopic [M + H]4 for 30 min and 25°C . The activity was determined with the chromogenic 2,2′-azinobis(3-ethylbenzthiazolinesulfonic acid) (ABTS) assay 420 = 43.2 mM−1 cm−1). One Unit of GalOx activity was defined as the amount of enzyme that is necessary for the oxidation of 2 µmol of ABTS per min, which equals the consumption of 1 µmol of O2 per min, under the conditions described above. A substantially identical activity assay was used for determination of the substrate specificity of GalOx, however, galactose was replaced by 100 µmol of the respective substrate in the reaction mixture. Protein concentrations were determined by the dye-binding method of Bradford Prior to activity measurements GalOx was activated by incubation with 1 mM CuSOE. coli at 30°C in 20 mM potassium phosphate buffer (pH 7.0) for different sugar substrates. D-galactose (1–500 mM), 1-methyl-β-D-galactopyranoside (5–200 mM), melibiose (1–250 mM), as well as raffinose, lactose, 1-methyl-α-D-pyranoside, lactulose, 2-deoxy-D-galactose and lactitol, each varied over a range of 10–250 mM, were used as substrates. All kinetic constants were calculated using non-linear least-square regression by fitting the observed data to the Michaelis-Menten equation . Turnover numbers (catk) were calculated using a theoretical molecular mass of 73.9 kDa.Kinetic constants were determined for GalOx produced in A pH-activity profile was determined in the range of pH 2.5 to 10.0 using the buffer system citric acid (pH 2.5–6.0), potassium phosphate (pH 6.0–8.0) and Tris (pH 8.0–10.0), each at a concentration of 50 mM. Activity measurements were performed otherwise as described above for the standard assay.4. Differential scanning calorimetric (DSC) measurements were performed using a MicroCal VP-Capillary DSC system  controlled by the VP-viewer program and equipped with an active cell volume of 130 µL. To check the influence of different buffers on the stability of the protein the studies were made with 4 µM GalOx in 20 mM citric acid, phosphate, and Tris buffer each at pH 7. Samples were analyzed using a heating scan rate of 60°C h−1 over a temperature range of 15–100°C. Collected DSC data were corrected for baseline with the corresponding buffer. Data analysis was performed with the MicroCal Original software.Determination of the temperature optimum of GalOx was achieved by measuring the activity with the standard assay at different temperatures in the range of 30 to 65°C. Thermal stability of GalOx was determined by incubating the protein at 30, 40, 50 and 60°C. Samples were taken at various time points, cooled on ice, and residual GalOx activity was measured using the standard ABTS assay after reactivating the enzyme by incubation with CuSOgao gene encoding GalOx was successfully amplified together with its signal sequence from genomic DNA of the fungus F. oxysporum G12. The gene consists of an open reading frame of 2043 bp encoding a polypeptide of 681 amino acids. The sequence (accession number KF601698) contains no introns and shows 99% similarity to the published amino acid sequence for GalOx from F. oxysporumgao gene has 96%, 62%, and 67% similarity with the gaoA (BK007071), gaoB (BK007067) and gaoC (BK007074) gene from F. oxysporum f. sp. lycopersici, respectively gao gene from F. oxysporum belongs to the gaoA cluster of GalOx genes. The similarity to the protein sequence of GalOx from F. graminearumgao gene was found under the conditions used.The F. oxysporum gao gene was used to generate a sequence alignment with the published sequence of F. graminearum using Clustal Omega F. graminearumF. oxysporum (F. graminearum (data not shown) F. graminearumThe amino acid sequence derived from the xysporum  and compgao gene in E. coli and P. pastoris was compared to find a suitable host for the production of recombinant GalOx. For expression in E. coli suitable restriction sites were introduced, and the full-length gene including the prepro sequence was cloned into the vector pET21a, which adds a C-terminal His-tag. After transformation of the plasmid into E. coli BL21 (DE3) the expression host was cultivated in double-concentrated LB medium and 5% lactose was used as inducer for expression of the gao gene. Routinely 5.7 mg L−1 of active, soluble GalOx  were obtained in shaking flask cultivations after incubation at 25°C for 16 h. This translates to a space-time yield of 0.36 mg L−1 h−1, which is tenfold higher than the yield reported for native GalOx from F. graminearum expressed in E. coligao gene without its prepro sequence in E. coli (data not shown).Expression of the P. pastoris two different strategies were used. To test whether P. pastoris is able to recognize the secretion signal of F. oxysporum the full-length gao cDNA  was cloned into the vector pPIZB, adding again a C-terminal His-tag. After 8 days of growth in BMMY medium with methanol feed the activity in the supernatant reached a maximum volumetric activity of 700 U L−1, corresponding to 10.6 mg of recombinant enzyme per L medium. This translates to a space-time yield