02605nas a2200253 4500008004100000022001400041245018200055210006900237260005700306300001200363490000700375520164900382653001602031653002202047653001602069653001202085653002902097100002402126700001402150700001302164700001602177700002602193856013202219 2013 eng d a0175-759800aCombinatorial approach of statistical optimization and mutagenesis for improved production of acidic phytase by Aspergillus niger NCIM 563 under submerged fermentation condition0 aCombinatorial approach of statistical optimization and mutagenes a233 SPRING ST, NEW YORK, NY 10013 USAbSPRINGERcJAN a673-6790 v973 a

Combination of statistical optimization and mutagenesis to isolate hypersecretory strains is studied to maximize phytase production from Aspergillus niger NCIM 563 under submerged fermentation. The overall results obtained show a remarkable 5.98-fold improvement in phytase production rates when compared to that using basal medium. Optimization of culture conditions from parent strain is studied first by the Plackett-Burman technique to evaluate the effects of 11 variables for phytase production. The results showed that glucose, MgSO4, KCl, incubation period, and MnSO4 are the most significant variables affecting enzyme production. Further optimization in these variables, using a central composite design technique, resulted in 3.74-fold increase in the yield of phytase production to 254,500 U/l when compared with the activity observed with basal media (68,000 U/l) in shake flask. Our experiments show that the phytase from A. niger NCIM 563 exhibits desirable activity in simulated gastric fluid conditions with low pH and also improved thermostability when compared to commercial phytase. The improved yield demonstrates the potential applicability of phytase enzyme as a source of phytase supplement for phosphorus nutrition and environmental protection in animal feed industry. Physical and chemical mutagenesis experiments were carried out in parallel to isolate hypersecretory mutants that could possibly further enhance the enzyme production. Using optimized media conditions of the parent strain, our results show that mutant strain A. niger NCIM 1359 increased the phytase activity by another 1.6-fold to 407,200 U/l.

10aAnimal feed10aAspergillus niger10aMutagenesis10aPhytase10aStatistical optimization1 aBhavsar, Kavita, P.1 aGujar, P.1 aShah, P.1 aKumar, Ravi1 aKhire, Jayant, Malhar uhttps://library.ncl.res.in/content/combinatorial-approach-statistical-optimization-and-mutagenesis-improved-production-acidic-002192nas a2200241 4500008004100000022001400041245010200055210006900157260005700226300001200283490000700295520130800302653002201610653001501632653001601647653002301663653002701686100002601713700002601739700002801765700002501793856013201818 2013 eng d a1615-759100aTwo-stage fermentation process for enhanced mannitol production using Candida magnoliae mutant R90 aTwostage fermentation process for enhanced mannitol production u a233 SPRING ST, NEW YORK, NY 10013 USAbSPRINGERcFEB a193-2030 v363 a

Mutants of Candida magnoliae NCIM 3470 were generated by treatment of ultra-violet radiations, ethyl methyl sulphonate and N-methyl-N'-nitro-N-nitrosoguanidine. Mutants with higher reductase activity were screened by means of 2,3,5-triphenyl tetrazolium chloride agar plate assay. Among the screened mutants, the mutant R9 produced maximum mannitol (i.e. 46 g l(-1)) in liquid fermentation medium containing 250 g l(-1) glucose and hence was selected for further experiments. Preliminary optimization studies were carried out on shake-flask level which increased the mannitol production to 60 g l(-1) in liquid fermentation medium containing 300 g l(-1) glucose. A two-stage fermentation process comprising of growth phase and production phase was employed. During the growth phase, glucose was supplemented and aerobic conditions were maintained. Thereafter, the production phase was initiated by supplementing fructose and switching to anaerobic conditions by discontinuing aeration and decreasing the speed of agitation. The strategy of two-stage fermentation significantly enhanced the production of mannitol up to 240 g l(-1), which is the highest among all fermentative production processes and corresponds to 81 % yield and 4 g l(-1) h(-1) productivity without formation of any by-product.

