Development of a novel Pichia pastoris expression platform via genomic integration of lipase gene for sustained release of methanol from methyloleate.

A novel Lip+ Pichia pastoris expression platform was developed by integrating lipase Lip2 from Yarrowia lipolytica under constitutive Glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. Effective expression of reporter protein amylase from Bacillus licheniformis was achieved utilizing methyloleate in Lip+Amy+host. Lipase hydrolyzed methyloleate into methanol that sustained PAOX1 induction, and oleic acid, which was readily utilized as a carbon source. The protein expression achieved in presence of methyloleate was comparable to methanol-induced cells, along with an increase in productive biomass. In Lip+Amy+ host, total amylase production of 220.9 ± 13 U/mg biomass was achieved at 96 h using methyloleate supplemented every 24 h. While 206.0 ± 17 U/mg biomass was obtained at 108 h in an Amy+ host induced with methanol every 12 h. Further, lipase expression neither affected growth nor added additional burden on the cellular machinery and no oleic acid accumulation was observed at any time point due to its emulsification and efficient utilization by lipase positive host. Similar results obtained with the second reporter protein γ-cyclodextrin glycosyltransferase (CGTase) from Evansella caseinilytica validated the platform. An alternate lipase Lip11 from Y. lipolytica was also employed in developing a Lip+ host to validate disparity between lipase background and PAOX1 induction in presence of methyloleate.

Functional characterization of hormone sensitive-like lipase from Bacillus halodurans: synthesis and recovery of pNP-laurate with high yields.

The HSL-like lipase encoding gene (Blip) from the polyextremophile Bacillus halodurans C-125 has been heterologously expressed in E.coli BL21(DE3). The enzyme is a monomer of ~42 kDa. It has extremely high thermal stability with a t 1/2 of 35 min at 100 °C. Thermal denaturation/renaturation studies by CD and fluorescence analysis revealed complete refolding of the protein back to its native conformation even after 30 min at 90 °C. Blip prefers substrates with mid to long chain fatty acids. It has a higher catalytic efficiency on para-nitrophenyl fatty acyl esters as opposed to triacylglycerides (k cat/K m with pNP-palmitate as a substrate was 2.52 × 105 mM-1 min-1 while that with glyceryl tripalmitin was 4.06 × 102 mM-1 min-1, respectively). The enzyme also has a unique selectivity for hydrolysis of unsaturated fatty acyl esters. The enzyme catalyses the synthesis of pNP-laurate with an optimized conversion of 95.94 ± 0.24%. A simple procedure for purification of the product has been developed that led to 89.91 ± 0.33% product recovery.

Heterologous expression of lipases YLIP4, YLIP5, YLIP7, YLIP13, and YLIP15 from Yarrowia lipolytica MSR80 in Escherichia coli: Substrate specificity, kinetic comparison, and enantioselectivity.

Five lipase genes, ylip4, ylip5, ylip7, ylip13, and ylip15, from Yarrowia lipolytica MSR80 were cloned and expressed in the pEZZ18-HB101 system. The lipases shared maximum sequence identity with Candida galli lipase, whereas they shared structural similarity with YLIP2 of Y. lipolytica CLIB122. The enzymes, purified using IgG sepharose, had specific activities in the range of 7-25 U mg-1 . Biochemical characteristics of all the lipases varied with respect to thermostability, substrate specificity, and enantioselectivity. All the enzymes were most active at neutral or slightly alkaline pH and were stable in the pH range 3.0-8.0, except YLIP4, which showed 50% stability at pH 10.0. Temperature optima of all the lipases varied from 30 to 50 ºC. YLIP15 and YLIP13 were most thermostable with a t1/2 of 138 and 112 Min, respectively, at 60 °C. The lipases exhibited varied substrate specificity on p-nitrophenyl esters ranging from short-chain specificity (YLIP15), mid-chain specificity (YLIP4, YLIP5, YLIP7), and long-chain specificity (YLIP13). Catalytic efficiency on p-nitrophenylcaprate was highest for YLIP13 (67 × 103 mM-1 min-1 ) and lowest for YLIP15 (6.7 × 103 mM-1 min-1 ). YLIP13 was S-enantioselective, and YLIP15 was R-enantioselective with enantiomeric excess of 53 and 36%, respectively. Of all five lipases, YLIP13 and YLIP15 could be considered as industrially important enzymes as they were thermostable and enantioselective.

