Purification and Characterization of Endoxylanases Cloned from Fibrobacter Succinogenes S85 and Expressed in Escherichia Coli HB101

The xylanase enzyme from Escherichia coli HB 101 containing the xylanolytic recombinant plasmid pBX6 was purified to homogeneity using ultrafiltration, DEAESepharose, CM-Sepharose and Sephadex 0-200 chromatography. Three xylanases, namely, Xyn A, Xyn BI and Xyn BII were obtained and were found to have the same molecular weight and optimum pH which were estimated to be 60.3 kDa and pH 7.0 respectively. The optimum assay temperature for both Xyn A and Xyn BI was 50°C, while for Xyn BII, it was 40°C. The xylanases were stable up to 45°C at pH 7.2 for 30 min. Approximately 80% of the enzyme activity was retained at the pH range of 5.0 to 8.0. The isoelectric point for Fraction A, Fraction BI and Fraction BII was 8.2, 8.5 and 5.5, respectively. The respective apparent K", and Vmax value on oat-spelt xylan was 12.2 mg/ml and 47.9 µmol xylose/min/mg protein for Xyn A; 10.8 mglml and 52.1 µmol xylose/min/mg protein for Xyn BI; 8.7 mg/ml and 54.2 µmol xylose/min/mg protein for Xyn BII. From the hydrolysis products of oat-spelt xylan analysed on thinlayer chromatography, the xylanases hydrolysed xylan through an endo-acting mechanism as no xylose, xylobiose or arabinose was detected. Thus, the xylanases were classified as an endoxylanase. The xylanases showed no activity toward carboxymethyl cellulose (CMC), crystalline cellulose (Avicel) and cellulose filter paper. The xylanases were not affected by potassium chloride, EDT A and EGTA at concentrations of 10 mM. Calcium chloride and magnesium chloride at the same concentrations enhanced the xylanase activities by 50%. Mercury chloride at 1.0 mM concentration completely inhibited the activities of all the purified xylanases. From zymogram analysis and characteristics of the xylanases investigated, multiplicity of xylanases in E. coli HB 101 (pBX6) was probably due to post-translational modification of a single gene product

Structure and boosting activity of a starch-degrading lytic polysaccharide monooxygenase.

Lytic polysaccharide monooxygenases (LPMOs) are recently discovered enzymes that oxidatively deconstruct polysaccharides. LPMOs are fundamental in the effective utilization of these substrates by bacteria and fungi; moreover, the enzymes have significant industrial importance. We report here the activity, spectroscopy and three-dimensional structure of a starch-active LPMO, a representative of the new CAZy AA13 family. We demonstrate that these enzymes generate aldonic acid-terminated malto-oligosaccharides from retrograded starch and boost significantly the conversion of this recalcitrant substrate to maltose by β-amylase. The detailed structure of the enzyme's active site yields insights into the mechanism of action of this important class of enzymes.This work was supported by a grant from the European Research Agency—Industrial Biotechnology Initiative as financed by the national research councils: Biotechnology and Biological Sciences Research Council (grant number BB/L000423) and Agence Française de l'Environnement et de la Maîtrise de l'Energie (grant number 1201C102). The Danish Council for Strategic Research (grant numbers 12-134923 and 12-134922). The Danish Ministry of Higher Education and Science through the Instrument Center DANSCATT and the European Community’s Seventh Framework Programme (FP7/2007-2013) under BioStruct-X (grant agreement N°283570) funded travel to synchrotrons. P.H.W. acknowledges the experimental assistance of Rebecca Gregory and Dr Victor Chechik. L.L.L. acknowledges the experimental assistance of Dorthe Boelskifte and the ESRF and MAXLAB staff for assistance with data collection.This is the final version of the article. It first appeared from NPG via http://dx.doi.org/10.1038/ncomms696

DNA methylation subgroups and the CpG island methylator phenotype in gastric cancer: A comprehensive profiling approach

10.1186/1471-230X-14-55BMC Gastroenterology141-BGMA

微生物における環状アミド化合物変換の生化学的および応用研究

京都大学0048新制・課程博士博士(農学)甲第8422号農博第1106号新制||農||799(附属図書館)学位論文||H12||N3379(農学部図書室)UT51-2000-F326京都大学大学院農学研究科応用生命科学専攻(主査)教授 清水 昌, 教授 江﨑 信芳, 教授 熊谷 英彦学位規則第4条第1項該当Doctor of Agricultural ScienceKyoto UniversityDA