10aCandida magnoliae10aD-Mannitol10aMutagenesis10aStrain improvement10aTwo-stage fermentation1 aSavergave, Laxman, S.1 aGadre, Ramchandra, V.1 aVaidya, Bhalchandra, K.1 aJogdand, Vitthal, V. uhttps://library.ncl.res.in/content/two-stage-fermentation-process-enhanced-mannitol-production-using-candida-magnoliae-mutant-000712nas a2200205 4500008004100000022001400041245008800055210006900143260000800212300000900220490000700229653002400236653001600260653002600276100001800302700002600320700002600346700001900372856011500391 2014 eng d a2053-273300aStructure of highly active BSH enzyme with subordinated post-translational excision0 aStructure of highly active BSH enzyme with subordinated posttran cMAR aC2720 v7010aBile salt hydrolase10aMutagenesis10aX-ray crystallography1 aChand, Deepak1 aPanigrahi, Priyabrata1 aRamasamy, Sureshkumar1 aSuresh, C., G. uhttps://library.ncl.res.in/content/structure-highly-active-bsh-enzyme-subordinated-post-translational-excision02452nas a2200265 4500008004100000022001400041245015600055210006900211260009500280300001000375490000800385520139300393653002201786653002801808653002301836653001601859653002801875100002901903700002601932700001801958700002001976700003401996700002602030856013002056 2016 eng d a1047-847700aStructural analysis of a penicillin V acylase from pectobacterium atrosepticum confirms the importance of two Trp residues for activity and specificity0 aStructural analysis of a penicillin V acylase from pectobacteriu a525 B ST, STE 1900, SAN DIEGO, CA 92101-4495 USAbACADEMIC PRESS INC ELSEVIER SCIENCEcFEB a85-940 v1933 a

Penicillin V acylases (PVA) catalyze the deacylation of the beta-lactam antibiotic phenoxymethylpenicillin (Pen V). They are members of the Ntn hydrolase family and possess an N-terminal cysteine as the main catalytic nucleophile residue. They form the evolutionarily related cholylglycine hydrolase (CGH) group which includes bile salt hydrolases (BSH) responsible for bile deconjugation. Even though a few PVA and BSH structures have been reported, no structure of a functional PVA from Gram-negative bacteria is available. Here, we report the crystal structure of a highly active PVA from Gram-negative Pectobacterium atrosepticum (PaPVA) at 2.5 angstrom resolution. Structural comparison with PVAs from Gram-positive bacteria revealed that PaPVA had a distinctive tetrameric structure and active site organization. In addition, mutagenesis of key active site residues and biochemical characterization of the resultant variants elucidated the role of these residues in substrate binding and catalysis. The importance of residue Trp23 and Trp87 side chains in binding and correct positioning of Pen V by PVAs was confirmed using mutagenesis and substrate docking with a 15 ns molecular dynamics simulation. These results establish the unique nature of Gram-negative CGHs and necessitate further research about their substrate spectrum. (C) 2015 Elsevier Inc. All rights reserved.

