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.

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.

Draft Genome Sequence of a Poly-γ-Glutamic Acid-Producing Isolate, Bacillus paralicheniformis Strain bcasdu2018/01.

Bacillus paralicheniformis bcasdu2018/01 was isolated from the indoor environment of a chemistry laboratory. As part of the extracellular matrix, this isolate produces copious amounts of poly-γ-glutamic acid (γ-PGA). Here, we report the 4.25-Mbp draft genome assembly of the organism with an average G+C content of 45.92%.

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.

Hippocampal atrophy and cognitive function in transient ischemic attack and minor stroke patients over three years.

Introduction: Transient ischemic attack (TIA) and minor ischemic stroke (IS) is associated with a increased risk of late life dementia. In this study we aim to study the extent to which the rates of hippocampal atrophy in TIA/IS differ from healthy controls, and how they are correlated to neuropsychological measurements. Methods: TIA or minor stroke patients were tested with a neuropsychological battery including tests of executive function, and verbal and non-verbal memory at three time points out to 3 years. Annualized rates of hippocampal atrophy in TIA/IS patients were compared to controls. A linear-mixed regression model was used to assess the difference in rates of hippocampal atrophy after adjusting for time and demographic characteristics. Results: TIA/IS patients demonstrated a higher hippocampal atrophy rate than healthy controls over a 3-year interval: the annual percentage change of the left hippocampal volume was 2.5% (78 mm3 per year (SD 60)) for TIA/IS patients compared to 0.9% (29 mm3 per year (SD 32)) for controls (p < 0.01); and the annual percentage change of the right hippocampal volume was 2.5% (80 mm3 per year (SD 46)) for TIA/IS patients compared to 0.5% (17 mm3 per year (SD 33)) for controls (P < 0.01). Patients with higher annual hippocampal atrophy were more likely to report higher TMT B times, but lower ROC total score, lower California Verbal Learning Test-II total recall, and lower ROC Figure recall scores longitudinally. Conclusion: TIA/IS patients experience a higher rate of hippocampal atrophy independent of TIA/IS recurrence that are associated with changes in episodic memory and executive function over 3 years.

Characterization of a novel thiol activated phospholipase TAPLB1 from Trichosporon asahii MSR 54.

Phospholipases are hydrolytic enzymes that play crucial roles in vivo and also possess immense biotechnological potential. In the present study, the phospholipase B of Trichosporon asahii MSR54 was overexpressed in E. coli and characterized. The 68-kDa enzyme was monomeric in solution and possessed phospholipase, lysophospholipase, esterase and acyltransferase activities. It was maximally active at pH 8.0 and 40 °C. The enzyme retained >50% activity between pH 3.0-8.0 and had a half-life of 30 min at 60 °C. Its activity was not metal dependent and was stable in the presence of most metal ions. Its catalytic efficiency on lysophosphatidyl choline was 1.0 × 103 mM-1 h-1. Site directed mutagenesis revealed R121 (present in the GYRAMV motif), S194 (present in the conserved GLSGG motif) and D420 (present in LVDXGE motif) to be the crucial amino acid residues for esterolytic activity. S194 and D420 were also the catalytic amino acids for lysophospholipase and phospholipase activities of the enzymes, while R121 was not involved in catalysis of phospholipid substrates. Further, it was found that cysteine residues in C61 and C354 were involved in disulphide linkages that imparted the properties of thiol activation and thermostability, respectively.

High level extracellular production of recombinant γ-glutamyl transpeptidase from Bacillus licheniformis in Escherichia coli fed-batch culture.

Increasing demand of microbial γ-glutamyl transpeptidase (GGT) in food and pharmaceutical sectors raised the need for process development for high level production of the enzyme. In this respect, GGT from Bacillus licheniformis ER15 (SBLGGT) was cloned along with its native secretion signal and expressed in E. coli using different expression vectors. Native signal of the enzyme assistedits extracellular translocationin E. coli.Maximum enzyme expression was shown by construct pET51b-sblggt,in comparison to other clones, in E. coli. Shake-flask cultivation and expression using Luria-Bertani (LB) medium resulted in 2800 U/l enzyme titers in 48 h which was furtherenhancedto 4.3-fold after optimizing various cultivation conditions viz. inducer concentration, agitation, medium and induction optical density. High cell density cultivation using fed-batch fermentation strategy resulted in 20-fold increase over shake flask studies to a level of 61250 U/l. After 24 h,the specific product yield was 2355 U/g dry cell weight (DCW)with volumetric productivity of 2552 U/l/h. Of the total enzyme expressed,40% was translocated extracellularly during high cell density fed-batch fermentation resulting in an enzyme activity of 24500 U/l in the extracellular medium after 24 h. This is the highest reported enzyme titers of bacterial GGT enzyme in E. coli expression system. Thus, the current study provides a cost-effective method for the over-expression and preparation of bacterial GGT enzyme for its industrial applications.

