Banka Alison L, Guralp Saadet Albayrak, Gulari Erdogan
Department of Chemical Engineering, University of Michigan, 3074 H.H.Dow Buil., 2300 Hayward St, Ann Arbor, MI, 48109, USA.
Appl Biochem Biotechnol. 2014 Dec;174(8):2702-10. doi: 10.1007/s12010-014-1219-1. Epub 2014 Sep 16.
Microbial hydrolysis of lignocellulosic biomass is becoming increasingly important for the production of renewable biofuels to address global energy concerns. Hemicellulose is the second most abundant lignocellulosic biopolymer consisting of mostly xylan and other polysaccharides. A variety of enzymes is involved in complete hydrolysis of xylan into its constituent sugars for subsequent biofuel fermentation. Two enzymes, endo-β-xylanase and β-xylosidase, are particularly important in hydrolyzing the xylan backbone into xylooligosaccharides and individual xylose units. In this study, we describe the cloning, expression, and characterization of xylanase and β-xylosidase isolated from Bacillus subtilis M015 in Escherichia coli. The genes were identified to encode a 213 amino acid protein for xylanase (glycoside hydrolase (GH) family 11) and a 533 amino acid protein for β-xylosidase (GH family 43). Recombinant enzymes were produced by periplasmic-leaky E. coli JE5505 and therefore secreted into the supernatant during growth. Temperature and pH optima were determined to be 50 °C and 5.5-6 for xylanase and 35 °C and 7.0-7.5 for β-xylosidase using beech wood xylan and p-nitrophenyl-β-D-xylopyranoside as the substrates, respectively. We have also investigated the synergy of two enzymes on xylan hydrolysis and observed 90 % increase in total sugar release (composed of xylose, xylobiose, xylotriose, and xylotetraose) for xylanase/β-xylosidase combination as opposed to xylanase alone.
为解决全球能源问题，微生物对木质纤维素生物质的水解在可再生生物燃料生产中变得越来越重要。半纤维素是第二丰富的木质纤维素生物聚合物，主要由木聚糖和其他多糖组成。多种酶参与木聚糖完全水解为其组成糖，用于后续生物燃料发酵。内切-β-木聚糖酶和β-木糖苷酶这两种酶在将木聚糖主链水解为木寡糖和单个木糖单元方面尤为重要。在本研究中，我们描述了从枯草芽孢杆菌M015中分离的木聚糖酶和β-木糖苷酶在大肠杆菌中的克隆、表达和特性。这些基因被鉴定为分别编码一种213个氨基酸的木聚糖酶蛋白（糖苷水解酶（GH）家族11）和一种533个氨基酸的β-木糖苷酶蛋白（GH家族43）。重组酶由周质渗漏型大肠杆菌JE5505产生，因此在生长过程中分泌到上清液中。分别以山毛榉木聚糖和对硝基苯基-β-D-木吡喃糖苷为底物，测定木聚糖酶的最适温度和pH为50°C和5.5 - 6，β-木糖苷酶的最适温度和pH为35°C和7.0 - 7.5。我们还研究了两种酶对木聚糖水解的协同作用，观察到与单独使用木聚糖酶相比，木聚糖酶/β-木糖苷酶组合的总糖释放量（由木糖、木二糖、木三糖和木四糖组成）增加了90%。