Hyperproduction of γ-glutamyl transpeptidase from Bacillus licheniformis ER15 in the presence of high salt concentration.

BACKGROUND: Microbial γ-glutamyl transpeptidases (GGTs) have been exploited in biotechnological, pharmaceutical, and food sectors for the synthesis of various γ-glutamyl compounds. But, till date, no bacterial GGTs are commercially available in the market because of lower levels of production from various sources. In the current study, production of GGT from Bacillus licheniformis ER15 was investigated to achieve high GGT titers. RESULTS: Hyperproduction of GGT from B. licheniformis ER15 was achieved with 6.4-fold enhancement (7921.2 ± 198.7 U/L) by optimization of culture medium following one-variable-at-a-time strategy and statistical approaches. Medium consisting of Na2HPO4: 0.32% (w/v); KH2PO4: 0.15% (w/v); starch: 0.1% (w/v); soybean meal: 0.5% (w/v); NaCl: 4.0% (w/v), and MgCl2: 5 mM was found to be optimal for maximum GGT titers. Maximum GGT titers were obtained, in the optimized medium at 37°C and 200 rpm, after 40 h. It was noteworthy that GGT production was a linear function of sodium chloride concentration, as observed during response surface methodology. While investigating the role of NaCl on GGT production, it was found that NaCl drastically decreased subtilisin concentration and indirectly increasing GGT recovery. CONCLUSION: B. licheniformis ER15 is proved to be a potential candidate for large-scale production of GGT enzyme and its commercialization.

Selective disruption of disulphide bonds lowered activation energy and improved catalytic efficiency in TALipB from Trichosporon asahii MSR54: MD simulations revealed flexible lid and extended substrate binding area in the mutant.

TALipB (33 kDa) is a solvent stable, enantioselective lipase from Trichosporon asahii MSR54. It is cysteine-rich and shows activation in presence of thiol reducing agents. DIANNA server predicted three disulphide bridges C53-C195 (S1), C89-C228 (S2) and C164-C254 (S3) in the enzyme. Selective disruption of disulphide bonds by cysteine to alanine mutations at Cys53 and Cys89 of S1 and S2 bonds resulted in enzyme activation. Mutant mTALipB (S1+S2) showed increase in specific activity by ∼4-fold (834 mM/mg) and improved Vmax of 6.27 µmol/ml/min at 40 °Con pNP caprate. Temperature optima of mTALipB shifted from 50 to 40 °C and activation energy decreased by 0.7 kcal mol(-1). However, the mutant was less thermostable with a t1/2 of 18 min at 60 °C as compared to t1/2 of 38 min for the native enzyme. Mutant also displayed an improved activity on all pNP esters and higher enantiomeric excess (61%) during esterification of (±) 1-phenylethanol. Far-UV CD analysis showed significant changes in secondary structure after S-S bridge disruption with 7.16% decrease in α-helices and 1.31% increase in ß-sheets. In silico analysis predicted two lids (α5 and α9) in TALipB. Molecular dynamic simulations at 40 °C and 50 °C revealed that in the mTALipB, both the lids opened at 40 °C with clockwise and anticlockwise rotations in Lid1 and Lid2, respectively. In the native protein, however, the lid was only partially open even at 50 °C. Concomitant to lid flexibility, there was an extension of accessible catalytic triad surface area resulting in improved catalytic efficiency of the mutant enzyme.

Autonomic Function Tests in Prehypertensive Young Adult Males of Uttarakhand Region.

