Department of Biotechnology, Lund University, P. O. Box 124, SE-221 00 Lund, Sweden.
Enzyme Microb Technol. 2011 Jul 10;49(2):177-82. doi: 10.1016/j.enzmictec.2011.05.012. Epub 2011 May 27.
Site-directed mutagenesis of a thermostable alkaline phytase from Bacillus sp. MD2 was performed with an aim to increase its specific activity and activity and stability in an acidic environment. The mutation sites are distributed on the catalytic surface of the enzyme (P257R, E180N, E229V and S283R) and in the active site (K77R, K179R and E227S). Selection of the residues was based on the idea that acid active phytases are more positively charged around their catalytic surfaces. Thus, a decrease in the content of negatively charged residues or an increase in the positive charges in the catalytic region of an alkaline phytase was assumed to influence the enzyme activity and stability at low pH. Moreover, widening of the substrate-binding pocket is expected to improve the hydrolysis of substrates that are not efficiently hydrolysed by wild type alkaline phytase. Analysis of the phytase variants revealed that E229V and S283R mutants increased the specific activity by about 19% and 13%, respectively. Mutation of the active site residues K77R and K179R led to severe reduction in the specific activity of the enzyme. Analysis of the phytase mutant-phytate complexes revealed increase in hydrogen bonding between the enzyme and the substrate, which might retard the release of the product, resulting in decreased activity. On the other hand, the double mutant (K77R-K179R) phytase showed higher stability at low pH (pH 2.6-3.0). The E227S variant was optimally active at pH 5.5 (in contrast to the wild type enzyme that had an optimum pH of 6) and it exhibited higher stability in acidic condition. This mutant phytase, displayed over 80% of its initial activity after 3h incubation at pH 2.6 while the wild type phytase retained only about 40% of its original activity. Moreover, the relative activity of this mutant phytase on calcium phytate, sodium pyrophosphate and p-nitro phenyl phosphate was higher than that of the wild type phytase.
对来自芽孢杆菌 MD2 的耐热碱性植酸酶进行了定点突变，目的是提高其比活性和在酸性环境中的活性和稳定性。突变位点分布在酶的催化表面（P257R、E180N、E229V 和 S283R）和活性位点（K77R、K179R 和 E227S）上。选择这些残基是基于这样的想法，即酸性活性植酸酶在其催化表面周围带更多的正电荷。因此，碱性植酸酶的催化区域中带负电荷的残基含量减少或正电荷增加，被认为会影响酶在低 pH 值时的活性和稳定性。此外，扩大底物结合口袋有望提高对野生型碱性植酸酶不能有效水解的底物的水解效率。对植酸酶变体的分析表明，E229V 和 S283R 突变体分别使比活性提高了约 19%和 13%。活性位点残基 K77R 和 K179R 的突变导致酶的比活性严重降低。对植酸酶突变体-植酸盐复合物的分析表明，酶与底物之间形成了更多的氢键，这可能会阻碍产物的释放，从而导致活性降低。另一方面，双突变体（K77R-K179R）植酸酶在低 pH（pH 2.6-3.0）下表现出更高的稳定性。E227S 变体在 pH 5.5 时表现出最佳活性（与最适 pH 为 6 的野生型酶相反），并且在酸性条件下表现出更高的稳定性。这种突变体植酸酶在 pH 2.6 孵育 3 小时后保留了初始活性的 80%以上，而野生型植酸酶仅保留了约 40%的原始活性。此外，这种突变体植酸酶对钙植酸盐、焦磷酸钠和对硝基苯膦酸的相对活性高于野生型植酸酶。
