Shibata Nozomu, Suetsugu Mari, Kakeshita Hiroshi, Igarashi Kazuaki, Hagihara Hiroshi, Takimura Yasushi
Biological Science Research, Kao Corporation, 1334 Minato, Wakayama, Wakayama 640-8580 Japan.
Biotechnol Biofuels. 2017 Nov 21;10:278. doi: 10.1186/s13068-017-0970-2. eCollection 2017.
is considered a candidate fungal enzyme producer for the economic saccharification of cellulosic biomass. However, performance of the saccharifying enzymes produced by is insufficient. Therefore, many attempts have been made to improve its performance by heterologous protein expression. In this study, to increase the conversion efficiency of alkaline-pretreated bagasse to sugars, we conducted screening of biomass-degrading enzymes that showed synergistic effects with enzyme preparations produced by recombinant .
sp. strain KSM-F532 produced the most effective enzyme to promote the saccharification of alkaline-pretreated bagasse. Biomass-degrading enzymes from strain KSM-F532 were fractionated and analyzed, and a xylanase, named PspXyn10, was identified. The amino acid sequence of PspXyn10 was determined by cDNA analysis: the enzyme shows a modular structure consisting of glycoside hydrolase family 10 (GH10) and carbohydrate-binding module family 1 (CBM1) domains. Purified PspXyn10 was prepared from the supernatant of a recombinant strain. The molecular weight of PspXyn10 was estimated to be 55 kDa, and its optimal temperature and pH for xylanase activity were 75 °C and pH 4.5, respectively. More than 80% of the xylanase activity was maintained at 65 °C for 10 min. With beechwood xylan as the substrate, the enzyme had a of 2.2 mg/mL and a of 332 μmol/min/mg. PspXyn10ΔCBM, which lacked the CBM1 domain, was prepared by limited proteolysis. PspXyn10ΔCBM showed increased activity against soluble xylan, but decreased saccharification efficiency of alkaline-pretreated bagasse. This result indicated that the CBM1 domain of PspXyn10 contributes to the enhancement of the saccharification efficiency of alkaline-pretreated bagasse. A recombinant strain, named X2PX10, was constructed from strain X3AB1. X3AB1 is an β-glucosidase-expressing PC-3-7. X2PX10 also expressed PspXyn10 under the control of the promoter. An enzyme preparation from X2PX10 showed almost the same saccharification efficiency of alkaline-pretreated bagasse at half the enzyme dosage as that used for an enzyme preparation from X3AB1.
Our results suggest that PspXyn10 promotes the saccharification of alkaline-pretreated bagasse more efficiently than TrXyn3, a GH10 family xylanase from , and that the PspXyn10-expressing strain is suitable for enzyme production for biomass saccharification.
被认为是纤维素生物质经济糖化的候选真菌酶生产者。然而，其所产生的糖化酶性能不足。因此，人们进行了许多尝试，通过异源蛋白表达来提高其性能。在本研究中，为提高碱预处理甘蔗渣向糖的转化效率，我们对与重组产生的酶制剂具有协同作用的生物质降解酶进行了筛选。
sp. 菌株KSM-F532产生了促进碱预处理甘蔗渣糖化的最有效酶。对菌株KSM-F532的生物质降解酶进行了分级分离和分析，鉴定出一种木聚糖酶，命名为PspXyn10。通过cDNA分析确定了PspXyn10的氨基酸序列：该酶具有由糖苷水解酶家族10（GH10）和碳水化合物结合模块家族1（CBM1）结构域组成的模块化结构。从重组菌株的上清液中制备了纯化的PspXyn10。PspXyn10的分子量估计为55 kDa，其木聚糖酶活性的最佳温度和pH分别为75°C和pH 4.5。超过80%的木聚糖酶活性在65°C下保持10分钟。以山毛榉木聚糖为底物，该酶的Km为2.2 mg/mL，Vmax为332 μmol/min/mg。通过有限蛋白酶解制备了缺乏CBM1结构域的PspXyn10ΔCBM。PspXyn10ΔCBM对可溶性木聚糖的活性增加，但碱预处理甘蔗渣的糖化效率降低。该结果表明PspXyn10的CBM1结构域有助于提高碱预处理甘蔗渣的糖化效率。从菌株X3AB1构建了一个重组菌株，命名为X2PX10。X3AB1是表达β-葡萄糖苷酶的PC-3-7。X2PX10也在启动子的控制下表达PspXyn10。来自X2PX10的酶制剂在酶用量为X3AB1酶制剂一半的情况下，对碱预处理甘蔗渣的糖化效率几乎相同。
我们的结果表明，PspXyn10比来自的GH10家族木聚糖酶TrXyn3更有效地促进碱预处理甘蔗渣的糖化，并且表达PspXyn10的菌株适用于生物质糖化的酶生产。