Pre-hypertensives are at increased risk for rise in blood pressure and cardio vascular morbidities. Autonomic dysfunction is both a cause and effect of high blood pressure. Considering its serious prognosis the current study was undertaken with an aim to assess autonomic functions in pre-hypertensive young adult males. Male volunteers of 20-40 years were divided into normotensives (n=44) and clinically healthy pre-hypertensives (n=44). They were subjected to anthropometric measurements, Heart Rate variability (HRV) analysis along with Standard Isometric Handgrip Test (HGT) and Sinus arrhythmia test. Analysis showed a significant increase in sympathetic activity among prehypertensives characterized by lower rise in DBP (mmHg) in response to HGT: [11.27±3.76 mmHg vs 23.95±6.29 mmHg, (p<0.05)]. Parasympathetic activity was significantly decreased among prehypertensives (HFnu: 30.02±14.43 vs 41.62±14.91, p<0.05); E/I ratio: 1.20±0.10 vs 1.32±0.10, p<0.05). Sympathovagal balance showed prominent sympathetic activity (2.85±1.78 vs 1.75±0.98; (p<0.01) among prehypertensives. Salt intake among prehypertensives was significantly higher than normotensives (21.2±1.2 gm/day vs 9.0±0.5 gm/day: P<0.001). Conclusion: Autonomic dysfunction is evident in prehypertensives with an enhanced sympathetic activity, decreased parasympathetic activity and an altered sympathovagal balance in prehypertensives, salt intake was found on a higher side. Thus lifestyle changes in the form of balanced diet and moderate exercise may be advised to attenuate the risk progression of prehypertension to hypertension.

Extracellular expression of YlLip11 with a native signal peptide from Yarrowia lipolytica MSR80 in three different yeast hosts.

Lipase YlLip11 from Yarrowia lipolytica was expressed with a signal peptide encoding sequence in Arxula adeninivorans, Saccharomyces cerevisiae and Hansenula polymorpha using the Xplor®2 transformation/expression platform and an expression module with the constitutive Arxula-derived TEF1 promoter. The YlLip11 signal peptide was functional in all of the yeast hosts with 97% of the recombinant enzyme being secreted into the culture medium. However, recombinant YlLip11 with His Tag fused at C-terminal was not active. The best recombinant YlLip11 producing A. adeninivorans G1212/YRC102-YlLip11 transformant cultivated in shake flasks produced 2654 U/L lipase, followed by S. cerevisiae SEY6210/YRC103-YlLip11 (1632U/L) and H. polymorpha RB11/YRC103-YlLip11 (1144U/L). Although the biochemical parameters of YlLip11 synthesized in different hosts were similar, their glycosylation level and thermo stability differed. The protein synthesized by the H. polymorpha transformant had the highest degree of glycosylation and with a t1/2 of 60min at 70°C, exhibited the highest thermostability.

Functional characterization of a novel aspartic acid rich lipase, TALipC, from Trichosporon asahii MSR54: solvent-dependent enantio inversion during esterification of 1-phenylethanol.

A novel lipase gene TAlipC was isolated from Trichosporon asahii MSR54 and functionally expressed in Pichia pastoris. The protein was His-tagged and purified to homogeneity by affinity chromatography. Sequence comparison revealed a high homology with lipases from Cryptococcus sp. It had a GX type oxyanion hole and a GHSLG-type conserved penta-peptide similar to those in the lipases from Yarrowia lipolytica. The enzyme had optimal activity at pH 8 and 50 °C. It was specific for long chain fatty acid groups of p-nitrophenol esters and triacylglycerols, showing regio- and enantio-selectivity. It was activated by Mg(2+) ions (20 mM) and had a predicted Mg-binding domain at the aspartic acid-rich C-terminal. Solvent-based enantio- inversion was the key feature of the enzyme where it showed (S)-selectivity in 1,4-dioxane and 2-propanol and (R)-selectivity in hexane during chiral separation of (±)1-phenylethanol by esterification.

Revisiting microbial keratinases: next generation proteases for sustainable biotechnology.