of 0.06 mg L−1 h−1, which is significantly lower than the yield obtained in E. coli. No detectable GalOx activity was found in the biomass after cell disruption. In a second approach the native signal sequence of GalOx was replaced with that of the α-factor of S. cerevisiae in the pPICZαA vector. In this case a volumetric activity of 200 U L−1  was found in the supernatant after 8 days of cultivation. Again no GalOx activity was detectable in the biomass. P. pastoris is therefore not only able to recognize the GalOx signal peptide but the production of active enzyme is also >3-fold increased compared to when the α-factor signal sequence is used. This is in contrast to a previous report on the expression of recombinant GalOx from F. graminearum in P. pastorisF. graminearum in different host a threefold increase in volumetric productivity was found for expression in P. pastoris compared to E. coliFor expression in Based on the His-tag, which is provided by all three expression vectors, the protein could be conveniently purified by IMAC followed by a polishing step of size-exclusion chromatography. The His-tag was added C-terminally since this will position the tag away from the active site of the enzyme  in contrE. coli is summarized in −1 was obtained for the purified enzyme. The two-step purification procedure yielded an apparently homogenous protein, as judged by SDS-PAGE .A typical purification procedure of GalOx expressed in SDS-PAGE . Full-leE. coli rather than a 74-kDa band predicted by the DNA sequence including the prepro signal sequence and the His-tag  as well as 266–274/312–323 (2237.9 m/z) were observed when a trypsin/Asp-N digestion was applied . The ideF. oxysporum expressed and purified from the cell extract of E. coli. GalOx exhibited a broad pH optimum  of GalOx calculated from these data was 52°C. Additionally, calorimetric studies of the thermal denaturation of GalOx were performed. Using phosphate buffer at pH 7 the DSC gave a single endothermic peak with a Tm of 64.6°C. To test if different buffer systems have an influence on the temperature stability of the enzyme DSC measurements were performed with citric acid and Tris buffer, each at pH 7. A Tm of 63.7°C and 63.9°C was measured for citric acid and Tris buffer, respectively. Hence these buffer systems have only a small effect on the stability of the enzyme. Based on our studies, the thermostability of GalOx from F. oxysporum expressed in E. coli is significantly lower than the thermostability of the enzyme from other Fusarium strains kcat/Km) was found for 1-methyl-β-D-galactopyranoside (2.2 mM−1 s−1) followed by D-galactose (2.0 mM−1 s−1). Both substrates give similar Km values . The Km value for D-galactose of recombinant GalOx from F. oxysporum is comparable to that of recombinant GalOx from F. graminearum (35 mM) E. coli. These values are lower than the data for native GalOx from F. graminearum with 67 mM m) than to D-galactose but due to an increased kcat value the catalytic efficiency is lower for raffinose. Previous studies P. circinatus  as well as the recombinant GalOx from F. oxysporum  although the Km value for D-galactose is five-times higher for P. circinatus (240 mM). The activity of the enzyme with raffinose, lactose, melibiose, 1-methyl-β-D-galactopyranoside, 1-methyl-α-D-galactopyranoside, lactitol and lactulose indicates that GalOx can oxidize galactose derivatives with substitutes at the carbon-1 site, as previously reported by Alberton et al.F. graminearum produced by a fungal host against D-talose and D-galactosamine, respectively. Because of the high sensitivity of GalOx to the stereo configuration of the C4 hydroxyl group D-glucose is not a substrate for the enzyme. The here reported substrate specificity is in good agreement with previously published results F. oxysporum does not interfere with the catalytic activity. The relatively high Km values for different substrates for GalOx seem to be a consequence of the broad substrate specificity of the enzyme resulting in an active site capable of binding a range of different substrates, but therefore being relatively weak at binding any particular substrate GalOx has a broad substrate specificity, which is one of the most important characteristics of the enzyme gao gene coding for GalOx from F. oxysporum can be easily expressed in the preeminent microbial expression hosts E. coli and P. pastoris even without codon optimization or further amino acid substitutions that have been reported to improve expression in E. coliF. oxysporum is very well comparable in its biochemical and catalytic properties to other fungal GalOx, which is not surprising when considering the well-conserved geometry of the active site and the substrate-binding site in these different enzymes.GalOx is of interest for various biotechnological applications, ranging from biosensors to diagnostic use in medicine as well as biocatalysis Mass spectrometry as a tool for the detection of the Tyr-Cys crosslink could find wider application in the characterization of this unique protein cofactor in GalOx but also in related enzymes. Mass spectrometry does not provide quantitative data for these crosslinks, but is a rapid and standard methodology widely established by now, and thus could replace the indirect methods that are commonly used to characterize unequivocally the formation of the unique protein cofactor in GalOx as well as in related enzymes.


