Shivange Amol V, Dennig Alexander, Schwaneberg Ulrich
Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 1, D-52074 Aachen, Germany; School of Engineering and Science, Jacobs University Bremen gGmbH, Campus Ring 1, 28759 Bremen, Germany.
Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 1, D-52074 Aachen, Germany.
J Biotechnol. 2014 Jan 20;170:68-72. doi: 10.1016/j.jbiotec.2013.11.014. Epub 2013 Dec 4.
Directed evolution of Yersinia mollaretii phytase (Ymphytase) yielded an improved variant SM2P3E4 (also named M1; D52N, T77K, K139E, G187S, V298M) in our previous study. Variant M1 retained high specific activity (993U/mg; equivalent to 93% of wild-type activity) and improved thermal resistance (T50 improved by 1.5°C compared to wild-type at 58°C; 20min incubation time), making variant M1 an attractive enzyme for industrial applications. Recently, the OmniChange method was developed for multi-site saturation mutagenesis. The five sites identified in variant M1 were subjected to OmniChange saturation in order to explore whether a variant with higher activity, higher thermal resistance, and higher resistance at low pH (2-3h incubation was performed to mimic the gastric residence time of phytase) could be identified. Screening of a small library of 1100 clones, covering <0.004% of the theoretical sequence space of 3.35×10(7) variants, yielded a Ymphytase variant with 32% improved residual activity (58°C for 20min), 2°C increased apparent melting temperature (Tm), and 2-fold higher pH stability (pH 2.8; 3h incubation time) when compared to the wild-type Ymphytase. Compared to the M1 variant, the pH stability (pH 2.8; 3h incubation time) was improved by 3-fold, and thermal resistance as well as activity was improved slightly (residual activity: 32% compared to 20%; apparent Tm: 2°C compared to 1.5°C; activity difference <4%).
在我们之前的研究中，莫拉氏耶尔森菌植酸酶（Ymphytase）的定向进化产生了一个优化变体SM2P3E4（也称为M1；D52N、T77K、K139E、G187S、V298M）。变体M1保留了高比活性（993U/mg；相当于野生型活性的93%）并提高了耐热性（在58°C下孵育20分钟时，T50比野生型提高了1.5°C），这使得变体M1成为工业应用中一种有吸引力的酶。最近，开发了OmniChange方法用于多位点饱和诱变。对在变体M1中鉴定出的五个位点进行了OmniChange饱和诱变，以探索是否能鉴定出一种具有更高活性、更高耐热性以及在低pH值下（进行2 - 3小时孵育以模拟植酸酶在胃中的停留时间）具有更高抗性的变体。对一个包含1100个克隆的小文库进行筛选，该文库覆盖的理论序列空间为3.35×10⁷个变体的不到0.004%，结果得到了一个Ymphytase变体，与野生型Ymphytase相比，其残余活性提高了32%（58°C下20分钟），表观解链温度（Tm）升高了2°C，pH稳定性提高了2倍（pH 2.8；3小时孵育时间）。与M1变体相比，pH稳定性（pH 2.8；3小时孵育时间）提高了3倍，耐热性和活性也略有提高（残余活性：与20%相比为32%；表观Tm：与1.5°C相比为2°C；活性差异<4%）。
