黑酵母Hortaea werneckii GH10家族耐热木聚糖酶的异源表达及其降解桑皮特性  

Heterologous Expression of GH10 Xylanase from Black Yeast Hortaea werneckii and Its Characteristics in Mulberry Bark Degradation

作　　者：谢晨 游帅 张温馨 白致远 吴福安 王俊[1,2] Xie Chen;You Shuai;Zhang Wenxin;Bai Zhiyuan;Wu Fuan;Wang Jun(School of Biotechnology,Jiangsu University of Science and Technology,Zhenjiang Jiangsu 212100,China;Sericul-tural Research Institute,Chinese Academy of Agricultural Sciences,Zhenjiang Jiangsu 212000,China)

机构地区：[1]江苏科技大学生物技术学院,江苏镇江212100 [2]中国农业科学院蚕业研究所,江苏镇江212100

出　　处：《蚕业科学》2021年第6期559-567,共9页ACTA SERICOLOGICA SINICA

基　　金：国家自然科学基金项目(21978121);江苏省自然科学基金项目(BK20190957)。

摘　　要：我国桑树种植面积广,桑园管理每年需要伐枝,其桑皮除了含有活性物质还含有丰富的木质纤维素,急需进行高值化开发利用,因此选用高比活耐热木聚糖酶是桑皮降解及其转化应用的关键酶。本文从黑酵母Hortaea werneckii中克隆得到糖苷水解酶HwXYL10A基因,在毕赤酵母中进行了异源表达,探究其酶学性质,为利用其降解桑皮木质纤维素提供可行性。该基因编码的蛋白与GH10家族高温木聚糖酶XYL10C_△N(GenBank:ACS96449.1)的同源性为60%。该酶的最适温度为75℃,最适pH为4.0,T_(50)值为76℃,在75℃和80℃下半衰期分别为70 min和11 min,表明HwXYL10A具有良好的热稳定性。在最适条件下,以榉木木聚糖、甘蔗木聚糖和玉米芯木聚糖为底物测定,其比活分别是3 260 U/mg、3 160 U/mg和3 870 U/mg,催化效率分别为2 970 mL/(s·mg)、2 320 mL/(s·mg)和975 mL/(s·mg)。采用该木聚糖酶与纤维素酶协同水解经氨水或氢氧化钙预处理的桑皮,协同处理组均在12 h产糖量达到最高,分别为20.38μmol/mL和22.13μmol/mL;最大协同率分别为1.59和1.47。这些优良的酶学性质使HwXYL10A在桑皮以及其他木质纤维素降解中具有潜在的应用价值,同时为耐热木聚糖酶的研究和应用提供了新的素材。The large amount of mulberry bark cut down every year in mulberry fields of China is abundant in lignocellulosic besides active substances, which is in urgent need of high value development and utilization. Therefore, high-specific active and heat-resistant xylanase is the key enzyme for mulberry bark degradation and its transformation application. In this study, glycoside hydrolase HwXYL10 A coding gene was cloned from Hortaea werneckii and heterologous expressed in Pichia pastoris to explore its enzymatic properties, so as to provide feasibility for its degradation of mulberry lignocellulose. The protein encoded by this gene has 60% homology with GH10 family thermophilic xylanase XYL10 C_△N(GenBank: ACS96449.1). The optimal temperature for the enzyme is 75 ℃, and the optimal pH is 4.0. HwXYL10 A has an excellent thermostability with T_(50)value of 76 ℃, half-life of 70 min and 11 min at 75 ℃ and 80 ℃, respectively. Under the optimum conditions, beech xylan, sugarcane xylan and corn cob xylan were used as substrates. The specific activity was 3 260 U/mg, 3 160 U/mg and 3 870 U/mg, respectively. The catalytic efficiency was 2 970 mL/(s·mg), 2 320 mL/(s·mg) and 975 mL/(s·mg), respectively. For the first time, the xylanase and cellulase were used to synergistically hydroxide the mulberry bark pretreated with ammonia or calcium hydroxide. It was found that the sugar yield of mulberry bark in the synergistically-treated group reached the highest point at 12 h, which was 20.38 μmol/mL and 22.13 μmol/mL, respectively. The maximum synergy rates were highest at 1.59 and 1.47, respectively. With these excellent enzymatic properties, HwXYL10 A have potential application value in the degradation of mulberry bark and other lignocellulosic, and provide new materials for the research and application of thermophilic xylanase.

关 键 词：GH10木聚糖酶 高比活 耐热 桑皮 协同降解 

分 类 号：Q814.9[生物学—生物工程] S886.9[农业科学—特种经济动物饲养]