Fusarium sambucinum cultures and overexpressed in Escherichia coli yielding 4.4 mg enzyme per L of growth culture with a specific activity of 159 U mg−1. By adding a C-terminal His-tag the enzyme could be easily purified with a single affinity chromatography step with high recovery rate (90%). The enzyme showed a single band on SDS–PAGE with an apparent molecular mass of 68.5 kDa. The pH optimum for the oxidation of galactose was in the range of pH 6–7.5. Optimum temperature for the enzyme activity was 35 °C, with a half-life of 11.2 min, 5.3 min, and 2.7 min for incubation at 40 °C, 50 °C, and 60 °C, respectively. From all tested substrates, the highest relative activity was found for 1-methyl-β-galactopyranoside (226 U mg−1) and the highest catalytic efficiency (kcat/mK) for melibiose (2700 mM−1 s−1). The enzyme was highly specific for molecular oxygen as an electron acceptor, and showed no appreciable activity with a range of alternative acceptors investigated. Different chemicals were tested for their effect on GalOx activity. The activity was significantly reduced by EDTA, NaN3, and KCN.A gene encoding a galactose oxidase  was isolated from  Because of this specificity, various analytical techniques are based on GalOx, such as the determination of lactose in milk and dairy products d-galactose to food-grade cross-linking agents Galactose oxidase  is the most extensively studied Aspergillus nidulansAspergillusoryzae and Fusarium venenatumPichia pastorisEscherichia coliThe enzyme is secreted by a number of fungal species, of which gao gene without its prepro sequence from Fusarium sambucinum in E. coli. Furthermore, the purification and biochemical characterization of the enzyme are reported. Alternative electron acceptors, and possible activators as well as inhibitors were tested for their effect on GalOx activity.In the present paper we describe cloning and recombinant expression of a new E.coli strain BL21 (DE3) was purchased from Invitrogen , the cloning vector pJET 1.2 was from Fermentas and the expression vector pET21a was from Novagen . The HisPrep FF 16/10 column was from GE Healthcare Bioscience AB . SDS–PAGE protein standard (Precision Plus Protein prestained standard) was from BioRad . The electron acceptors ferrocenium (FcPF6), guaiacol, 2,6-dimethoxyphenol, caffeic acid, p-coumaric acid, ferulic acid, sinapic acid, Thioflavin T, 2-(4′-methylaminophenyl)benzothiazole, 1,1′-diethyl-2,2′-carbocyanine iodide, 1,4-benzoquinone, 2,6-dichloro-indophenol, and ferricyanide were purchased from Sigma–Aldrich. F.sambucinum (synonym Gibberella pulicaris) strain MA1886 was kindly provided by Gerhard Adam .Chemicals for enzyme assays, buffers and media were purchased from Sigma–Aldrich  and were of the highest purity available. 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) was purchased from Amresco . Restriction enzymes, dNTP mix, Rapid DNA Ligation Kit and standard for Agarose gelelectrophoresis (GeneRuler DNA Ladder Mix) were from Fermentas  and the Phusion polymerase was from New England BioLabs . Synthetic oligonucleotides were synthesized by VBC-Biotech . F. sambucinum MA1886 was cultivated in 50 mL Sabouraud medium  in shaken flasks at 25 °C and 110 rpm for 3 days. Fungal mycelia were collected by centrifugation at 4 °C and 5000×g for 15 min and the pellet was washed in 50 mL saline solution . Genomic DNA was isolated from 100 mg of frozen mycelia ground in liquid nitrogen by the phenol–chloroform-extraction as described by Chomczynski and Sacchi gao gene coding for GalOx was amplified by PCR using degenerated primers based on the published sequences from related organisms (Accession Number: FGSG_11032.3/M86819/FOXG_09956.2/FVEG_08555.3): 5′-GCCTCAGCA/TCCC/TA/CTCGG-3′ and 5′-CTGAGTAACGA/CGAAG/TA/CGT-3′, purified by agarose gel electrophoreses and subcloned into the pJET 1.2 cloning vector using the CloneJET PCR Cloning Kit (Fermentas). Restriction sites were introduced using the following forward primers: 5′-TCGCACATATGTACCTTTTGTCACTCGCTC-3′ and 5′-GCTGACATATGGCCTCAGCACCCATTGGA-3′ for gao with and without the prepro sequence, respectively, and 5′-GCTACGCGGCCGCCTGAGTAACGCGAAT-3′ as the reverse primer (restriction sites underlined). Subsequent, the PCR product was digested with NdeI and NotI and cloned in the equally treated expression vector pET21a in frame with the C-terminal His6-tag by the Rapid DNA Ligation Kit. The resulting plasmid was transformed into E. coli BL21 (DE3) by electroporation. DNA sequencing was performed as a commercial service . The amino acid sequence derived from the GalOx gene was used to generate a three-dimensional model based on the published structure