Keratinases are special proteases which attack the highly recalcitrant keratin substrates. They stand apart from the conventional proteases due to their broad substrate specificity towards a variety of insoluble keratin rich substrates like feather, wool, nail, hair. Owing to this ability, keratinases find immense applications in various environmental and biotechnological sectors. The current boost in keratinase research has come up with the discovery of the ability of keratinases to address the challenging issue of prion decontamination. Here we present a comprehensive review on microbial keratinases giving an account of chronological progress of research along with the major milestones. Major focus has been on the key characteristics of keratinases, such as substrate specificity, keratin degradation mechanisms, molecular properties, and their role in prion decontamination along with other pharmaceutical applications. We conclude by critically evaluating the present state of the keratinases discussing their commercial status along with future research directions.

A hydrolytic γ-glutamyl transpeptidase from thermo-acidophilic archaeon Picrophilus torridus: binding pocket mutagenesis and transpeptidation.

γ-Glutamyl transpeptidase of a thermo-acidophilic archaeon Picrophilus torridus was cloned and expressed using E. coli Rosetta-pET 51b(+) expression system. The enzyme was expressed at 37 °C/200 rpm with γ-GT production of 1.99 U/mg protein after 3 h of IPTG induction. It was improved nearby 10-fold corresponding to 18.92 U/mg protein in the presence of 2 % hexadecane. The enzyme was purified by Ni(2+)-NTA with a purification fold of 3.6 and recovery of 61 %. It was synthesized as a precursor heterodimeric protein of 47 kDa with two subunits of 30 kDa and 17 kDa, respectively, as revealed by SDS-PAGE and western blot. The enzyme possesses hydrolase activity with optima at pH 7.0 and 55 °C. It was thermostable with a t (1/2) of 1 h at 50 °C and 30 min at 60 °C, and retained 100 % activity at 45 °C even after 24 h. It was inhibited by azaserine and DON and PMSF. Ptγ-GT shared 37 % sequence identity and 53 % homology with an extremophile γ-GT from Thermoplasma acidophilum. Functional residues identified by in silico approaches were further validated by site-directed mutagenesis where Tyr327 mutated by Asn327 introduced significant transpeptidase activity.

Biotechnological applications and prospective market of microbial keratinases.

Keratinases are well-recognized enzymes with the unique ability to attack highly cross-linked, recalcitrant structural proteins such as keratin. Their potential in environmental clean-up of huge amount of feather waste has been well established since long. Today, they have gained importance in various other biotechnological and pharmaceutical applications. However, commercial availability of keratinases is still limited. Hence, to attract entrepreneurs, investors and enzyme industries it is utmost important to explicitly present the market potential of keratinases through detailed account of its application sectors. Here, the application areas have been divided into three parts: the first one is dealing with the area of exclusive applications, the second emphasizes protease dominated sectors where keratinases would prove better substitutes, and the third deals with upcoming newer areas which still await practical documentation. An account of benefits of keratinase usage, existing market size, and available commercial sources and products has also been presented.

The influence of the 2:1 yogic breathing technique on essential hypertension.

In 2:1 breathing exhalation is twice of inhalation. The study was performed to study the influence of 2:1 yogic breathing technique on patients of essential hypertension. 30 patients of essential hypertension between ages of 20-50 years were selected. After a rest of 15-20 minutes in a comfortable sitting posture their baseline physiological parameters recorded on a digital polygraph were, Electromyogram (EMG), Galvanic skin response (GSR), Finger tip temperature (FTT), Heart rate(HR) and Respiratory rate(RR). Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were recorded by automated digital Sphygmomanometer. Then they were guided to do 2:1 breathing maintaining respiratory rate of around 6/min. Subjects were then instructed to do 2:1 breathing twice a day for 5-7 minutes for next 3 months. Subjects reported back weekly for recording of BP. The physiological parameters of the subjects were assessed again by polygraph at the end of three months of practicing 2:1 yogic breathing. The mean fall of SBP over 12 weeks was 12 mm Hg (8%) and DBP was 7 mm Hg (7%). P value < 0.001 in both. After practicing 2:1 breathing for 3 months there was statistically significant reduction of SBP, DBP, HR RR, EMG, GSR and rise in FTT. The study showed that 2:1 breathing technique caused a comprehensive change in body physiology by altering various parameters that are governed by the autonomic nervous system. It is an effective modality for management of essential hypertension.

