Recombinant Bacillus amyloliquefaciens xylanase A expressed in Pichia pastoris and generation of xylooligosaccharides from xylans and wheat bran.
Recombinant Bacillus amyloliquefaciens xylanase A expressed in Pichia pastoris and generation of xylooligosaccharides from xylans and wheat bran.
复制标题
DOI：
10.1016/j.ijbiomac.2017.07.073
复制
发表时间：
2017-12
影响因子：
8.2
通讯作者：
Ming-qi Liu;Wen-kang Huo;Xin Xu;X. Weng
中科院分区：
文献类型：
--
作者：
Ming-qi Liu;Wen-kang Huo;Xin Xu;X. Weng
在本研究中，将DETAILED A（GenBank：KM 624029）编码的解淀粉芽孢杆菌木聚糖酶A（Bacillusamyloliquefaciens glycanase A，简称AMA）在毕赤酵母GS115中得到高效表达。重组木聚糖酶rePBaxA经Ni亲和树脂纯化，分子量为35.0kDa。rePBaxA的最适温度为50 °C，最适pH为5.0。反应动力学参数为米氏常数Km = 5.41 mg/mL，最大反应速率Vmax = 22.42 μmol/min/mL。高效液相色谱分析结果表明，rePBaxA水解山毛榉木聚糖和桦木木聚糖6 h后释放出木糖-木六糖（X1-X6）混合物，主要产物分别为木二糖（X2）和木三糖（X3）。燕麦斯卑尔脱酶、麦麸不溶性木聚糖和经rePBaxA预处理的麦麸的酶解产物均含有X2 ~ X6，其中X6的浓度最高。作用模式分析表明rePBaxA是一种具有转糖基活性的内切木聚糖酶。X2可能是rePBaxA水解的最小寡聚体。预处理后的麦麸和麦麸不溶性木聚糖可直接被rePBaxA水解。本研究为利用农业废弃物作为生产低聚木糖的高附加值益生菌提供了基础。
In this study,BaxA(GenBank: KM624029), which encodes theBacillus amyloliquefaciensxylanase A (BaxA), was highly expressed inPichia pastorisGS115 under the control of the AOX1 promoter. The recombinant xylanase, namely rePBaxA, was purified to homogeneity by using Ni-affinity resin and its molecular weight was 35.0 kDa. The optimum temperature and pH of rePBaxA were 50 °C and 5.0, respectively. The kinetic parameters Michaelis–Menten constant (Km) and maximum reaction rate (Vmax) of rePBaxA were 5.41 mg/mL and 22.42 μmol/min/mL, respectively. High–performance liquid chromatography results showed that after 6 h of hydrolysis, rePBaxA released xylose–xylohexaose (X1–X6) mixture from beechwood and birchwood xylan, with xylobiose (X2) and xylotriose (X3) as the major products, respectively. The hydrolyates from oat spelt, wheat bran insoluble xylan and pretreated wheat bran by rePBaxA included X2–X6, with X6 having the highest concentration. The mode of action analysis revealed that rePBaxA was anendo-acting xylanase with transglycosylation activity. X2 might be the minimum oligomer hydrolyzed by rePBaxA. The pretreated wheat bran and wheat bran insoluble xylan could be directly hydrolyzed by rePBaxA. This study provided a basis for using agricultural waste by-products as substrates for manufacting value-added probiotics, namely, xylooligosaccharides.