of GalOx from F. graminearumE. coli BL21 (DE3) for production of the recombinant enzyme was performed in 30 mL of double concentrated LB medium  with 50 mg L−1 ampicillin in 125-mL baffled flasks. Cells were grown at 37 °C and 120 rpm until reaching an OD600 of 0.4–0.6. Then recombinant protein expression was induced by addition of 5% lactose and cultivation was continued at 25 °C and 130 rpm overnight. The cell pellet after centrifugation was resuspended in 20 mM potassium phosphate buffer pH 7.0, and an aliquot of 500 μL was homogenized by Precellys24 . The cell homogenate was tested for the presence of GalOx activity. Large scale cultivation was done in 1-L baffled flasks containing 300 mL medium Cultivation of g for 20 min and 4 °C, and resuspended in phosphate buffer . After disruption in a French Press at 100 MPa the crude cell extract was separated from cell debris by centrifugation  and used for protein purification by Immobilized Metal Affinity Chromatography (IMAC) with a 20 mL Ni-charged Sepharose 6 Fast Flow column . Before loading the sample the column was equilibrated with 10 column volumes (CV) of buffer A . After the protein sample was applied to the column, it was washed with 3 CV of the same buffer, and eluted in a linear gradient from 0.01 to 1 M imidazole in 10 CV. Fractions containing GalOx activity were pooled and the purity of the purified GalOx was checked by electrophoresis. SDS–PAGE was performed in principle as described by Laemmli The biomass from these cultivations was harvested by centrifugation at 4000×4 for 30 min at 800 rpm and 25 °C. GalOx was measured with the chromogenic ABTS ) assay ε420 = 43.2 mM−1 cm−1) was recorded at 30 °C for 180 s. The standard assay mixture  contained 1 μmol of ABTS in 20 mM potassium phosphate buffer (pH 7.0), 2 U horseradish peroxidase, 100 μmol d-galactose, and a suitable amount of GalOx sample. One Unit of GalOx activity was defined as the amount of enzyme that is necessary for the oxidation of 2 μmol of ABTS per min, which equals the consumption of 1 μmol of O2 per min, under the conditions described above. Protein concentrations were determined at 595 nm by the Bradford assay Prior to activity measurement GalOx was activated by incubation with 1 mM CuSOA pH–activity profile was determined in the range of pH 2.5–10.0 using the buffer systems citric acid (pH 2.5–6.0), potassium phosphate (pH 6.0–8.0) and Tris (pH 8.0–10.0), each at a concentration of 50 mM. Activity measurements were performed otherwise as described for the standard assay.4.Determination of the temperature optimum of GalOx was achieved by measuring the activity with the standard assay at different temperatures in the range of 30–70 °C. Thermal stability of GalOx was determined by incubating the protein at 30, 40, 50 and 60 °C. Samples were taken at various time points, cooled on ice, and residual GalOx activity was measured using the standard ABTS assay after reactivating the enzyme by incubation with CuSOd-Galactose (1–500 mM), 1-methyl-β-galactopyranoside (5–200 mM), melibiose (1–250 mM), raffinose (10–250 mM), and lactose (5–250 mM) were used as substrate. Kinetic constants were calculated by nonlinear least-square regression, fitting the data to the Henri–Michaelis–Menten equation .Steady-state kinetic constants were measured for GalOx for different electron donor substrates. All kinetic measurements were performed at 30 °C in 20 mM phosphate buffer (pH 7.0). Measurement of kinetic constants for various sugar substrates were done with oxygen (air saturation) and the standard ABTS assay. The enzyme reactions were carried out in a glove box  and were followed spectrophotometrically using an Agilent 8453 UV–visible spectrophotometer  at 30 °C, and quantified at wavelengths indicated later in the manuscript . To elim6), 1,4-benzoquinone, 2,6-dichloro-indophenol (DCIP), and ferricyanide. The activity test was performed with 0.2 U GalOx as with the standard assay, but using the respective electron acceptor instead of oxygen. The reaction stoichiometry is one for the two-electron acceptors  and two for the one-electron acceptors (ferrocenium ion and ferricyanide). Furthermore, the oxidized, radical forms of the ABTS cation, the phenols guaiacol, 2,6-dimethoxyphenol (DMP), caffeic acid, p-coumaric acid, ferulic acid and sinapic acid, the benzothiazoles thioflavin T and 2-(4′-methylaminophenyl)benzothiazole (BTA-1) and the cyanine dye 1,1′-diethyl-2,2′-carbocyanine iodide were tested as electron acceptors. Due to the short lifetime of these radicals they were produced immediately before the analyses. The oxidation of the corresponding compound to the radical was performed by recombinant laccase from Botrytis aclada expressed in P.pastorisd-galactose and 1.5 U laccase in 20 mM potassium phosphate buffer pH 6.5 in a final volume of 2.7 mL. The reaction was followed spectrophotometrically. After the oxidation was completed, laccase was inhibited by adding 300 μL 10 mM NaF solution, oxygen was removed by flushing with nitrogen, and the reaction was started