嗜热子囊菌JCM12803的α-半乳糖苷酶基因tcgal27A在毕赤酵母中的表达

发布时间：2018-03-07 14:51

本文选题：嗜热真菌　切入点：α-半乳糖苷酶　出处：《生物技术通报》2017年06期 　论文类型：期刊论文

【摘要】：从嗜热子囊菌(Thermoascus crustaceus)JCM12803克隆α-半乳糖苷酶基因,并对其酶学性质进行系统的研究,旨为获得工业应用性质优良的α-半乳糖苷酶。通过RT-PCR方法从T.crustaceus JCM12803中克隆α-半乳糖苷酶基因序列,并对其酶学性质进行了系统的分析。结果显示,tcgal27A属于糖苷水解酶27家族,基因全长1 918 bp,含有4个内含子,c DNA全长1 419 bp,编码472个氨基酸,预测tcgal27A N端含有24个氨基酸为其可能的信号肽。将TCGal27A在毕赤酵母中成功地进行了表达,所获得的重组TCGal27A具有高比活(336.5 U/mg)及宽泛的底物特异性,对蜜二糖(14.4 U/mg)、棉子糖(9.1 U/mg)、角豆胶(3.6 U/mg)、魔芋粉(1.6 U/mg)、瓜尔豆胶(1.3 U/mg)、水苏糖(0.7 U/mg)都有着不同程度的降解作用。以pNPG为底物时的Km值为1.6mol/m L,Vmax为536.8μmo L/(min·mg)。TCGal27A的最适作用pH为4.5,最适温度为65℃,50℃处理1 h之后酶活力还能保持86.0%。这些结果表明耐高温TCGal27A性质优良,可添加到饲料中,在消化和提高豆粕蛋白能量利用率方面有良好的潜在应用价值。
[Abstract]:From Thermoascus aurantiacus (Thermoascus crustaceus) JCM12803 cloning of alpha galactosidase gene, and systematic study on its enzymatic properties, in order to obtain excellent industrial application properties of alpha galactosidase. Cloning of alpha galactosidase gene sequence from T.crustaceus JCM12803 by RT-PCR method, and its enzymatic properties were analyzed. The results show that tcgal27A belongs to glycoside hydrolase family 27, gene is 1918 BP, containing 4 introns, C DNA was 1419 BP, encoding 472 amino acids, prediction of tcgal27A N terminal 24 amino acids for its possible signal peptide. TCGal27A in Pichia pastoris was carried out successfully the expression of the obtained recombinant TCGal27A with high specific activity (336.5 U/mg) and broad substrate specificity, two of honey sugar (14.4 U/mg), raffinose (9.1 U/mg), carob (3.6 U/mg), konjac powder (1.6 U/mg), guar gum (1.3 U/mg) Stachyose, (0.7 U/mg) have different degrees of degradation. Using pNPG as the substrate when the Km value is 1.6mol/m L, Vmax 536.8 Mo L/ (min mg) the optimum effect of.TCGal27A pH was 4.5, the optimum temperature is 65 DEG, 50 DEG C after treatment for 1 h enzyme activity these results indicate that 86.0%. can maintain excellent properties of high temperature resistant TCGal27A, can be added to the feed, the digestion and improvement have good potential application value of the soybean protein energy utilization.

【作者单位】：工业发酵微生物教育部重点实验室天津市工业微生物重点实验室天津科技大学生物工程学院;中国农业科学院饲料研究所;北京林业大学生物科学与生物技术学院;
【基金】：国家杰出青年科学基金(31225026)
【分类号】：Q78;Q936

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