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来源于革兰氏阳性短乳杆菌Lactobacillus brevis ATCC 367的乙醇脱氢酶LbADH是催化手性醇的模式酶,以四聚体的形式发挥催化活性.本研究通过计算机分子模拟进行理性设计,确定并设计LbADH中与单体间疏水相互作用及盐桥键形成相关的关键位点,通过定点突变在原始序列中引入相应的氨基酸,以改变单体间的结合能力,定向调节其催化活性和热稳定性.研究获得一个疏水突变体LbADH F146L,其在37℃下的酶活力为野生酶的8.5倍,在50℃下的酶活力为野生酶的5.6倍;其动力学参数Kcat/Km(1.99×106 L·mol-1·s-1)为野生酶(1.40×104 L·mol-1·s-1)的142倍,表明其催化效率明显提高.该研究结果表明,通过将LbADH中146位的苯丙氨酸(F146)突变为亮氨酸(L)增大疏水作用可以提高其催化活性.
The alcohol dehydrogenase LbADH, which is derived from Lactobacillus brevis ATCC 367, is a model enzyme that catalyzes the chiral alcohol and exerts its catalytic activity in the form of a tetramer. In this study, rational design was performed by computer molecular simulation to determine The key sites related to the hydrophobic interaction between monomers and salt bridge formation in LbADH were designed, and corresponding amino acids were introduced into the original sequence by site-directed mutagenesis to change the binding ability between monomers and to regulate the catalytic activity and thermal stability The hydrophobic mutant LbADH F146L was obtained and its activity was 8.5 times that of the wild enzyme at 37 ℃ and 5.6 times that of the wild enzyme at 50 ℃. The kinetic parameters Kcat / Km (1.99 × 106 L · mol-1 · s-1) was 142-fold that of wild-type enzyme (1.40 × 104 L · mol-1 · s-1), indicating that the catalytic efficiency was significantly improved. The mutation of phenylalanine (F146) to leucine (L) increases hydrophobicity and enhances its catalytic activity.