immediately by adding 0.2 U GalOx to 1 mL of the radical solution. The negative control was performed without GalOx. For a positive control ascorbate (1.25 mM) was added instead of GalOx.As possible alternative electron acceptors for GalOx, the following compounds were tested: ferrocenium ion  contains no introns and a 37 amino acid prepro sequence. The similarity to the protein sequences of GalOx from F. graminearumFusarium oxysporumF. sambucinum gao gene was used to generate a three-dimensional homology model based on the published structure of mature GalOx (1gog) from F. graminearumMycelium of NdeI and NotI were constructed. The gene was cloned with and without its prepro sequence into the expression vector pET21a, adding a C-terminal His6 tag to the protein. After transformation of the plasmids into E. coli BL21(DE3) different clones were selected, cultivated on a small scale in double-concentrated LB medium, and 5% of lactose was used as inducer for expression of the gao gene with and without its prepro sequence. No active enzyme was found in the clones containing the full-length gao gene containing its prepro sequence. From the clones containing the gao gene without its prepro sequence the best producer was selected and used for larger scale production of GalOx in 1-L shaking flasks. Routinely, 4.4 mg L−1 of active, soluble GalOx  were obtained in shaking flask cultivation after incubation at 25 °C for 16 h. This translates to a space–time yield of 0.28 mg h−1 L−1.Based on the determined nucleotide sequence, modified oligonucleotide primers containing restriction sites for −1. The purification procedure yielded an enzyme preparation that was apparently homogenous as judged by SDS–PAGE  was found for melibiose (2700 M−1 s−1) followed by raffinose (2500 M−1 s−1) whereas the corresponding value for d-galactose was 3-fold lower. The lowest catalytic efficiency was measured for lactose as a result of an unfavorably high Michaelis constant of 683 mM. The lowest measured mK value, 16 mM for melibiose, is still rather high when compared to other carbohydrate-active enzymes, which seems to be due to the broad substrate specificity for GalOx GalOx has a broad substrate specificity, which is one of the most interesting characteristics of the enzyme It is of interest to know whether GalOx can transfer electrons to other acceptors than oxygen. To answer this question we tested a range of alternative electron acceptors, some of which are used by other copper-containing oxidoreductases. The one-electron acceptors included also different organic radicals. Due to the short life span of these radicals they were produced directly prior to their use by oxidation with laccase, which was inhibited with fluoride when the radical-forming reaction was completed. GalOx did not show significant activity with any of the tested electron acceptors . These r2+, K+, Na+, NH4+, and Mn2+ showed no significant effect on the enzyme activity. The nonionic detergent Tween80 and fluoride had also no effect on GalOx activity. The enzyme activity was reduced to less than 50% by EDTA. This result is different to published data Fusarium acuminatum, Gibberella fujikuroi and Polyporus circinatus, respectively, where EDTA did not inhibit activity significantly. As expected other metallo-enzyme inhibitor such as azide and cyanide completely inactivated GalOx.The effect of various compounds, mainly various metal ions, on GalOx activity was determined . Monovalgao gene coding for GalOx from F. sambucinum can be easily expressed in the bacterial expression host E. coli even without codon optimization. A simple one step affinity purification is sufficient to purify the protein with a yield of 90%. GalOx from F. sambucinum is very well comparable in its biochemical and catalytic properties to other fungal GalOx, which is not surprising when considering the well-conserved geometry of the active site and the substrate-binding site in these enzymes. Because of its similar biochemical properties and its simple and efficient purification protocol this new enzyme could be an alternative for GalOx from other sources.GalOx is of interest for a number of biotechnological applications. Because of this interest, more detailed knowledge about GalOx from different sources is important since this might reveal novel or improved areas of application. The The authors declare no conflict of interest.


