Li Jianfang, Zhang Huimin, Wu Minchen, Wang Chunjuan, Dong Yunhai, Zhu Lijuan, Zhang Peng
State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu, 214122, People's Republic of China.
Appl Biochem Biotechnol. 2014 Apr;172(7):3476-87. doi: 10.1007/s12010-014-0786-5. Epub 2014 Feb 19.
Both Syxyn11P and Syxyn11E, two codon-optimized genes encoding glycoside hydrolase (GH) family 11 hyperthermotolerant xylanases (designated SyXyn11P and SyXyn11E), were synthesized and inserted into pPIC9K(M) and pET-28a(+) vectors, respectively. The resulting recombinant expression vectors, pPIC9K(M)-Syxyn11P and pET-28a(+)-Syxyn11E, were transformed into Pichia pastoris GS115 and Escherichia coli BL21, respectively. The maximum activities of two recombinant xylanases (reSyXyn11P and reSyXyn11E) expressed in P. pastoris and E. coli reached 30.9 and 17.8 U/ml, respectively. The purified reSyXyn11P and reSyXyn11E displayed the same pH optimum at 6.5 and pH stability at a broad range of 4.5-9.0. The temperature optimum and stability of reSyXyn11P were 85 and 80 °C, higher than those of reSyXyn11E, respectively. Their activities were not significantly affected by metal ions tested and EDTA, but strongly inhibited by Mn(2+) and Ag(+). The K m and V max of reSyXyn11P toward birchwood xylan were 4.3 mg/ml and 694.6 U/mg, whose K m was close to that (4.8 mg/ml), but whose V max was much higher than that (205.6 U/mg) of reSyXyn11E. High-performance liquid chromatography analysis indicated that xylobiose and xylotriose as the major products were excised from insoluble corncob xylan by reSyXyn11P.
合成了两个密码子优化的基因Syxyn11P和Syxyn11E，它们编码糖苷水解酶（GH）家族11的超耐热木聚糖酶（分别命名为SyXyn11P和SyXyn11E），并分别插入pPIC9K(M)和pET-28a(+)载体中。得到的重组表达载体pPIC9K(M)-Syxyn11P和pET-28a(+)-Syxyn11E分别转化到毕赤酵母GS115和大肠杆菌BL21中。在毕赤酵母和大肠杆菌中表达的两种重组木聚糖酶（reSyXyn11P和reSyXyn11E）的最大活性分别达到30.9和17.8 U/ml。纯化后的reSyXyn11P和reSyXyn11E在pH 6.5时具有相同的最适pH值，在4.5 - 9.0的较宽范围内具有pH稳定性。reSyXyn11P的最适温度和稳定性分别为85和80℃，高于reSyXyn11E。测试的金属离子和EDTA对它们的活性没有显著影响，但Mn(2+)和Ag(+)强烈抑制它们的活性。reSyXyn11P对桦木木聚糖的K m和V max分别为4.3 mg/ml和694.6 U/mg，其K m与reSyXyn11E的K m（4.8 mg/ml）接近，但其V max远高于reSyXyn11E的V max（205.6 U/mg）。高效液相色谱分析表明，reSyXyn11P从不溶性玉米芯木聚糖中切下木二糖和木三糖作为主要产物。
