Study On Enzymatic Property And Orthogenesis Of Malolatic Enzyme

Malolatic enzyme (MLE) is the key enzyme in the process of malolactic fermentation (MLF), and it can directly catalyse L-malic acid into L-lactic acid. Many researchers have constructed malic acid-reducing yeast by genetic engineering methods, but the conversion efficiency of malic acid is very low as the presence of malic acid transport barrier. In order to solve this problem, our laboratory has constructed the yeast with surface display of MLE, named Fm5, but the conversion efficiency of malic acid is not very high, the reason of the low activity maybe is that this enzyme is not acid-resistant.This study firstly explores the reasons of the low activity of MLE displaying on the surface of Fm5. Through the study of general properties of this enzyme, the result is that the acidic environment is the reason of the low enzyme activity. Moreover, Escherichia coli has advantages of short growth period and high conversion efficient, relative to yeast.Our laboratory has constructed the E.coli efficiency expression system of MLE. In order to improve the acid-resistance of MLE, error-prone PCR is used for the directed evolution of MLE, then E.coli mutant library and high-throughput screening method are constructed, in order to select the acid-resistant MLE from the mutant library. Main results are as follows:1. Study on enzyme property of MLE.The first chapter was the verification of Fm5. Based on the observation of morphology and growth status, this engineering strain and original strain had no significant differences. The plasmid pADH1-mle was extracted from this engineering strain, the corresponding PCR band was obtained after PCR verification, indicating that this Saccharomyces cerevisiae strain was correct. Study on the general properties of MLE, HPLC was used to detect its activity. The results revealed that the optimum NAD+concertation was 2 mmol/L, the optimum MnCl2 concertation was 5 mmol/L, the optimum pH value was 5.7, the optimal reaction temperature was 30℃,the stability of MLE was not good in the extremely acidic conditions.2. Establishment of high-efficiency screening method of acid-resistant MLE.In this chapter, the verification of PMS-NBT colorimetric method was done. In the medium containing malic acid, under 28℃, the E. coli BL21 containing pET28a(+)-mle was induced to produce MLE for 3 hours by adding IPTG. The obtained supernatant was used for the PMS-NBT reaction. Compared with E.coli BL21 and E.coli BL21 containing pET28a(+), the supernatant of E.coli BL21 containing pET28a(+)-mle, Fm5 and Oenococcus oeni could turned the color from yellow into lyons blue, which showed that this method could be used to screen lactic acid producing strains. After induced expression of MLE and handle for two hours under pH 1.0,1.5,2.0,2.5,3.0,3.5,4.0,4.5,5.0 or 5.7, then proceeded the degradation reaction of malic acid, the obtained supernatant was used to do color reaction of PMS-NBT in 96 microplates, thus the high-efficiency screening method of acid-resistant MLE was established. The supernatant did not make the color of liquid changed, indicating that MLE was inactivated, and the critical pH value of MLE inactivation is 3.5.3. Directed evolution of acid-resistant MLEThis chapter was about two-round random mutation of mle gene using error-prone PCR with GeneMorph Ⅱ Random random mutagenesis kit. Then the P-glucanase signal peptide gene was cloned from Bacillus subtilis 168 and linked to the upstream of mle gene using fusion PCR. The obtained fragment was inserted into expression vector pET28a (+) to construct pET28a(+)-mle. The constructed plasmid was transform into E.coli BL21 to construct mutant library. Four potential mutants producing acid-resistant MLE were screened from mutant library using high-throughput screening method. The mutational mle gene was been sequenced.