Since some bacteria are capable of vigorous γ-PGA biosynthesis from renewable biomass, γ-PGA is considered a promising bio-based chemical and is already widely used in the food, medical, and wastewater industries due to its biodegradable, non-toxic, and non-immunogenic properties. In this review, we consider the properties, biosynthetic pathway, production strategies, and applications of γ-PGA. Microbial biosynthesis of γ-PGA and the molecular mechanisms regulating production are covered in particular detail. Genetic engineering and optimization of the growth medium, process control, and downstream processing have proved to be effective strategies for lowering the cost of production, as well as manipulating the molecular mass and conformational/enantiomeric properties that facilitate screening of competitive γ-PGA producers. Finally, future prospects of microbial γ-PGA production are discussed in light of recent progress, challenges, and trends in this field.Poly-γ-glutamic acid (γ-PGA) is a naturally occurring biopolymer made from repeating units of The online version of this article (doi:10.1186/s13068-016-0537-7) contains supplementary material, which is available to authorized users. At present, there exist four methods for γ-PGA production: chemical synthesis, peptide synthesis, biotransformation, and microbial fermentation [Bacillus anthracis, γ-PGA has since been found in species from all three domains of life  [Poly-γ-glutamic acid (γ-PGA) is an unusual anionic homopolyamide made from d groups  (Additioentation . Comparearyotes) , 4. MostUnlike most proteinaceous materials, γ-PGA is synthesized in a ribosome-independent manner; thus, substances that inhibit protein translation (such as chloramphenicol) have no effect on the production of γ-PGA . FurtherAlthough the microbial production of γ-PGA is well established, the cost of production, including the cost of substrates as well as process costs, remains high. Most recent research on γ-PGA production is therefore focused on optimizing growth conditions to increase yield, manipulate enantiomeric composition, and alter the molecular mass. Surprisingly, only a small number of mini reviews on the biosynthesis and applications of γ-PGA have been published to date , 6–9. Thl or d enantiomers is soluble in ethanol, whereas γ-PGA containing equimolar amounts of l and d precipitates in ethanol [Generally, γ-PGA adopts five conformations; α-helix, β-sheet, helix-to-random coil transition, random coil, and enveloped aggregate. The conformation can be changed by altering environmental conditions such as pH, polymer concentration, and ionic strength . For exa ethanol . Manipul ethanol .5–8 × 106 Da), which can limit industrial applications due to high viscosity, unmanageable rheology, and difficult modification [The molecular mass of γ-PGA can also influence its properties and efficacy for specific applications. Microbial-derived γ-PGA generally has a relatively high molecular weight  [Recently, information about the genes and enzymes involved in γ-PGA synthesis has been reported and has contributed to the design of production systems , 8. As secursor) . Biosyntd- or l-glutamate alone, or from both l and d enantiomers together [d-glutamate into the growing l-chain, l-glutamate (exogenous or endogenous) is first converted into d-glutamate by a racemization reaction. In B. subtilis, two homologs of the glutamate racemase gene (racE/glr and yrpC) have been identified, and glr is essential for converting l-glutamate into d-glutamate for the synthesis of γ-PGA [l-form, but neither are responsible for the synthesis of γ-PGA [Generally, γ-PGA is synthesized from together , 20. Howof γ-PGA . Interesof γ-PGA . The funof γ-PGA , 23.pgsB, C, A, and E) and their homologs in Bacillus species are ywsC, ywtAB, and capBCA [2+ in B. subtilis [As shown in Fig. d capBCA , 24. Recd capBCA . PgsB and capBCA . The rold capBCA . Howeversubtilis . This masubtilis .Fig. 2ArdegQ prevents the synthesis of γ-PGA and effectively downregulates the production of degradation enzymes [γ-PGA synthesis is regulated by two signal transduction systems: the ComP-ComA regulator, and the two-part DegS-DegU, DegQ, and SwrA system . The rol enzymes . However enzymes . In cont enzymes . Overall enzymes .Bacilli: endo-γ-glutamyl peptidase and exo-γ-glutamyl peptidase [B. subtilis and B. licheniformis, where it is able to cleave high molecular weight γ-PGA into fragments of 1000 Da to 20 kDa, which decreases dispersity as a function of depolymerization time [B. subtilis, the genes encoding endo-γ-glutamyl peptidase  are located directly downstream of, and in the same orientation as, the pgsBCA operon  and a cleavage site (30A-E-A32) proximal to the N-terminus, indicating that the mature enzyme is secreted into the medium [There are two enzymes capable of degrading γ-PGA in eptidase . Endo-γ-ion time , 34, 35.e medium .B. anthracis, but not for γ-PGA synthesis [2O, resulting in transpeptidation or hydrolysis, respectively [B. subtilis, ggt and capD are located on the chromosome distant from the pgsBCA cluster and expressed during the stationary phase under the control of the ComQXPA quorum-sensing system, but are located on a plasmid directly downstream from the pgsBCA cluster in B. anthracis [Exo-γ-glutamyl peptidase (Ggt) is a key enzyme in glutathione metabolism, and catalyzes the formation of γ-glutamic acid di- and tripeptides in vitro, but does not appear to be involved in γ-PGA synthesis in vivo , 37. Forynthesis . As a meectively . GTTs diectively . In B. snthracis .B. subtilis strains deficient in exopeptidase are unable to cleave γ-PGA into fragments smaller than 105 kDa, and they sporulate earlier than