不同类型连接肽对β-甘露聚糖酶AuMan5A酶学性质的影响

Effects of Different Types of Peptide Linkers on the Enzymatic Properties of β-Mannanase

DOI：10.3969/j.issn.1673-1689.2017.09.008

中文关键词: 连接肽 β-甘露聚糖酶 融合酶 底物亲和力 热稳定性

英文关键词: peptide linker,β-Mannanase,fusion enzyme,substrate affinity,thermostability

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中文摘要:

以柔性肽F（GGGGS）3或刚性肽R（EAAAK）3为连接肽，将海栖热胞菌27家族碳水化合物结合域（CBM27） 与宇佐美曲霉5家族β-甘露聚糖酶（AuMan5A）的C末端融合，探讨不同类型连接肽对AuMan5A酶学性质的影响，获取具有优良酶学性质的融合酶。采用重叠PCR技术扩增融合酶基因Auman5A-F-cbm27和Auman5A-R-cbm27，分别将Auman5A和融合酶基因在毕赤酵母GS115中进行表达，分析表达产物reAuMan5A、reAuMan5A-F-C和reAuMan5A-R-C的酶学性质。结果表明：该3种β-甘露聚糖酶对角豆胶的Km值分别为1.7、1.9和0.9 mg/mL。3种酶的最适温度Topt分别为70、65和70 ℃；reAuMan5A-F-C和reAuMan5A-R-C在70 ℃的半衰期t701/2分别为36 min和124 min，较reAuMan5A的（t701/2=9 min）延长了3倍和12.8倍。reAuMan5A-R-C具有底物亲和力强、热稳定性高和pH稳定范围广等特点，在食品、饲料、医药和能源等领域有着巨大的应用潜力。

英文摘要:

A family 27 carbohydrate-binding module（CBM27） of Thermotoga maritima MSB8 was linked to the C-terminus of a glycoside hydrolase family 5 β-mannanaseof Aspergillus usamii YL-01-78（AuMan5A） by using a flexible peptide linker F（GGGGS）3 ora rigid peptide linker R（EAAAK）3. By overlapping PCR technique，twofusion enzyme genes，Auman5A-F-cbm27 and Auman5A-R-cbm27，was amplified. The gene Auman5A and two fusion genes were expressed in Pichia pastoris GS115，respectively. And then，the enzymatic properties of the recombinant β-mannanases，reAuMan5A，reAuMan5A-F-C and reAuMan5A-R-C，were analyzed. As a result，the Km ofreAuMan5A-R-C towards locust bean was 0.9 mg/mL，which was much smaller than those of reAuMan5A（1.7 mg/mL） and reAuMan5A-F-C（1.9 mg/mL）. The temperature optimum（Topt） of reAuMan5A-F-C was 65 ℃，which was 5 ℃ lower than that（70 ℃） of reAuMan5A or reAuMan5A-R-C. The half-lives at 70℃（t701/2） of reAuMan5A-F-C and reAuMan5A-R-C were 36 and 124 min，respectively，which were about 4- and 13.8 folds longer than that（t701/2=9 min） of reAuMan5A. In this study，the fusionenzymereAuMan5A-R-C was obtained with strong substrate affinity，high thermostability and wide range of pH stability. It will be a potential candidate for industrial processes，such as food，animal feed，energy，pharmaceuticals and so on.

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