【摘要】 L-天冬氨酸α-脱羧酶活性较低,稳定性较差,使得其在工业应用中受到限制。该研究旨在提高L-天冬氨酸α-脱羧酶的催化性能,促进生物法生产β-丙氨酸的工业化进程。依据嗜热蛋白酶的氨基酸内在进化趋势,对赤拟谷盗来源L-天冬氨酸α-脱羧酶进行分子改造,以期提高稳定性。实验共构建21个突变体,获得催化性能优良的突变体K221R,该突变体的比酶活较野生型提高20. 3%;野生型经50℃处理30 min,残余酶活接近0,而突变体K221R的残余酶活为43%。建立了基因工程菌全细胞催化天冬氨酸生成β-丙氨酸的工艺,K221R菌株的产量达到134. 72 g/L,摩尔转化率为94. 52%,是迄今为止的最高产量。该研究构建的基因工程菌具有工业应用潜力,同时也为生物法制备β-丙氨酸提供理论与技术基础。更多还原

【Abstract】 Low catalytic ability and poor stability limit industrial applications of L-aspartate α-decarboxylase.This study was therefore conducted to improve the catalytic activity of L-aspartate α-decarboxylase to promote biological production of β-alanine in industries. Based on evolutionary information of thermophilic bacteria,L-aspartate α-decarboxylase from Tribolium castaneum was molecularly modified to improve its enzyme stability. The mutant strain K221 R was screened,as it had improved thermal stability and enzymatic activity. Compared with the wild type,the specific enzyme activity of K221 R increased 20. 3%. Moreover,after incubating the enzyme at 50 ℃ for 30 min,the residual activity of the wild type was 0,while K221 R remained 43% activity. Furthermore,up to 134. 72 g/L β-alanine was produced using K221 R-expression whole cells,which was the highest production level achieved up-to-date,with 94. 52% molar conversion rate. In conclusion,the engineered strain containing K221 variant has great potential for industrial production of β-alanine.更多还原