化学修饰提高谷氨酰胺转胺酶活性与热稳定性

Chemical Modification of Transglutaminase for Improving Specific Activity and Thermal Stability

DOI：10.3969/j.issn.1673-1689.2015.11.003

中文关键词: 谷氨酰胺转胺酶 邻苯二甲酸酐 比活力 热稳定性 表面疏水度

英文关键词: transglutaminase, phthalic anhydride, specific activity, thermal stability, surface hydrophobicity

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中文摘要:

谷氨酰胺转氨酶（EC 2.3.2.13，简称TGase） 是一种催化转酰基反应，导致蛋白质分子间发生交联的酶，广泛应用于食品、纺织、皮革、医药等领域。为提高TGase比活力与热稳定性，作者以邻苯二甲酸酐为修饰剂，对重组吸水链霉菌（Streptomyces hygroscopicus） TGase分子赖氨酸残基进行化学修饰。确定最佳修饰条件为：pH 8.0、20 ℃修饰反应2 h、TGase表面氨基与PA摩尔比为1∶2。无胱胺（TGase的竞争性抑制剂）存在时，PA修饰使TGase比活力提高48.3%；10 mmol/L胱胺存在时，PA修饰使TGase热稳定性提高31.3%。晶体模拟结构显示，21个赖氨酸残基位于S. hygroscopicus TGase分子表面，其中5个位于催化活性中心附近区域。表面疏水性分析发现，不同条件的PA修饰使TGase分子表面疏水度降低9.46%~21.13%。因此，PA可能通过改变TGase分子不同区域的疏水性来提高其比活力和热稳定性的。上述结果表明，PA修饰是一种改进TGase酶学性质的有效策略，为其后续的分子改造提供了一定的理论参考。

英文摘要:

Transglutaminase（EC 2.3.2.13，TGase） can catalyze the acyl transfer reaction and leads to the crosslinking among proteins. It has been widely used in food，textile，leather，and pharmaceutical industry. In order to improve the specific activity and thermal stability，a recombinant Streptomyces hygroscopicus TGase chemically was modified by phthalic anhydride（PA） in this study. The optimal modification condition of the cultivation was at pH 8.0，20 ℃ for 2 h，and the molar ratio of surface-amine to phthalic anhydride 1∶2. In the presence of 10 mmol/L of cystamine，which is a competitive inhibitor of the TGase，the thermal stability of TGase was increased 31.3% after the PA modification，whereas specific activity of the TGase increased 48.3% in the absence of the cystamine. The modeling crystal structure showed that 21 lysine residues existed in surface of S. hygroscopicus TGase，and five of them located at region close the active site. The analysis of surface hydrophobicity indicated that PA modification reduced the surface hydrophobicity from 9.46% to 21.13%. Thus，PA may improve the specific activity and thermal stability of TGase by modifying the surface hydrophobicity at different regions. These results suggested that PA modification was an effective way to improve the catalytic properties of TGase，which provided a theoretical reference for molecular modification.

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