10aAromatic stacking10aCholylglycine hydrolase10amolecular dynamics10aMutagenesis10aOligomeric interactions1 aAvinash, Vellore, Sunder1 aPanigrahi, Priyabrata1 aChand, Deepak1 aPundle, Archana1 aSuresh, Cheravakattu, Gopalan1 aRamasamy, Sureshkumar uhttps://library.ncl.res.in/content/structural-analysis-penicillin-v-acylase-pectobacterium-atrosepticum-confirms-importance-003019nas a2200265 4500008004100000022001300041245014800054210006900202260000800271300001200279490000900291520209300300653002902393653002402422653002302446653001602469653002602485653002602511100001402537700001802551700001702569700001702586700001902603856013102622 2018 eng d a1570963900aStructure and function of a highly active Bile Salt Hydrolase (BSH) from Enterococcus faecalis and post-translational processing of BSH enzymes0 aStructure and function of a highly active Bile Salt Hydrolase BS cJAN a507-5180 v18663 aBile Salt Hydrolase (BSH), a member of Cholylglycine hydrolase family, catalyzes the de-conjugation of bile acids and is evolutionarily related to penicillin V acylase (PVA) that hydrolyses a different substrate such as penicillin V. We report the three-dimensional structure of a BSH enzyme from the Gram-positive bacteria Enterococcus faecalis (EfBSH) which has manifold higher hydrolase activity compared to other known BSHs and displays unique allosteric catalytic property. The structural analysis revealed reduced secondary structure content compared to other known BSH structures, particularly devoid of an anti-parallel β-sheet in the assembly loop and part of a β-strand is converted to increase the length of a substrate binding loop 2. The analysis of the substrate binding pocket showed reduced volume owing to altered loop conformations and increased hydrophobicity contributed by a higher ratio of hydrophobic to hydrophilic groups present. The aromatic residues F18, Y20 and F65 participate in substrate binding. Thus, their mutation affects enzyme activity. Docking and Molecular Dynamics simulation studies showed effective polar complementarity present for the three hydroxyl (-OH) groups of GCA substrate in the binding site contributing to higher substrate specificity and efficient catalysis. These are unique features characteristics of this BSH enzyme and thought to contribute to its higher activity and specificity towards bile salts as well as allosteric effects. Further, mechanism of autocatalytic processing of Cholylglycine Hydrolases by the excision of an N-terminal Pre-peptide was examined by inserting different N-terminal pre-peptides in EfBSH sequence. The results suggest that two serine residues next to nucleophile cysteine are essential for autocalytic processing to remove precursor peptide. Since pre-peptide is absent in EfBSH the mutation of these serines is tolerated. This suggests that an evolution-mediated subordination of the pre-peptide excision site resulted in loss of pre-peptide in EfBSH and other related Cholylglycine hydrolases.10aautocatalytic processing10aBile salt hydrolase10amolecular dynamics10aMutagenesis10aPolar complementarity10aX-ray crystallography1 aChand, D.1 aPanigrahi, P.1 aVarshney, N.1 aRamasamy, S.1 aSuresh, C., G. uhttps://library.ncl.res.in/content/structure-and-function-highly-active-bile-salt-hydrolase-bsh-enterococcus-faecalis-and-post02450nas a2200289 4500008004100000022001400041245012900055210006900184260000800253300001200261490000700273520149600280653001301776653002401789653002301813653001601836653001601852100001801868700002001886700002301906700001801929700002701947700001601974700002101990700002102011856012802032 2019 eng d a0959-665800aSemirational design and engineering of grapevine glucosyltransferases for enhanced activity and modified product selectivity0 aSemirational design and engineering of grapevine glucosyltransfe cNOV a765-7750 v293 a

Uridine diphosphate-dependent glycosyltransferases (UGTs) catalyze the transfer of a diversity of sugars to several acceptor molecules and often exhibit distinct substrate specificity. Modulation of glycosyltransferases for increased catalytic activity and altered substrate or product specificity are the key manipulations for the biotechnological use of glycosyltransferases in various biosynthetic processes. Here, we have engineered the binding pocket of three previously characterized Vitis vinifera glycosyltransferases, UGT88F12, UGT72B27 and UGT92G6, by structure-guided in silico mutagenesis to facilitate the interactions of active site residues with flavonol glucosides and thus modify substrate specificity and activity. Site-directed mutagenesis at selected sites, followed with liquid chromatography-mass spectrometry based activity assays, exhibited that mutant UGTs were altered in product selectivity and activity as compared to the wild-type enzymes. Mutant UGTs produced larger amounts of flavonol di-monosaccharide glucosides, which imply that the mutations led to structural changes that increased the volume of the binding pocket to accommodate a larger substrate and to release larger products at ease. Mutants showed increased activity and modified product specificity. Thus, structure-based systematic mutations of the amino acid residues in the binding pocket can be explored for the generation of engineered UGTs for diverse biotechnological applications.

10aflavonol10aGlycosyltransferase10ain silico analysis10aMutagenesis10aselectivity1 aJoshi, Rakesh1 aTrinkl, Johanna1 aHaugeneder, Annika1 aHaertl, Katja1 aFranz-Oberdorf, Katrin1 aGiri, Ashok1 aHoffmann, Thomas1 aSchwab, Wilfried uhttps://library.ncl.res.in/content/semirational-design-and-engineering-grapevine-glucosyltransferases-enhanced-activity-and