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2025年4月4日 星期五
  2017, Vol. 25 Issue (11): 1851-1859    
  研究论文与报告 本期目录 | 过刊浏览 | 高级检索 |
His159突变对嗜热革节孢EGⅡ活性及热稳定性的影响
高金鹏1,刘玉姣2,刘宁1,王欢2,李多川3
1. 山东农业大学
2.
3. 山东农业大学植保学院
Effect of His159 Mutations on the Activity and Thermostability of EGⅡ from Scytalidium thermophilum
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摘要 嗜热革节孢(Scytalidium thermophilum)内切葡聚糖酶Ⅱ(endoglucanase Ⅱ, EGⅡ)的第159位组氨酸(His159)在糖苷水解酶第五家族中是高度保守的,但其在酶催化中的作用尚不清楚。本研究将His159分别突变为丙氨酸、天冬氨酸、苯丙氨酸、精氨酸、色氨酸和酪氨酸,突变酶H159A、H159D、H159F、H159R、H159W和H159Y与原始酶(wild type, WT)在毕赤酵母(Pichia pastoris)中表达,经亲和层析纯化后进行特性分析。结果表明,6个突变酶的米氏常数(Km)均高于WT,突变酶H159D、H159F、H159R、H159W和H159Y的催化常数 (kcat)降低,但H159A的kcat有显著提高(P<0.05)。WT的最适反应温度为70 ℃,突变酶H159A、H159D 和H159F最适温度为60 ℃,H159R、H159W和H159Y最适温度为55 ℃。突变酶H159A在60 ℃ 10 min处理后有80%以上的活性,突变酶H159D和H159W的热稳定性明显下降,突变酶H159F、H159R和H159Y在60 ℃处理10 min几乎丧失全部活性。上述研究结果表明,159位的组氨酸与酶的活性和热稳定性有密切关系。本研究在前人研究的基础上,对保守位点His159进行定点突变和性质测定与比较,获得了更具体的数据,进一步分析H159氨基酸影响酶活性的分子与结构机制,为下一步的纤维素酶的分子改造提供参考。
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高金鹏
刘玉姣
刘宁
王欢
李多川
关键词 内切葡聚糖酶,定点突变,动力学参数,释放效率    
Abstract:As the major structural polysaccharide of plant cell walls, cellulose is the most abundant organic material on earth. Cellulose biodegradation offers the potential to produce fuels and other chemicals from renewable substrates. In cellulase biodegradation system, endogluconase attacks cellulose at random places, to break the long-chain polyer of glucose into short chains, exoglucanase attacks the ends of the cellulose, to release cellobiose, and β-glucosidase cuts cellobiose and cello-oligosaccharide into glucose. Endoglucanases are classified by the Carbohydrate-active Enzyme data into 13 glycosyl hydrolase families (5, 6, 7, 8, 9, 12, 44, 45, 48, 51, 74, 124 and 131) based on protein sequence similarity and catalytic domain structure. Glycosyl hydrolase family 5 (GH5) is one of the largest GH families, historically known as “cellulase family A” as it was the first cellulase family described. GH5 members are commonly found to be encoded as parts of multi-modular polypeptide chains mainly containing a catalytic domain and a cellulose-binding domain and play an important role in the cellulase biodegradation enzyme system. In this study, an endoglucanase (EGII) was isolated from thermophilic fungus Scytalidium thermophilum. Based on sequence alignment analysis, two catalytic residues of EGⅡ were identified by homology to the other GH5 structures as Glu 199 (acid-base) and Glu 306 (nucleophile), and 6 residues conserved among the glycoside hydrolase family 5 were also found. The 6 residues include Arg 115, His 159, Asn 198, His 264, Yyr 266, Trp 399. The His159 of the EGⅡ is highly conserved in the family 5 of glycoside hydrolases, but its function is unclear so far. To determine His159 function of catalysis, we cloned EGⅡ gene from S. thermophilum and constructed pPIC9K/egII recombinant plasmid and then mutated the His159 into Ala, Asp, Phe, Trp, Arg and Tyr by site-directed mutation, respectively. The mutant enzymes H159A, H159D, H159F, H159R, H159W, H159Y and wild-type enzyme WT (EGⅡ) were expressed in Pichia pastoris and purified using nickel affinity chromatography and characterized. Activity measurements indicated that the specific activity of all mutant enzymes was lower than that of WT. The specific activity of WT, H159A, H159D, H159F, H159R, H159W, H159Y is (11.02±0.41), (6.42±0.10), (2.09±0.07), (0.32±0.02), (0.66±0.01), (1.89±0.13), (0.19±0.02) U/mg, respectively. Further kinetic measurements indicated that Km values of all the mutant enzymes were higher than that of WT, indicating that the affinity of the mutant enzymes to the substrate is reduced. Compared with WT, kcat values of H159D, H159F, H159R, H159W and H159Y decreased but kcat value of H159A was significantly increased, indicating that His159 is involved in the binding of enzymes to substrates. The optimum reaction temperature was 70 ℃ for WT, 60 ℃ for H159A, H159D and H159F, and 55 ℃ for H159R, H159W and H159Y. After incubation at 60 ℃ for 10 minutes, H159A still retained more than 80% activity, H159D and H159W had a significant decrease, H159F, H159R and H159Y were substantially inactivated, indicating that His159 is also involved in EGⅡ thermostability. Our data provide insight into understanding of the function of His159 in the family 5 of glycoside hydrolases.
Key wordsEndoglucanase, Site-directed mutagenesis, Kinetic parameters, Release efficiency
收稿日期: 2017-05-04      出版日期: 2017-11-01
基金资助:国家863计划课题;国家科技支撑计划课题;国家自然科学基金项目
通讯作者: 李多川     E-mail: lidc20@sdau.edu.cn
引用本文:   
高金鹏 刘玉姣 刘宁 王欢 李多川. His159突变对嗜热革节孢EGⅡ活性及热稳定性的影响[J]. , 2017, 25(11): 1851-1859.
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http://journal05.magtech.org.cn/Jwk_ny/CN/     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2017/V25/I11/1851
 
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