D-乳酸生产菌菊糖芽孢乳杆菌来源的D-乳酸脱氢酶同工酶

Characterization of D-lactate dehydrogenase isozymes from a D-lactic acid producing bacterium Sporolactobacillus inulinus

摘要:

[目的] 菊糖芽孢乳杆菌（Sporolactobacillus inulinus）作为典型的同型发酵产D-乳酸的优势菌株，能够高效生产高纯度的D-乳酸。该菌株发酵受到多方面环境因素影响。糖代谢的关键酶例如葡萄糖激酶、磷酸果糖激酶、丙酮酸激酶以及乳酸脱氢酶均为由葡萄糖代谢成为乳酸的关键酶，该菌中相关代谢酶的研究是发酵调控至关重要的基础。分析S.inulinus的基因组表明有3个推测为D-乳酸脱氢酶的基因，其中已有报道研究了1个双功能蛋白[bifunctional protein（BP）]。本研究分别克隆并解析了另2个D-乳酸脱氢酶同工酶的性质。[方法] 本研究以S.inulinus Y2-8基因组DNA为模板，克隆得到2个D-ldh基因（dldh、dhdh），经测序分别为D-乳酸脱氢酶[D-lactic acid dehydrogenase（DLDH）]和D-羟基酸脱氢酶[D-isomerspecific 2-hydroxyacid dehydrogenase（DHDH）]的基因。构建的重组菌表达蛋白DLDH，DHDH均具有催化丙酮酸生成D-乳酸的功能。[结果] 重组菌表达的蛋白经镍柱亲和层析达到电泳纯。SDS-PAGE分析表明DLDH的表观分子量为37 kDa，DHDH的表观分子量为39 kDa。此外，DLDH以丙酮酸为底物时K_m值为（0.58±0.04）mmol/L，对底物有较高的亲和力，最适反应温度为35℃，最适pH为6.5；而DHDH以丙酮酸为底物时K_m值为（1.70±0.08）mmol/L最适反应温度为30℃，最适pH为7.5。另有报道的BP以丙酮酸为底物时K_m值为（3.40±0.02）mmol/L，最适反应温度为30℃，最适pH为5.5。[结论] 根据对底物丙酮酸的亲和力，最适温度及最适pH，推测DLDH是乳酸发酵中产D-乳酸的主导催化剂。结合相关酶学性质的分析可为今后的发酵调控提供理论依据。

关键词:菊糖芽孢乳杆菌;D-乳酸脱氢酶;克隆;酶学性质

Abstract:

[Objective] Sporolactobacillus inulinus, a typical homofermentative lactic acid bacterium, is an efficient D-lactic acid producer. Various environment factors affect the productivity of S. inulinus. Glucokinase, phosphofructokinase, pyruvate kinase and lactic dehydrogenase are the key enzymes of D-lactic acid production from glucose by S. inulinus. The characteristics of these enzymes are important in controlling and regulating the fermentation process. According to the genome bioinformatics analysis of S. inulinus CASD, three putative D-lactate dehydrogenases were identified, among which the bifunctional protein had been reported. In this study, we provided insights into the characteristics of the other two D-lactate dehydrogenase isozymes.[Methods] S. inulinus Y2-8 genome was used as the template to amplify D-lactate dehydrogenase gene (dldh) and D-isomer specific 2-hydroxyacid dehydrogenase gene (dhdh). The two recombinant strains E-pET-28a/dldh and E-pET-28a/dhdh were constructed for enzyme expression. Both recombinants DLDH and DHDH could convert pyruvic acid into D-lactic acid.[Results] Enzymes expressed by recombinant strains were purified by Ni-NTA chromatography. The apparent molecular mass of DLDH was approximately 37 kDa by SDS-PAGE analysis, and DLDH showed a high affinity to pyruvate with the K_m value of (0.58±0.04) mmol/L. The optimal reaction temperature and pH for DLDH was 35℃ and 6.5, respectively. The apparent molecular mass of DHDH was approximately 39 kDa, and the K_m of DHDH toward pyruvate was (1.70±0.08) mmol/L. The optimum catalysis temperature and pH of DHDH were 30℃ and 7.5, respectively.[Conclusion] According to the K_m and optimal reaction pH, DLDH was suggested as the main catalyst in formation D-lactic acid from pyruvate during the fermentation. The enzymatic properties would contribute to the regulation of the fermentation of S. inulinus.

Key words:Sporolactobacillus inulinus;D-lactate dehydrogenase;cloning;characteristics