02308nas a2200241 4500008004100000022001400041245015900055210006900214260007900283300001000362490000700372520137500379653002001754653001901774653001401793653001401807653001901821100002501840700003401865700002301899700001401922856013001936 2010 eng d a0196-978100aMolecular cloning, over expression, and activity studies of a peptidic HIV-1 protease inhibitor: designed synthetic gene to functional recombinant peptide0 aMolecular cloning over expression and activity studies of a pept a360 PARK AVE SOUTH, NEW YORK, NY 10010-1710 USAbELSEVIER SCIENCE INCcJAN a16-210 v313 a

The aspartic protease inhibitor (ATBI) purified from a Bacillus sp. is a potent inhibitor of several proteases including recombinant HIV-1 protease, pepsin, and fungal aspartic protease. In this study, we report the cloning, and over expression of a synthetic gene coding for ATBI in Escherichia coli and establish a purification Protocol. The ATBI molecule consists of eleven amino acids and is peptidic in nature. We used the peptide sequence data of ATBI to synthesize complementary oligonucleotides, which were annealed and subsequently cloned in-frame with the gene for glutathione-S-transferase (GST). The expression of the resulting fusion protein was induced in E. coli BL21-A1 cells using arabinose. The recombinant peptide was Purified using a reduced glutathione column, and cleaved with Factor Xa to remove the GST tag. The resultant product was further purified to homogeneity using RP-HPLC. Mass spectroscopy analysis revealed that the purified peptide had a molecular weight of 1186 Da which matches the theoretical molecular weight of the amino acids present in the synthetic gene. The recombinant peptide was found to be active in vitro against HIV-1 protease, pepsin, and fungal aspartic protease. The protocol described in this study may be used to clone pharmaceutically important peptide molecules. (C) 2009 Elsevier Inc. All rights reserved.

10aGateway cloning10aHIV-1 protease10ainhibitor10aProteases10aSynthetic gene1 aVathipadiekal, Vinod1 aUmasankar, Perunthottathu, K.1 aPatole, Milind, S.1 aRao, Mala uhttps://library.ncl.res.in/content/molecular-cloning-over-expression-and-activity-studies-peptidic-hiv-1-protease-inhibitor-002101nas a2200253 4500008004100000022001400041245015000055210006900205260009900274300001400373490000800387520111000395653001901505653003601524653004101560653001201601653001101613100001801624700002301642700002101665700002001686700001401706856012701720 2011 eng d a0960-852400aMicrobial production of xylitol from D-xylose and sugarcane bagasse hemicellulose using newly isolated thermotolerant yeast Debaryomyces hansenii0 aMicrobial production of xylitol from Dxylose and sugarcane bagas aTHE BOULEVARD, LANGFORD LANE, KIDLINGTON, OXFORD OX5 1GB, OXON, ENGLANDbELSEVIER SCI LTDcFEB a3304-33080 v1023 a

A thermotolerant yeast capable of fermenting xylose to xylitol at 40 C was isolated and identified as a strain of Debaryomyces hansenii by ITS sequencing. This paper reports the production of xylitol from D-xylose and sugarcane bagasse hemicellulose by free and Ca-alginate immobilized cells of D. hansenii. The efficiency of free and immobilized cells were compared for xylitol production from D-xylose and hemicellulose in batch culture at 40 degrees C. The maximum xylitol produced by free cells was 68.6 g/L from 100 g/L of xylose, with a yield of 0.76 g/g and volumetric productivity 0.44 g/L/h. The yield of xylitol and volumetric productivity were 0.69 g/g and 0.28 g/L/h respectively from hemicellulosic hydrolysate of sugarcane bagasse after detoxification with activated charcoal and ion exchange resins. The Ca-alginate immobilized D. hansenii cells produced 73.8 g of xylitol from 100 g/L of xylose with a yield of 0.82 g/g and volumetric productivity of 0.46 g/L/h and were reused for five batches with steady bioconversion rates and yields. (C) 2010 Elsevier Ltd. All rights reserved.

10aImmobilization10aSugarcane bagasse hemicellulose10aThermotolerant Debaryomyces hansenii10axylitol10aXylose1 aPrakash, Gyan1 aVarma, Anjanikumar1 aPrabhune, Asmita1 aShouche, Yogesh1 aRao, Mala uhttps://library.ncl.res.in/content/microbial-production-xylitol-d-xylose-and-sugarcane-bagasse-hemicellulose-using-newly-002035nas a2200229 4500008004100000022001400041245012600055210006900181260007500250300001200325490000700337520117600344653002701520653001601547653002001563653002701583653001401610653002301624100001801647700001401665856012601679 2013 eng d a1053-050900aMechanistic insights into the inhibition of endo-beta 1,4 xyloglucan hydrolase by a classical aspartic protease inhibitor0 aMechanistic insights into the inhibition of endobeta 14 xylogluc a233 SPRING ST, NEW YORK, NY 10013 USAbSPRINGER/PLENUM PUBLISHERScMAR a311-3210 v233 a

This is the first report of inactivation of xyloglucanase from Thermomonospora sp by pepstatin A, a specific inhibitor towards aspartic proteases. The steady state kinetics revealed a reversible, competitive, two-step inhibition mechanism with IC (50) and K (i) values of 3.5 +/- 0.5 mu M and 1.25 +/- 0.5 mu M respectively. The rate constants determined for the isomerization of EI to EI* and the dissociation of EI* were 14.5 +/- 1.5 x 10(-5) s(-1) and 2.85 +/- 1.2 x 10(-8) s(-1) respectively, whereas the overall inhibition constant K (i) (*) was 27 +/- 1 nM. The conformational changes induced upon inhibitor binding to xyloglucanase were monitored by fluorescence analysis and the rate constants derived were in agreement with the kinetic data. The abolished isoindole fluorescence of o-phthalaldehyde (OPTA)-labeled xyloglucanase and far UV analysis suggested that pepstatin binds to the active site of the enzyme. Our results revealed that the inactivation of xyloglucanase is due to the interference in the electronic microenvironment and disruption of the hydrogen-bonding network between the essential histidine and other residues involved in catalysis.

10a4 xyloglucan hydrolase10aEndo-beta 110aEnzyme kinetics10aInactivation mechanism10aPepstatin10aSlow-tight binding1 aMenon, Vishnu1 aRao, Mala uhttps://library.ncl.res.in/content/mechanistic-insights-inhibition-endo-beta-14-xyloglucan-hydrolase-classical-aspartic-0