Extracellular expression and characterization of thermostable lipases, LIP8, LIP14 and LIP18, from Yarrowia lipolytica.

Two new lipases, LIP14 and LIP18, along with LIP8 from Yarrowia lipolytica MSR80 were functionally expressed as extracellular proteins with an IgG tag using Escherichia coli HB101 pEZZ18 host vector system. Each enzyme had an optimal activity at pH 7 and 40 °C and was activated by 6 mM Ca(2+) and 90 % (v/v) non-polar solvents but inhibited by 10 mM of each 1,10-phenanthraline, DTNB, PMSF and N-bromosuccinamide. All the enzymes were thermostable with t(1/2) of 52 min, 49 min and 68 min for LIP8, LIP14 and LIP18 at 80 °C, respectively. LIP18 was most thermostable among all with a high arginine: lysine ratio and proline content. All the three lipases showed a preference for oleic acid rich triacylglycerols and oils.

Comparative biochemical characterization and in silico analysis of novel lipases Lip11 and Lip12 with Lip2 from Yarrowia lipolytica.

Novel lipases lip11 and lip12 from Yarrowia lipolytica MSR80 were cloned and expressed in E. coli HB101 pEZZ18 system along with lip2. These enzymes were constitutively expressed as extracellular proteins with IgG tag. The enzymes were purified by affinity chromatography and analyzed by SDS-PAGE with specific activity of 314, 352 and 198 U/mg for Lip2, Lip11 and Lip12, respectively on olive oil. Biochemical characterization showed that all were active over broad range of pH 4.0-9.0 and temperature 20-80 °C with optima at pH 7 and 40 °C. All the three lipases were thermostable up to 80 °C with varying t(½). Activity on various substrates revealed that they were most active on oils > triacylglycerides > p-np-esters. Relatively Lip2 and Lip11 showed specificity for mid to long chain fatty acids, while Lip12 was mid chain specific. GC analysis of triolein hydrolysis by these lipases revealed that Lip2 and Lip11 are regioselective, while Lip12 is not. Effect of metal ions showed that Lip2 and Lip12 were activated by Ca²âº whereas Lip11 by Mg²âº. All were thiol activated and inhibited by PMSF and N-bromosuccinimide. All were activated by non polar solvents and inhibited by polar solvents. Detailed sequence analysis and structural predictions revealed Lip11 and Lip12 shared 61 and 62 % homology with Lip2 (3O0D) and three dimensional superimposition revealed Lip2 was closer to Lip11 than to Lip12 as was observed during biochemical characterization. Finally, thermostability and substrate specificity has been explained on the basis of detailed amino acid analysis.

Effects of ergoreflex on respiration and other efferent effects in adult male patients with chronic obstructive pulmonary disease.

In COPD, overactivation of ergoreceptors may be a link between peripheral changes, sympathetic over activation and increased hemodynamic and ventilatory responses to exercise. The current study was undertaken to test the hypothesis that, ergoreflex in COPD patients is hyperactive and contributes to breathlessness and exercise limitation. The aim of study was to (1) demonstrate its hyperactivity in COPD (2). To record other efferent effects. Twenty stable COPD male patients aged 50 +/- 2.5 years and twenty healthy male subjects aged 48 +/- 3.5 years were studied under experimental and control groups. The ergoreflex contribution to cardio respiratory parameters was assessed by post-handgrip regional circulatory occlusion method (PH-RCO) and computed as the difference in heart rate and respiratory rate response between PH-RCO and control run without PH-RCO. Results were analyzed for significance between two groups by repeated measures ANOVA. COPD patients showed over activation of ergoreflex as compared to control subjects in terms of heart rate during sustained hand grip (SHG) exercise (117 +/- 1.22 versus 89 +/- 0.89) beats/min, recovery heart rate (p < 0.001), and respiratory rate during SHG (24 +/- 0.54 versus 19 +/- 03.24) breaths/min and recovery respiratory rate (p < 0.001). Degree of overactivation of ergoreflex was significant in COPD patients (p < 0.001). In COPD, overactivity of ergoreflex is associated with abnormal cardio respiratory reflex control. COPD patients showed overactivation of sympathetic nervous system as evidenced by heart rate changes during exercise and delayed recovery.