Localized surface plasmon resonance-based fiber-optic sensor for the detection of triacylglycerides using silver nanoparticles.

A label-free technique for the detection of triacylglycerides by a localized surface plasmon resonance (LSPR)-based biosensor is demonstrated. An LSPR-based fiber-optic sensor probe is fabricated by immobilizing lipase enzyme on silver nanoparticles (Ag-NPs) coated on an unclad segment of a plastic clad optical fiber. The size and shape of nanoparticles were characterized by high-resolution transmission electron microscopy and UV-visible spectroscopy. The peak absorbance wavelength changes with concentration of triacylglycerides surrounding the sensor probe, and sensitivity is estimated from shift in the peak absorbance wavelength as a function of concentration. The fabricated sensor was characterized for the concentration of triacylglyceride solution in the range 0 to 7 mM. The sensor shows the best sensitivity at a temperature of 37°C and pH 7.4 of the triacylglycerides emulsion with a response time of 40 s. A sensitivity of 28.5 nm/mM of triacylglyceride solution is obtained with a limit of detection of 0.016 mM in the entire range of triacylglycerides. This compact biosensor shows good selectivity, stability, and reproducibility in the entire physiological range of triacylglycerides and is well-suited to real-time online monitoring and remote sensing.

Rational design of drug-like compounds targeting Mycobacterium marinum MelF protein.

The mycobacterial mel2 locus (mycobacterial enhanced infection locus, Rv1936-1941) is Mycobacterium marinum and M. tuberculosis specific, which can withstand reactive oxygen species (ROS) and reactive nitrogen species (RNS) induced stress. A library of over a million compounds was screened using in silico virtual ligand screening (VLS) to identify inhibitors against the modeled structure of MelF protein expressed by melF of mel2 locus so that M. marinum's ability to withstand ROS/RNS stress could be reduced. The top ranked 1000 compounds were further screened to identify 178 compounds to maximize the scaffold diversity by manually evaluating the interaction of each compound with the target site. M. marinum melF was cloned, expressed and purified as maltose binding protein (MBP)-tagged recombinant protein in Escherichia coli. After establishing the flavin dependent oxidoreductase activity of MelF (~ 84 kDa), the inhibitors were screened for the inhibition of enzyme activity of whole cell lysate (WCL) and the purified MelF. Amongst these, 16 compounds could significantly inhibit the enzyme activity of purified MelF. For the six best inhibitory compounds, the minimal inhibitory concentration (MIC) was determined to be 3.4-19.4 µM and 13.5-38.8 µM for M. marinum and M. tuberculosis, respectively. Similarly, the minimal bactericidal concentration (MBC) was determined to be 6.8-38.8 µM and 27-38.8 µM against M. marinum and M. tuberculosis, respectively. One compound each in combination with isoniazid (INH) also showed synergistic inhibitory effect against M. marinum and M. tuberculosis with no cytotoxicity in HeLa cells. Interestingly, these inhibitors did not display any non-specific protein-structure destabilizing effect. Such inhibitors targeting the anti-ROS/RNS machinery may facilitate the efficient killing of replicating and nonreplicating mycobacteria inside the host cells.

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.

Targeted mutations and MD simulations of a methanol-stable lipase YLIP9 from Yarrowia lipolytica MSR80 to develop a biodiesel enzyme.

Biodiesel, an environment friendly alternative for fuels, contains methyl esters of long-chain fatty acids. Our group has reported a methanol-stable YLIP9 from Yarrowia lipolytica MSR80 that shows poor catalysis of long-chain fatty acids. To shift its substrate specificity, residues within lid and binding pocket were identified for sequential mutations using YLIP2 as the template. Of the two point mutations (Glu116Leu and Ser119Val) introduced in the lid, the former mutation (YLIP9L1) increased the catalytic rate by ∼2-fold without any change in substrate specificity. In this mutant, six binding pocket residues (Bp2-Bp7) were further mutated to obtain six double mutants. YLIP9L1Bp3 showed significant shift in substrate specificity towards long-chain pNPesters with 11-fold increase in catalytic efficiency than YLIP9. Double mutations also led to increased thermostability and lowered activation energy of YLIP9L1Bp3 thereby shifting its optimum temperature from 60°C to 50°C. In silico molecular dynamics simulations revealed improved lid flexibility and increased catalytic triad volume in YLIP9L1Bp3. The enzyme YLIP9L1Bp3 was methanol-stable having selectivity for long-chain fatty acids with improved catalytic efficiency. Its application as a biodiesel enzyme was validated by transesterification of palm oil in presence of methanol, where it showed 8-fold increase in conversion of oil to methyl esters.

