Studies on the properties and application of xylanase from Bacillus subtilis

• Main Authors: Chien Li-Ling, 簡里玲
• Other Authors: Chen-Tein Chang, Ph. D
• Format: Others
• Language: zh-TW
• Published: 2000
• Online Access: http://ndltd.ncl.edu.tw/handle/41762766117731693238

碩士 === 靜宜大學 === 食品營養學系 === 89 === Xylan, a major component of the hemicellulose of plant cell wall, could be used for production of xylooligosaccharides by enzymatic hydrolysis method. Xylooligosaccharides stimulate the growth of benefical bifidobacteria in large intestine, and could be used as a effective component for preparation of functional foods. The purpose of this study is to investigate the possible use of inexpensive commercial xylanase for preparation of xylooligosaccharides from bagasse hemicellulose. In addition, xylanase was also purified and characterized from the preparation of crude xylanase. Crude hemicellulose with the highest amount of pentosan could be extracted from bagasse with 8% NaOH and precipitated with ethanol. About 23 g of crude hemicellulose was isolated from 100 g of bagasse. Crude hemicellulose could be further purified by precipitating with Felhing reagent. About 60g of purified hemicellulose were obtained from 100 g of crude hemicellulose. Both hemicelluloses were converted to xylooligosaccharides by hydrolyzing with xylanase (Puplzyme HC) at 50℃ for 24 hr. The main components of the xylooligosaccharides were xylobiose and xylotriose as analyzed by thin-layer chromatography and gel permeation chromatography. Two xylanases, XynA and XynB, were purified from commercial crude xylanase by sequential steps of Sephacryl S-100 HR gel filtration, Polybuffer exchanger PBE-94 chromatofocusing, Mono S ion-exchange chromatography and Superdex 75 HR gel filtration. Using these steps, the purity of XynA was increased by 33 fold, with a yield of 2.7%, while the purity of XynB was increased 29.4 fold, with a yield of 2.4%. For hydrolysis of xylan, XynA had an optimal pH of 6.2, an optimal temperature of 70℃, a Km of 0.15% and an activation energy of 14.6 kcal/mole, while XynB had an optimal pH of 6.2, an optimal temperature of 70℃, a Km of 0.32% and an activation energy of 18.25 kcal/mole. Both enzymes had a molecular mass of 24.3 kDa, as estimated by SDS-PAGE, a pI value of above 9.3, as estimated by isoelectro- focusing electrophoresis. Mental ions (1 mM), Na+, K+, Mg2+, Zn2+, Mn2+, Ca2+ stimulate both enzyme activities . Heavy metal ions (1 mM) Hg2+, Fe2+, Fe3+, EDTA (1 mM), and chemical modification agents (0.05 mM) N-bromosuccinimide (NBS) and p-hydroxymercuribenzoic acid (PHMB) significantly inhibited both enzyme activities. Both xylanase were active on birchwood xylan and bagasse hemicellulose. The products produced in the hydrolysates of both enzymes were xylobiose, xylotriose and some xylooligosaccharides had longer chain length.