Clinical validation of aneroid sphygmomanometer.

Recent concerns about the mercury toxicity and its ill effects on the environment and health has led to widespread use of aneroid manometers. Present study was conducted to analyse whether this change would lead to any systematic shift in measured blood pressure or consistency of blood pressure measurement in clinical setting. The clinical accuracy of the Welch Allyn aneroid sphygmomanometer model 7670-04 was studied against the mercury sphygmomanometer on 83 volunteers from Dehradun. Two blood pressure reading of each study subject was recorded with pretested instruments (aneroid and mercury sphygmomanometer). Data analysis showed the difference of means between the reading of two devices against mean of the observer reading for both systolic (-3.62 +/- 4.88) and diastolic (-2.36 +/- 3.77) blood pressure were not statistically different. The corresponding values of the SBP and DBP from both the instruments showed significant correlation. Regression analysis of mercury versus aneroid showed regression line (Y = 9.52 + 0.95X for SBP, Y = 0.36 + 0.96X for DBP) significantly different from line of equality (P < 0.001). The study has demonstrated that the aneroid device (model: 7670-04) achieved grade B performance according to the British Hypertensive Society criteria.

Cell surface expression of γ-CGTase from Evansella caseinilytica on E. coli: Application in the enzymatic conversion of starch to γ-cyclodextrin.

The γ-cyclodextrin glycosyltransferase (γ-CGTase) from Evansella caseinilytica was expressed on the cell surface of E. coli using pAIDA-I autotransporter and was further utilized in the conversion of starch to γ-cyclodextrins (CDs). Maximum cyclization activity of 2.28 ± 0.46 U/g biomass was achieved after 3 h of induction using 0.1 mM IPTG at 37 ºC. Surface expression of γ-CGTase was confirmed using flow cytometry employing a FITC-conjugated anti-HIS antibody. Biochemical characterization of surface-displayed γ-CGTase revealed optima at pH 10.0 and 40 ºC along with a t1/2 of 24.75 min at 50ºC. The Km and Vmax values on soluble potato starch were 10.94 mg/ml and 4.33 µmoles min-1 g-1 DCW respectively, and the activation energy was calculated to be 89.8 kJ/mol. The surface displayed γ-CGTase was further utilized for CD production and specifically, γ-CD conversion was obtained. The maximum conversion was achieved at 50 ºC, pH 9.0 using soluble potato starch (2.5%; w/v) taking a final enzyme concentration of 0.6 U/g starch. The surface-displayed γ-CGTase was able to convert soluble potato starch (2.5%) into γ-CDs with a 72.7% specific yield and no other peaks corresponding to α- and ß-CDs were observed on HPLC. The enzyme was found to be ~100% operationally stable for up to 2 consecutive cycles of 24 h, with > 75% storage stability at - 20 ºC even after 7 days.

Gamma cyclodextrin glycosyltransferase from evansella caseinilytica: production, characterization and product specificity.

Alkalohalophilic Evansella caseinilytica produced an extracellular cyclodextrin glycosyltransferase (CGTase) with cyclization activity of 43.5 ± 4.4 U/L in M1 medium containing 1% starch and 6% NaCl in nutrient broth at 37 ºC, pH 9.0, after 48 h. This is the first report of CGTase from this bacterium. 0.1% starch was found to induce CGTase, and further optimization using one variable at a time approach followed by statistical optimization led to 5.5-fold enhancement resulting in 240.5 ± 5.46 U/L. Six parameters were identified as positive signals using Plackett-Burman (PB). Of these, yeast extract, MgSO4 and tryptone were taken further for Response Surface Methodology (RSM) by disposing beef extract and fixing starch and soya peptone. The optimized M4 medium consisted of tryptone (0.1%, w/v), yeast extract (0.25%, w/v), MgSO4 (8 mM, w/v), potato starch (0.1%, w/v) and soya peptone (0.2%, w/v). CGTase was further purified with 6.44-fold purification and 19.32% yield employing starch affinity. It was found to be monomeric, corresponding to a size of 68 kDa as estimated by SDS-PAGE and was further confirmed to be 65 kDa by size exclusion chromatography. γ-Cyclodextrins were produced as the major product with a conversion of 5% soluble starch into 20.38% γ-cyclodextrins after 24 h reaction, as determined by HPLC. Peptide fingerprint after LC-MS analysis matched with IPT/TIG domain-containing protein within the genome of E. caseinilytica. Further blastp analysis revealed the closest homology with γ-CGTase from an alkalophilic E. clarkii, thereby confirming CGTase from E. caseinilytica as γ-CGTase.

