将来源于草酸青霉菌(Penicillium oxalicum sp.68)的阿拉伯呋喃糖苷酶基因poabf62a进行密码子优化后,在毕赤酵母GS115中重组表达PoAbf62A酶,并分析酶学性质。为进一步提高重组蛋白表达量,通过单因素及响应面分析法优化了重组菌株产PoAbf62A酶的发酵工艺。结果表明,以对硝基苯-α-L-阿拉伯呋喃糖苷(p-nitrobenzene-L-arabinofuranoside,pNPAF)为底物时,重组阿拉伯呋喃糖苷酶PoAbf62A的最适反应pH值为4.5,最适反应温度为40 ℃;该酶在pH 4.5和40~45 ℃下较稳定;Al3+、Co2+、Zn2+、Mg2+等金属离子对该酶活性有促进作用,Fe3+、Mn2、Cu2+和Fe2+可抑制其活性;以pNPAF为底物测得酶的Km值为2.46 mmol/L,最大反应速度Vmax为9.05 μmol/(min·g),kcat为0.22 s-1。最佳发酵参数为：BMMY培养基中接种量初始OD600值为2.4,添加诱导剂甲醇的体积分数为1.05%,每24 h补加甲醇使甲醇的体积分数恒定,30.5 ℃发酵120 h,发酵液酶活力为0.51 U/mL,相比未优化前提高了3.25倍。
The gene poabf62a encoding an arabinofurosidase from Penicillium oxalicum was codon optimized,chemically synthesized and expressed in Pichia pastoris GS115 and the recombinant enzyme was characterized. To further improve the expression level,the fermentation condition was optimized. The optimal reaction pH and temperature of the recombinant PoAbf62A on pNPAF were 4.5 and 40 ℃,respectively. The enzyme was stable at pH 4.5 and 40~45 ℃. Metal ions,such as Al3+,Co2+,Zn2+,Mg2+ could enhance its activity,while inhibited by Fe3+,Mn2+,Cu2+ and Fe2+. The Km value was 2.46 mmol/L,the Vmax was 9.05 μmol/(min·g),and the kcat was 0.22/s. The optimized fermentation parameters were as follows: inoculating size of the exponential growth phase cells from BMGY to BMMY with primary OD600 of 2.4,methanol supplemented with 1.05% of φ(methanol) every 24 h. As a result,the enzyme activity was 0.51 U/mL after 120 h fermentation at 30.5 ℃,which was 3.25 times higher.
[1] KALE M S,YADAV M P,CHAU H K,et al.Molecular and functional properties of a xylanase hydrolysate of corn bran arabinoxylan[J].Carbohydrate Polymers,2018,181:119-123.
[2] 过嫣丹. 麸质阿拉伯木聚糖的改性及应用[D].无锡:江南大学,2012.
[3] NUMAN M T,BHOSLE N B.Alpha-L-arabinofuranosidases: the potential applications in biotechnology[J].Journal of Industrial Microbiology and Biotechnology,2006,33(4):247-260.
[4] 张秋培. 啤酒糟制备阿魏酰低聚糖的研究[D].无锡:江南大学,2015.
[5] HENRISSAT B,DAVIES G J.Glycoside hydrolases and glycosyltransferases. Families,modules,and implications for genomics[J].Plant Physiology,2000,124(4):1 515-1 519.
[6] TAYLOR E J,SMITH N L,TURKENBURG J P,et al.Structural insight into the ligand specificity of a thermostable family 51 arabinofuranosidase,Araf51,from Clostridium thermocellum[J].Biochemistry Journal,2006,395(1):31-37.
[7] HU Y,YAN X,ZHANG H,et al.Cloning and expression of a novel α-1,3-arabinofuranosidase from Penicillium oxalicum sp. 68[J].AMB Express,2018,8(1):51.
[8] 解西柱,张明,林材,等.α-L-阿拉伯呋喃糖苷酶在毕赤酵母中的表达及其对大麦麦芽过滤性能的影响[J].食品与发酵工业,2018,44(2):48-54.
[9] 邢亚欣,黄火清,苏小运.一个来源于稻平脐蠕孢的α-阿拉伯呋喃糖苷酶[J].生物技术通报,2020,36(4):84-92.
[10] 薛业敏,毛忠贵,邵蔚蓝.热稳定性α-阿拉伯糖苷酶/木糖苷酶的纯化[J].食品与发酵工业,2003,29(9):22-26.
[11] DAMASCENO L M,HUANG C J,BATT C A.Protein secretion in Pichia pastoris and advances in protein production[J].Applied Microbiology and Biotechnology,2012,93(1):31-39.
[12] GAO J,XU W,FANG Q,et al.Efficient biotransformation for preparation of pharmaceutically active ginsenoside compound K by Penicillium oxalicum sp. 68[J].Annals of Microbiology,2013,63(1):139-149.
[13] DE L M M,GUAIS O,BONNIN E,et al.Molecular and biochemical characterization of three GH62 alpha-L-arabinofuranosidases from the soil deuteromycete Penicillium funiculosum[J].Enzyme and Microbial Technology,2013,53(5):351-358.
[14] CARTMELL A,MCKEE L S,PENA M J,et al.The structure and function of an arabinan-specific α-1,2-arabinofuranosidase identified from screening the activities of bacterial GH43 glycoside hydrolases[J].Journal of Biological Chemistry,2011,286(17):15 483-15 495.
[15] REN J,SUN R.Applications of hemicelluloses and of their derivatives in papermaking-A review[J].Cellulose Chemistry and Technology,2006,40(5):281-289.
[16] 过嫣丹. 麸质阿拉伯木聚糖的改性及应用[D].无锡:江南大学,2012.
[17] 闵兆升,郭会明,顾承真,等.木聚糖酶及其在食品工业中的应用[J].食品与发酵工业,2013,39(10):168-173.
[18] 王靖宇,刘玉春,韩伟,等.玉米皮纤维发酵裂褶菌的产酶分析及木聚糖酶基因克隆、表达和酶学性质测定[J].食品与发酵工业,2018,44(5):46-51.
[19] KANEZA P,杨林青,孙付保,等.甘蔗渣在添加剂和辅助酶作用下的浓醪酶解糖化[J].食品与发酵工业,2019,45(17):1-6.
[20] 袁小平. 酶解麦麸制备阿魏酰低聚糖及其生物活性的研究[D].无锡:江南大学,2006.