wild-type strains [As mentioned above, γ-PGA can be anchored to the bacterial surface or released into the medium, and CapD catalyzes the anchorage of γ-PGA to the peptidoglycan, whereas PgsS catalyzes its release. Therefore, inhibiting or knocking down γ-PGA hydrolase can result in the production of high molecular weight γ-PGA . Indeed, strains .Bacillus species, Fusobacterium nucleatum, and some archaea and eukaryotes [Bacillus species are used most widely to study biological γ-PGA production. Bacteria are either l-glutamate-dependent  or non-tilis C1  and B. aiens LL3 ) produceuctivity . TherefoB. amyloliquefaciens [B. subtilis [E. coli [To this end, most research on γ-PGA fermentation has focused on optimizing growth conditions to improve γ-PGA yield, alter the enantiomeric composition, and manipulate the molecular mass of γ-PGA . Additioefaciens , B. subtsubtilis , and E. [E. coli  has alsoBacillus species have been established as γ-PGA producers, and native strains can produce more than 20 g/L of γ-PGA in fermentation processes. As shown in Table Bacillales. Most γ-PGA producers can therefore be divided into two groups: Group I = Bacillus species; Group II = other bacteria.Numerous Bacillus subtilis is a Gram-positive, endospore-forming, rod-shaped bacteria that has generally been recognized as having a safe (GRAS) status and can therefore be used to produce enzymes such as alpha amylase and proteases that are used in the food and medicine industries. Isolation of B. subtilis strains with excellent γ-PGA production abilities has been achieved due to its ubiquitous and sporulating nature. As shown in Table B. subtilis strains have been widely used for producing γ-PGA, and B. subtilis CGMCC 1250 produces 101.1 g/L γ-PGA, demonstrating the potential this organism has for γ-PGA production [Bacillus licheniformis, Gram-positive, endospore-forming bacterium, shares many similarities with B. subtilis, and this non-pathogenic organism has also been exploited for the production of γ-PGA.oduction . More imBacillus species discussed above, Bacillus methylotrophicus SK19.001 should also be noted, because it yields a high level of γ-PGA with an ultrahigh molecular weight [B. anthracis and Bacillus thuringiensis also have the capacity for γ-PGA production [B. anthracis is not viable owing to its toxicity [Other than the two r weight . Other soduction , but thetoxicity .With the development of metabolic engineering, homologous hosts have been engineered for γ-PGA production Table . HoweverEscherichia coli is the most commonly used host for γ-PGA biosynthesis, and the γ-PGA synthase genes pgsBCA and racE from B. licheniformis NK-03 and B. amyloliquefaciens LL3 were, respectively, cloned and co-expressed in E. coli JM109 to evaluate γ-PGA production [l-glutamate, and co-expression of the racE gene further increased the production of γ-PGA to 0.65 g/L. Another similar study was carried out using Corynebacterium glutamicum as the host, clone, and expression of the γ-PGA synthase genes pgsBCA from Bacillus subtilis TKPG011. The production of γ-PGA reached 18 g/L when the combinant was cultured with the limitation of biotin [Expression of γ-PGA-producing genes in heterologous hosts has been attempted Table . Escherioduction . The engf biotin . Those sAs shown in Fig. Other than glucose which is the most successful carbon substrate for γ-GPA production from a variety of biomass materials, cane molasses, xylose, agro-industrial wastes, rapeseed meal, soybean residue, fructose,corncob fibers, hydrolysate, and crude glycerol have also been tested Tables , 2. Alth4)2SO4 or NH4Cl [2+ in particular can improve cell growth, prolong cell viability, and assist the utilization of different carbon sources, as well as significantly alter the stereochemical and enantiomeric composition of γ-PGA, and increase γ-PGA production [Additionally, much work has been carried out on the nutritional requirements for cell growth to improve γ-PGA productivity and modify the D/L composition of the polymer. For an exogenous glutamate-independent producer, yeast extract proved to be an excellent nitrogen source for bacterial cell growth and γ-PGA production, but the high cost is a barrier to commercial production . Therefoor NH4Cl  Table 142SO4 or oduction .Efficient and effective control of fermentation depends on an understanding of the key biological and chemical parameters , and disB. subtilis batch fermentation process, and 0.3 % n-heptane increased to 39.4 g/L and molecular weight 19.0 × 105 Da [Oxygen is essential in aerobic fermentation and affects cell growth, carbon source utilization, biosynthesis of products, and NAD(P)H recycling . Various× 105 Da .Table 3AB. licheniformis ATCC 9945A [B. subtilis IFO 3335 [Culture pH is another important environmental factor in γ-PGA fermentation . A pH ofCC 9945A , whereasIFO 3335 . HoweverIFO 3335 .In industrial fermentation, the choice of reactor operation mode may be vital for achieving optimal process design. A series of operation modes should be tested at small