Evolving transpeptidase and hydrolytic variants of γ-glutamyl transpeptidase from Bacillus licheniformis by targeted mutations of conserved residue Arg109 and their biotechnological relevance.

γ-Glutamyl transpeptidase (GGT) catalyzes the transfer of the γ-glutamyl moiety from donor compounds such as l-glutamine (Gln) and glutathione (GSH) to an acceptor. During the biosynthesis of various γ-glutamyl-containing compounds using GGT enzyme, auto-transpeptidation reaction leads to the formation of unwanted byproducts. Therefore, in order to alter the auto-transpeptidase activity of the GGT enzyme, the binding affinity of Gln should be modified. Structural studies of the Bacillus licheniformis GGT (BlGT) complexed with the glutamic acid has shown that glutamic acid has strong ionic interactions through its α-carboxlic group with the guanidine moiety of Arg109. This interaction appears to be an important contributor for the binding affinity of Gln. In view of this, six mutants of Bacillus licheniformis ER15 GGT (BlGGT) viz. Arg109Lys, Arg109Ser, Arg109Met, Arg109Leu, Arg109Glu and Arg109Phe were prepared. As seen from the structure of BlGT, the mutation of Arg109 to Lys109 may reduce the affinity for Gln to some extent, whereas the other mutations are expected to lower the affinity much more. Biophysical characterization and functional studies revealed that Arg109Lys mutant has increased transpeptidation activity and catalytic efficiency than the other mutants. The Arg109Lys mutant showed high conversion rates for l-theanine synthesis as well. Moreover, the Arg109Met mutant showed increased hydrolytic activity as it completely altered the binding of Gln at the active site. Also, the salt stability of the enzyme was significantly improved on replacing Arg109 by Met109 which is required for hydrolytic applications of GGTs in food industries.

Heterologous expression of γ-glutamyl transpeptidase from Bacillus atrophaeus GS-16 and its application in the synthesis of γ-d-glutamyl-l-tryptophan, a known immunomodulatory peptide.

Gamma-glutamyl transpeptidase from a mesophilic bacterium Bacillus atrophaeus GS-16 (BaGGT) was expressed heterologously in E. coli using pET-51b vector. Maximum production of BaGGT was obtained at 16°C after 16h of IPTG induction and the protein, in its native conformation, was active as a heterooctamer which was composed of four heterodimeric units combined together. One heterodimeric unit constituted two subunits with molecular masses of 45kDa and 21kDa, respectively. The recombinant enzyme was purified by one step His-tag affinity purification protocol with a specific activity of 90U/mg and 5.2 fold purity. The purified enzyme had a pH optimum of 10.0 and temperature optimum of 50°C. It exhibited broad pH stability (6.0-12.0) and was thermostable (t1/2 of 54min at 50°C). The enzyme was completely inactivated by Pb2+ ions and strongly inhibited in presence of N-bromosuccinimide, azaserine and 6-diazo-5oxo-l-norleucine. Kinetic characterization of BaGGT using GpNA as a donor and glycylglycine as acceptor revealed that it had a Km of 0.15mM and 0.37mM and Vmax of 23.09µmol/mg/min and 121.95µmol/mg/min for hydrolysis and transpeptidation reactions, respectively. BaGGT also displayed broad substrate specificity for various amino acids. It was studied for its prospective use in the synthesis of an immunomodulatory peptide, γ-d-glutamyl-l-tryptophan. After optimization of various process parameters, a conversion rate of 50%, corresponding to 25mM product yield, was achieved within 6h of incubation using 50mM d-glutamine as donor and 50mM l-tryptophan as acceptor and 0.3U/mL of BaGGT in the reaction, performed at pH 10.0 and 37°C. The product was purified to homogeneity using Dowex 1×2 column and its purity was confirmed by HPLC and H1 NMR.

High Resolution X-ray Diffraction Dataset for Bacillus licheniformis Gamma Glutamyl Transpeptidase-acivicin complex: SUMO-Tag Renders High Expression and Solubility.

Gamma glutamyl transpeptidase, (GGT) is a ubiquitous protein which plays a central role in glutathione metabolism and has myriad clinical implications. It has been shown to be a virulence factor for pathogenic bacteria, inhibition of which results in reduced colonization potential. However, existing inhibitors are effective but toxic and therefore search is on for novel inhibitors, which makes it imperative to understand the interactions of various inhibitors with the protein in substantial detail. High resolution structures of protein bound to different inhibitors can serve this purpose. Gamma glutamyl transpeptidase from Bacillus licheniformis is one of the model systems that have been used to understand the structure-function correlation of the protein. The structures of the native protein (PDB code 4OTT), of its complex with glutamate (PDB code 4OTU) and that of its precursor mimic (PDB code 4Y23) are available, although at moderate/low resolution. In the present study, we are reporting the preliminary analysis of, high resolution X-ray diffraction data collected for the co-crystals of B. licheniformis, Gamma glutamyl transpeptidase, with its inhibitor, Acivicin. Crystals belong to the orthorhombic space group P212121 and diffract X-ray to 1.45 Å resolution. This is the highest resolution data reported for all GGT structures available till now. The use of SUMO fused expression system enhanced yield of the target protein in the soluble fraction, facilitating recovery of protein with high purity. The preliminary analysis of this data set shows clear density for the inhibitor, acivicin, in the protein active site.

