源于厌氧真菌Orpinomyces sp.PC-2的xynA在毕赤酵母中的高效表达及其酶学性质分析  

High-level Expression and Characterization of xyn A Originated from Anaerobic Fungi Orpinomyces sp. PC-2 in Pichia pastoris

作　　者：汪艳[1] 罗艳丽[2] 胡玮[1,3] 张慧玲[1] 

机构地区：[1]新疆农业大学动物科学学院/新疆肉乳用草食动物营养与饲料重点实验室,乌鲁木齐830052 [2]新疆农业大学草业与环境科学学院,乌鲁木齐830052 [3]新疆农业大学食品科学与药学学院,乌鲁木齐830052

出　　处：《农业生物技术学报》2016年第5期708-717,共10页Journal of Agricultural Biotechnology

基　　金：新疆维吾尔自治区高技术研究发展项目(No.201211104);新疆肉乳用草食动物营养与饲料重点实验室开放课题(No.2013002)

摘　　要：β-D-1,4-内切木聚糖酶(endo-1,4-β-D-xylanase,xyn A,EC.3.2.1.8)简称β-木聚糖酶(β-xylanase),是木聚糖降解酶系中最重要的成员,广泛用于饲料、食品、造纸和生物能源等领域。厌氧真菌Orpinomyces sp.PC-2 xyn A具有潜在的开发价值,但是其异源表达的活性较低。本研究根据毕赤酵母(Pichia pastoris)对基因密码子偏好性,对厌氧真菌Orpinomyces sp.PC-2的xyn A进行密码子优化。通过全基因合成技术合成优化后的xyn Am,并克隆到毕赤酵母表达载体p PIC9K的多克隆位点,构建重组质粒p PIC9K-xyn Am,电击转化至毕赤酵母GS115菌株中,以29℃、0.5%甲醇诱导表达,获得重组xyn A,并对该酶的酶学特性进行了研究。结果表明,在摇瓶水平条件下,诱导表达108 h时重组β-木聚糖酶活性达到最大值,为612 IU/m L。在10 L发酵罐中诱导表达96 h后,xyn A的活性为3 515 IU/m L,比活性为2 411 IU/mg;菌体培养湿重、干重和培养上清总蛋白质的浓度分别为324、156和2.25 g/L。非变性十二烷基硫酸钠-聚丙烯酰胺(sodium dodecyl sulfate-polyacrylamide gel electropheresis,SDS-PAGE)凝胶电泳显示,重组木聚糖酶的分子量约为42.7 k D。酶学性质分析表明,重组β-木聚糖酶的最适反应温度为55℃,最适反应p H 6.0,在温度为30~50℃、pH 4.0~10.6之间该酶具有较好的稳定性,其Km=27.86 mg/m L,Vmax=277.78 mg/(m L·min)。底物特异性分析表明,重组xyn A可水解低粘度阿拉伯木聚糖、高粘度阿拉伯木聚糖、燕麦木聚糖、桦木木聚糖和4-O-甲基-D-葡萄糖醛酸-D-木聚糖,但不降解大麦(Hordeum vulgare)β-葡聚糖和地衣多糖。10 mmol/L的Co^(2+)和K^+对该酶的活性略有激活作用,Mn^(2+)、Fe^(2+)和SDS对该酶活性有一定的抑制作用。本研究为进一步工业化应用来源于厌氧真菌Orpinomyces sp.PC-2的β-内切木聚糖酶奠定了基础。Endo-1,4-β-xylanase （xynA, EC.3.2.1.8）, the most important member of xylanolytic enzymes, is widely used in feed, food, papermaking and bioenergy and other fields. The xynA from anaerobic fungi Orpinomyces sp. PC-2 has potential exploitation value, but its enzyme activity of heterologous expression is low. In the present research, according to the codon usage frequency of highly expressed genes in Pichia pastoris, a β- xylanase gene （xyrut） derived from anaerobic fungi Orpinomyces sp. PC-2 was optimized, and the modified gene （xynAm） was chemical synthesized and constructed a yeast expression vector pPIC9K-xynAm. Then, the xynAm gene was expressed in Pichia pastoris GS115, and enzymatic properties of the recombinant /3-xylanase were characterized. The results showed that the enzyme activity of the recombinant 13-xylanase reached the maximum of 612 IU/mL in shake-flask culture at 108 h of induction. In 10 L fermenter, the enzyme activity and specific activity of the recombinant 13-xylanase reached the maximum of 3 515 IU/mL and 2 411 IU/mg at 96 h of induction. The dry weight, wet weight of the yeast cells and the protein content in the culture supematant were reached 324, 156 and 2.25 g/L, respectively, at this time. The molecular weight of the enzyme was about 42.7 kD revealed by non-denatured sodium dodecyl sulfate-polyacrylamide gel electropheresis （SDS-PAGE）. Enzymatic properties analysis showed that the optimum pH and reaction temperature of the purification xylanase were 55℃ and 6.0, and relatively more stable at 30-50℃and pH 4.0-10.6, and the Km and Vmax were 27.86 mg/mL and 277.78 mg/（mL, rain）, respectively. Substrate specificity analysis showed that the xylanase could hydrolyze low and high viscosity arabinoxylans, oat spelt xylan, birch xylan and 4-O-methyl-D-glucurono-D-xylan but not barley （Hordeum vulgare） β-glucan and lichenan. The enzyme activity was slightly activated by 10 mmol/L Co2＋ and K＋, and inhibited by Mn2＋, Fe2＋ and SDS. This study lays a foundation

关 键 词：β-D-1 4-内切木聚糖酶A(xynA) 厌氧真菌PC-2 酶学性质 毕赤酵母 

分 类 号：Q939.97[生物学—微生物学]