Disrupting putative N-glycosylation site N17 in lipase Lip11 of Yarrowia lipolytica yielded a catalytically efficient and thermostable variant accompanying conformational changes.

Lip11 gene from oleaginous yeast Yarrowia lipolytica MSR80 was recombinantly expressed in Pichia pastoris X33. Native secretion signal present in its sequence resulted in 92 % expression in comparison to α-secretion factor which resulted to 900 U/L in the extracellular broth. Catalytic triad in Lip11, like most lipases, was formed by serine, histidine, and aspartate residues. While point mutation disrupting putative glycosylation site (N389) present towards the C-terminus ruinously effected its stability and catalytic activity, disruption of the first putative glycosylation site (N17) located towards the N-terminus presented interesting insights. Mutation resulted in a variant N1 exhibiting higher thermal and acid stability; a t1/2 of 198 min was obtained at 50 °C and it retained almost 80 % activity following incubation at pH 3. Catalytic efficiency was improved by 2.7 fold and a 10 °C rise in temperature optima was accompanied by higher relative activity in acidic range. Thermal stability corresponded to convoying structural modifications in the tertiary structure, findings of fluorescence spectroscopy suggested. Thermal fluorescence studies revealed a Tm of 65 °C for both Lip11 and N1 and λmax of Lip11 exhibited a blue shift upon refolding while no shift in the λmax of N1 was observed. A resilient tertiary structure which could fold back to its native confirmation upon thermal denaturation and increase in surface-exposed hydrophobic residues as revealed by ANS binding assay summed up to thermal stability of N1. Furthermore, circular dichroism data disclosed an alternate ratio of alpha-helices and beta-sheets; respective values changed from 36 % and 8%-27% and 19 %. Following mutation, substrate specificity remained unaffected and similar to native protein, N1 showed activation in presence of organic solvents and most divalent cations.

Bacterial Gamma-Glutamyl Transpeptidase, an Emerging Biocatalyst: Insights Into Structure-Function Relationship and Its Biotechnological Applications.

Gamma-glutamyl transpeptidase (GGT) enzyme is ubiquitously present in all life forms and plays a variety of roles in diverse organisms. Higher eukaryotes mainly utilize GGT for glutathione degradation, and mammalian GGTs have implications in many physiological disorders also. GGTs from unicellular prokaryotes serve different physiological functions in Gram-positive and Gram-negative bacteria. In the present review, the physiological significance of bacterial GGTs has been discussed categorizing GGTs from Gram-negative bacteria like Escherichia coli as glutathione degraders and from pathogenic species like Helicobacter pylori as virulence factors. Gram-positive bacilli, however, are considered separately as poly-γ-glutamic acid (PGA) degraders. The structure-function relationship of the GGT is also discussed mainly focusing on the crystallization of bacterial GGTs along with functional characterization of conserved regions by site-directed mutagenesis that unravels molecular aspects of autoprocessing and catalysis. Only a few crystal structures have been deciphered so far. Further, different reports on heterologous expression of bacterial GGTs in E. coli and Bacillus subtilis as hosts have been presented in a table pointing toward the lack of fermentation studies for large-scale production. Physicochemical properties of bacterial GGTs have also been described, followed by a detailed discussion on various applications of bacterial GGTs in different biotechnological sectors. This review emphasizes the potential of bacterial GGTs as an industrial biocatalyst relevant to the current switch toward green chemistry.