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.

Blood pressure reduction following accumulated physical activity in prehypertensive.

CONTEXT: Accumulated moderate physical activity (PA) for 30 min in a day is the only recommended treatment of prehypertension. OBJECTIVE: We investigated autonomic modulation as a possible mechanism for the decrease in blood pressure (BP) during the rest periods in each 10 min session of PA. DESIGN SETTING AND PARTICIPANTS: We conducted a single-blind randomized multi-arm control trial on 40 prehypertensive (pre-HT) young male adults. METHODS: Participants were randomly divided by using random number table into four groups. Control (no intervention); Group 1 (walking at 50% of predicted VO2 peak); Group 2 (walking at 60% of predicted VO2 peak); Group 3 (walking at 70% of predicted VO2 peak). BP, heart rate variability (HRV), and heart rate recovery 1 min (HRR 1 min) were measured at baseline and during the rest period after each session of 10 min over 30 min of accumulated physical activity (PAcumm). RESULTS: Significant diastolic BP (DBP) reduction (P < 0.001) was observed during the rest period after each session of PAcumm in intervention groups. An average reduction in DBP was more in pre-HT undertaking PAcumm at 70% of predicted VO2 Peak. Decrease in the mean value of low-frequency (LF) and LF/high-frequency ratio was observed following PAcumm in all intervention groups irrespective of the intensity of PA. No significant association of reduction of BP with HRV and HRR 1 s was observed. CONCLUSION: Reduction in BP was observed during the rest period after each 10 min session of PAcumm irrespective of the intensity of PA. Autonomic modulation does not seem to be the possible mechanism for the reduction in BP during the sessions.

Arterial Compliance and Autonomic Functions in Adult Male Smokers.

INTRODUCTION: Smoking is known to augment sympathetic activity and may lead to increased arterial stiffness. Several studies have reported association of increased sympathetic activity and arterial stiffness to cardiovascular risks among smokers. Pulse Wave Velocity (PWV) of peripheral arteries, instead of aorta can be used as a non-invasive indicator of arterial stiffness. AIM: To measure non-invasively, the autonomic functions and peripheral arterial stiffness in smokers, and to find out whether the aforementioned factors are modified by the level of physical activity in these smokers. MATERIALS AND METHODS: This cross-sectional analytical study was conducted in the Department of Physiology, HIMS, Dehradun, over a period of 12 months (2013-2014) on 100 adult males (20-40 years); 50 smokers and 50 non-smokers. The parameters analysed include relevant anthropometric and cardiovascular parameters, Pulse Wave Velocity (PWV), sustained Hand Grip Test (HGT) and Heart Rate Variability (HRV) domains. Data interpretation and analysis was carried out using SPSS 17.0. Comparison of the above mentioned parameters amongst groups was done with unpaired t-test. The relationship of pack-years & physical activity with vascular functions was assessed by Pearson's correlation. Interaction of various grades of smoking and physical activity with Cardiovascular System (CVS) parameters was assessed by one-way ANOVA. RESULTS: Smokers had higher values of PWV (5.7±0.5m/s) as compared to non-smokers (4.8±0.4m/s) (p<0.001). ΔDBP during HGT was lower (7±3.18mmHg) among smokers as compared to non-smokers (19.4±3.5mmHg) (p<0.001). Smoking (pack-years) was positively related to PWV (r= .03) but showed a weak negative relationship with change in Diastolic Blood Pressure (ΔDBP) (r= -0.084, p=0.56) showing that, more the frequency of smoking, the more was arterial stiffening and the lesser was the sympathetic response to the HGT. The smokers had significantly higher sympathetic activity; Low Frequency (LF) & Low Frequency: High Frequency ratio (LF: HF) (p<.001) whereas High Frequency (HF) was significantly lower (p<.001) showing a decline in parasympathetic activity. CONCLUSION: Smokers demonstrated higher peripheral PWV and higher intrinsic sympathetic activity and this increase in intrinsic sympathetic activity may lead to increased arterial stiffness. Interaction of autonomic function and PWV with levels of physical activity and grades of smoking showed no significant differences, suggesting the fact that increased physical activity or reduced smoking may not have any effect on the endothelial dysfunction or CVS morbidity caused by smoking.