保加利亚乳杆菌ATCC11842 L-乳酸脱氢酶同工酶基因的克隆与表达分析

通过对保加利亚乳杆菌（Lactobacillus delbrueckii subsp.bulgaricus）L-乳酸脱氢酶（L-lactate dehydrogenase，L-LDH）同工酶基因的异源表达、酶活测定和摇瓶发酵研究L-LDH在乳酸合成中的作用。将保加利亚乳杆菌ATCC11842中L-乳酸脱氢酶基因ldb0120和ldb0094分别克隆至载体pET28a(+)中，构建重组表达载体pET28aldb0120和pET28aldb0094，并转化到大肠埃希菌（Escherichia coli） BL21(DE3)中进行表达。进一步对重组蛋白进行Ni-NTA柱亲和层析和酶学活性测定，结果显示，LDB0120和LDB0094的比活力分别为0 和25 U/mg，表明LDB0094是具有低活性的L-乳酸脱氢酶，而LDB0120不具有活性。对两株重组菌分别进行好氧和微好氧发酵，重组菌E.coli BL21/pET28aldb0094在好氧和微好氧条件可以合成L-乳酸，浓度分别为41.9和227.9 mg/L，而菌株E.coli BL21/pET28aldb0120在两种培养条件下均基本不合成L-乳酸，推测保加利亚乳杆菌中L-乳酸脱氢酶LDB0094为催化L-乳酸合成的关键酶。首次对保加利亚乳杆菌的L-乳酸脱氢酶同工酶基因进行研究，通过基因异源表达、蛋白纯化、酶活测定和摇瓶发酵，揭示Ldb0094酶为保加利亚乳杆菌ATCC11842中催化L-乳酸合成的关键酶。

Cloning and Expression Analysis of L-Lactate Dehydrogenase Isoenzyme Genes from Lactobacillus delbrueckii subsp. bulgaricus ATCC11842

The role of L-Lactate dehydrogenase (L-LDH) isoenzymes of Lactobacillus delbrueckii subsp. bulagricus in L-lactic acid synthesis was studied adopting heterologous expressions, enzyme activity determination, and flask shaking fermentation. The L-lactate dehydrogenase gene ldb0120 and ldb0094 from L.bulgaricus ATCC11842 were cloned into vector pET28a(+) respectively, and the recombinant expression vectors pET28alldb0120 and pET28adb0094 were constructed, and then was transformed into E. coli BL21 (DE3) for gene expression. Further tests with Ni-NTA column affinity chromatography and enzymatic activities of recombinant protein, results showed that the specific activities of LDB0120 and LDB0094 respectively were 0 U/mg and 25 U/mg, indicating that LDB0094 had L-lactate dehydrogenase of low activity, while LDB0120 had no activity. Two recombinant strains were carried out aerobic and micro-aerobic fermentation, recombinant strain E.coli BL21/pET28aldb0094 could synthesize L-lactic acid under aerobic and micro-aerobic conditions at concentrations of 41.9 and 227.9 mg/L respectively. While E.coli BL21/pET28aldb0120 basically did not synthesize L-lactic acid under the two cultivation conditions. It was speculated that L-lactate dehydrogenase LDB0094 of L. bulgaricus ATCC11842 might be the key enzyme to catalyze the synthesis of L-lactic acid. Therefore, L-lactate dehydrogenase isozyme genes of L.bulgaricus were studied systematically for the first time. Through heterologous gene expression, protein purification, enzyme activity determination, and flask fermentation, it is revealed that Ldb0094 enzyme is a key enzyme catalyzing the synthesis of L-lactic acid in L.bulgaricus ATCC11842.