scale, such as batch, fed-batch, continuous culture, cell recycling, and cell immobilization, all of which may have their own advantages and disadvantages. For example, continuous culture can be operated at a steady state with continuous feeding, which can enhance productivity and/or lower labor intensity, but a high yield may be difficult to achieve. For γ-PGA production, batch and fed-batch are the most common fermentation strategies and, overall, the batch mode has tended to achieve a higher product yield and productivity and is the most promising method for industrial-scale γ-PGA fermentation Table .l-glutamic acid, symbiotic fermentation was also proposed and developed, in which the l-glutamate-dependent B. subtilis was co-cultured with Corynebacterium glutamicum using glucose and sucrose as a mixed carbon source. Thus, integrated bioprocesses have advantages that included shortening the fermentation time and reducing the production cost, and produced γ-PGA with an average molecular mass of 1.24 × 106 Da [To avoid the addition of exogenous × 106 Da .During microbial fermentation, downstream processing is always a key issue for improving process economy. As discussed above, γ-PGA fermentation is influenced by various nutritional and environmental parameters, and the effects of these variables on product recovery should be assessed. For example, excessive use of complex raw materials will pose difficulties for product isolation.2+, Al3+, Cr3+, and Fe3+, and Cu2+ is the most efficient metal ion for selectively precipitating γ-PGA, even at a low concentration [There exist three fundamentally different approaches to recovering γ-PGA from the culture broth: precipitation by complex formation, precipitation by reducing water solubility, and filtration . In all ntration . The resntration . Comparentration . Finallyntration . For exantration .Due to being water soluble, biodegradable, edible, and non-toxic, γ-PGA and its derivatives have been applied in a broad range of industrial fields, including food, cosmetics, agriculture, medicine, and bioremediation Table .Table 4Anatto (fermented soybeans), but also as a food supplement, osteoporosis-preventing agent, texture enhancer, cryoprotectant, and oil-reducing agent , and this significantly improved both the safety and efficiency of the drug (compared with standard paclitaxel) by enhancing its pharmacokinetic profile and water solubility. Furthermore, this improved tumor selectivity via enhanced accumulation and retention in tumor tissue [As shown in Table r tissue .6 Da appears to be superior to many conventional flocculants used in wastewater treatment plants operating downstream of food processing fermentation processes [5 Da could effectively remove 98 % of basic dyes from aqueous solution at pH 1 and could then be re-used [Due to its non-toxic and biodegradable properties, γ-PGA offers an eco-friendly alternative for wastewater treatment. γ-PGA with a molecular weight of ~5.8–6.2 × 10rocesses . More in re-used .γ-PGA has also been explored for use in cosmetics as a hydrophilic humectant to increase the production of natural moisturizing agents such as urocanic acid, pyrrolidone carboxylic acid, and lactic acid . Many otDuring more than 70 years of γ-PGA-related research, great insight has been gained regarding its production, metabolic regulation, and applications. Owing to its biodegradability and non-toxic and non-immunogenic properties, it is used widely in the food, medicine, and wastewater industries. Biotechnological production of natural γ-PGA from renewable biomass continues to be of significant interest, especially in the face of decreasing fossil fuels and a need to reduce carbon emissions.A lot of research has been carried out on the molecular biology  of γ-PGA and its biosynthesis in different organisms, some of which have been applied to improving its production , 8, 73. The specific properties of γ-PGA determine its applications, and γ-PGA produced by different bacteria or culture conditions may therefore be suited to different uses. Optimization of the cost of production, molecular mass, and conformational/enantiomeric properties is crucial if the potential of γ-PGA is to be fully realized . For insWith the increasing trend in using biomass as a carbon source for fermentation processes, much research into the biological production of γ-PGA has aimed at improving the cost-effectiveness and the efficiency of recovery. To realize better industrial production of γ-PGA from renewable biomass, further effort should be made in this area. For example, high-throughput screening of potential new producers should include thermo- and salt-tolerant bacterial extremophiles . AdditioA greater understanding of the molecular regulatory mechanisms of γ-PGA biosynthesis and control of stereoisomers would undoubtedly prove valuable. Therefore, a systems approach that combines synthetic biology, metabolic engineering, and traditional fundamental research will likely lead to improved fermentative production of γ-PGA from renewable biomass.