基于蛋白质折叠自由能分析的定点突变提高谷氨酰胺转胺酶热稳定性

Improvement of TGase Thermal Stability through Site-Directed Mutagenesis Based on Analysis of Folding Free Energy

谷氨酰胺转胺酶(Transglutaminase,EC 2.3.2.13,TGase)广泛应用于食品、纺织等领域。为提高TGase的热稳定性,通过PoPMuSiC-2.1预测了降低Streptomyces hygroscopicus TGase分子折叠能的氨基酸位点,并构建了相应的突变体。PoPMuSiC-2.1预测结果显示,替换P132的氨基酸引起TGase折叠自由能下降的幅度最大。基于此,通过定点突变分别构建了低折叠自由能的突变体P132I、P132G、P132M、P132Q。酶学分析表明,P132I在50℃下的半衰期达到5.0 min,较野生酶提高31%;其它突变体则较野生酶提高2%～13.7%。此外,P132I和P132G比酶活亦分别较野生酶提高24%和12.4%,其它突变比酶活变化不明显。作用力分析发现,突变体P132I中较野生TGase增加两个氢键。上述结果表明,基于蛋白质折叠自由能分析的定点突变能有效地提高TGase的热稳定性与催化活性,氢键的增加可能是P132I热稳定性提高的原因之一。

Transglutaminases(EC 2.3.2.13,TGase)is an industrial enzyme widely used in several domains that include food processing and textile industry.The amino acid sites that may reduce folding free energy were predicted by using PoPMuSiC-2.1 to improve the thermal stability of TGase,and the corresponding mutants were then constructed.As indicated by PoPMuSiC-2.1 analysis,mutation at P132 revealed the highest level of decreases in folding free energy of TGase.Based on this prediction,the mutants with decreased folding free energy was thus contracted,including P132I,P132G,P132M,and P132Q.The analysis of enzymatic property showed that the half-life of P132I can reach 5.0 min at 50℃which was 31%higher than that of wild-type TGase,and the other mutants also exhibited enhanced half-life.Moreover,P132I and P132G showed 24%and 12.4%enhanced specific activities in contrast to wild-type enzyme,respectively;while the specific activities of the rest mutants did not change significantly.And the structure analysis indicated that P132I mutant formed additional two hydrogen bonds as compared with wild-type enzyme.These results suggested that site-directed mutagenesis based on analysis of folding free energy is an effective method to improve the thermal stability of TGase,and newly formed hydrogen bond may be one of the reasons for enhanced thermal stability of P132I.