Identification Of A Thermophilic Lactobacillus And Cloning And Expression Of Its L-lactate Dehydrogenase (ldhL) Gene In Esherichia Coli

Lactic acid is an important organic acid, which is widely exists in most organisms such ashuman, animal, plant and microorganism. It is the important sour additive and preservative infood industry and is an important chemical material of medicine, printing, dyeing and leatherindustry. As rapidly developing of poli-lactic acid as biodegrading plastic, the domestic andforeign demand of high optical purity of L-lactic acid is becoming more and more.Many disadvantages have been exposed about the traditional lactic acid productionmethods, so microbial fermentation has become the main method to produce lactic acid. Thefirst key factor of influencing fermentation qaulity is that different lactic acid fermentationstrain being collected. The traditional method of strain improvement is mostly mutationscreening, but it is must to collect the higher L-lactic acid producing strains by geneticengineering. Now , constructing the high L-lactic acid producing strains by gene recombinationhas already become a focus in international research.Thermophilus lactobacillus have the advantages of high pollution resistant ability, realizathe synchronization of saccharification and fermentation, shorten the cooling time and reducethe quantity of cooling water for the course of the fermentation than common bacterium. Thestudy of the high L-lactic acid producing thermophilus lactobacillus not only can improve thepurity of L-lactic acid achieved but also can reduce the production cost. This paper is based onthe study: breeding of thermophilus strain producing L-lactic acid and optimization offermentation conditions.In this study, using the thermophilic lactobacillus BAE 0421 isolated from corn steepliquor as starting strain, its chemical, physical and biological characteristics was preliminarystudy by the conventional appraisal. The strain BAE 0421 was characterized by 16S rDNAsequence analysis and constructed the molecular evolutionary trees by DNAMAN soft. We designed the primers based on the ldhL gene sequence of Bacillus coagulans 36D1(GenBank accession number AAWV01000028)and genome DNA of the strain BAE 0421was used as template to amplify the gene encoding ldhL by polymerase chain reaction(PCR).Then the PCR products were cloned into pMD19-T simple vector and identified by PCRanalysis. Moreover, of the sequencing result, some futher bioinformatics analyses wereconducted.The DNA fragment of ldhL was double digested with restriction endonucleases, and theninserted into prokaryotic expression vector pET32a and transformed into E.coli BL21. After itwas induced to express ldhL with IPTG,the expression products were analysis by SDS-PAGE.1. After the conventional appraisal of the strain BAE 0421, the colony morphology issmall, yellowish or white, circular, smooth. Its cell is rod-shaped, the strain is gram positive,form spore, and is the optional anaerobic fermentation.The contact enzyme experimentation ofthe strain is positive,and it can calisthenics. In summary,the strain fall into Bacillus.The 16S rDNA sequence analysis showed that the strain belonged to the Bacilluscoagulans genus, GenBank accession number of the 16S rDNA is FJ392315.2. One pair of primer was designed and synthesized according to the ldhL gene sequenceof Bacillus coagulans 36D1. The total DNA was isolated from the strain BAE 0421 and wasused as PCR templates to amplify the ldhL gene. Then the PCR products were connected withthe pMD19-T simple vector. The recombinant pMD19-ldhL was identified by PCR, and thensequenced. The Blast sults showed the homology of this sequence and other reported ldhL genesequence of NADB_Rossmann family was very high. This ldhL gene consisting of 939 basepairs, was an integral open reading frame. Start codon was ATG and terminal codon was TAA.This sequence encoded 312 amino acids continuously. The molecular weight is approximately34083.8Da, the molecular formula is C1516H2415N409O464S9. It has 38 negatively chargedresidues(Asp+Glu), and 30 positively charged residues(Arg+Lys). Theoretical pI 5.44.Hydrophobicity analysis showed that ldhL is hydrophilic, and the figure of the 3D structure ofthe ldhL was constructed by Swiss-mode. The nucleotide sequence was submitted to the NCBIGenBank and the Accession Number is FJ392314.3. The DNA fragment of ldhL was double digested with restriction endonucleases, and then inserted into prokaryotic expression vector pET32a and transformed into E.coli BL21successfully. After it was induced by IPTG, SDS-PAGE showed that the specific fusion proteinwith molecular weight 37KD was expressed.Enzyme activity assay revealed that the ldhL activity of E.coli BL 21 with pET-ldhL was100.2 U/mL, outclassed the ldhL activity of starting strain(Bacillus coagulans BAE 0421)and E.coli BL 21 with pET 32a detected no ldhL activity.The thermophilic lactobacillus BAE 0421 belonged to the Bacillus coagulans genus. Thesequence results showed that ldhL gene is 939 bp,and the cloned ldhL was expressed in BL21.The results are expected to lay foundation for further studies on the gene engineering E.colistrain of high yield L(+)-lactic acid by fermentation.