3-磷酸甘油酸脱氢酶(3-phosphoglycerate dehydrogenase, PGDH)是L-丝氨酸生物合成途径的限速酶,是影响L-丝氨酸高效合成的关键。该研究以来源于谷氨酸棒杆菌(Corynebacterium glutamicum)的PGDH为研究对象,通过对其酶学性质进行测试分析发现,Cg-PGDH的酶活性仅为1.98 U/mg,而且热稳定性较差,严重制约了L-丝氨酸的高效生物合成。针对这一关键问题,该研究采用定向进化和高通量筛选的方法对Cg-PGDH进行改造,提高酶的催化活性和热稳定性。通过多轮筛选,最终获得了5个催化活性显著提升的突变体,P85Q、D365Y、A389T、A183V、I231V。其中,突变体P85Q的酶活性提升了1.55倍,并显著提高了酶的温度稳定性,最适催化温度达到60 ℃。随后该研究对P85Q、D365Y、A389T、A183V、I231V等5个突变体的突变位点进行组合突变,获得了温度稳定性进一步提高的突变体P85Q-D365Y。通过对Cg-PGDH突变位点的三维结构解析和分子动力学模拟实验发现,尽管突变体P85Q与D365Y的突变位点不位于Cg-PGDH催化活性中心,但是可通过蛋白结构形变增强Cg-PGDH活性中心130~140区间loop的稳定性,从而增加了酶的底物亲和性和催化活性。相关研究成果为进一步解析PGDH催化机制,提高PGDH催化活性提供了重要的研究基础和方向。
黄新燕
,
孙慕娇
,
杜穆花
,
潘越
,
张心语
,
张瑶函
,
徐宁
,
刘君
,
鞠建松
,
魏亮
. 谷氨酸棒杆菌3-磷酸甘油酸脱氢酶的定向进化改造研究[J]. 食品与发酵工业, 2025
, 51(3)
: 89
-96
.
DOI: 10.13995/j.cnki.11-1802/ts.038795
3-Phosphoglycerate dehydrogenase (PGDH) is the rate-limiting enzyme in the biosynthetic pathway of L-serine, playing a crucial role in efficient L-serine synthesis.In this study, the enzymatic characteristics of PGDH from Corynebacterium.glutamicum was investigated.The results indicated that the enzyme activity of Cg-PGDH was only 1.98 U/mg with poor thermal stability, which significantly hindered the high-efficiency biosynthesis of L-serine.To address this issue, directed evolution and high-throughput screening were employed to enhance the catalytic activity and thermal stability of Cg-PGDH.Through multiple rounds of screening, five mutants with significantly improved catalytic activity were obtained, including P85Q, D365Y, A389T, A183V, and I231V.Notably, the P85Q mutant exhibited a 1.55-fold increase in enzyme activity and significantly improved temperature stability, demonstrating an optimum catalytic temperature of 60 ℃.Subsequently, the combined mutations on the mutation sites of five mutants including P85Q, D365Y, A389T, A183V, and I231V were conducted, leading to the mutant P85Q-D365Y with enhanced temperature stability.Structural analysis and molecular dynamics simulations of the mutated sites within Cg-PGDH revealed that, despite P85Q and D365Y not being located in the catalytic center, they enhanced the stability of the loop in the 130-140 region, thereby increasing substrate affinity and catalytic activity.These research findings provided a significant foundation and direction for further elucidating the catalytic mechanism of Cg-PGDH and enhancing its catalytic activity.
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