Genome Scanning Analysis Of Two Lipase-producing Strains And The Gene Synthesis And Function Identification Of HS-BTLm

Lipases(triacylglycerol acylhydrolases EC 3.1.1.3)are a class of hydrolase to catalytic hydrolysis of triglycerides to glycerol and free fatty acids,which are widely existed in animals,plants and microorganisms.Lipases have the advantages of high catalytic activity,diversity rich resources,ease of industrial production,stability,low production cost,and short production cycle,which make them increasingly popular applicated in the food,fine chemistry,oil chemistry,separating of chiral compounds,synthesis of biodiesel,preparation of polymer materials,detergent and additive industries.Looking for highly-producing lipase strains is still the basis of all research and development work as important industrial application value,huge potential demand and high cost of the preparation of lipase.In this study,one lipase producing strain HS-BTL2 was isolated in the soil from the sediments from the Yasha hot spring of Yunnan,and its lipase activity was 3.87 U/mL.HS-BTL2 strain was preliminarily identified as Bacillus thermophiles by morphology observation,physiological and biochemical identification of 16S rDNA.The genomes of HS-BTL2 and HS-L6 were extracted and detected,and the gene scanning was taken by Illumina Hiseq 2000.The genome information showed that the total length of HS-BTL2 was 404170bp,which contained 5881 ORFs;and the total length of HS-L6 was 4496304bp,which contained 4976 ORFs.GO analysis showed that 2078 had annotation information in the genome of HS-BTL2,while 3821 had annotation information in HS-L6 genome.The genes related to lipase gene cluster in the whole genome were analyzed.The numbers of genes associated with fat degradation in HS-BTL2 and HS-L6 genome were 41 and 19,respectively.It is the important premise of lipase large-scale production to build efficient genetic engineering bacteria.Methanol nutritional pichia expression system is a set of good system.It has a high amount of exogenous protein expression,quick growth and low nutritional requirements and suitable for high density fermentation that has been widely used in production.The frequency of whole expression in pichia is low because of huge differences in codon usage frequency between pichia and Thermophilic bacillus.In this study,the thermophilic lipase gene in HS-BTL2 was optimally designed:the preference codon in yeast was choosed;yeast genes energy was higher with the lower stability of the mRNA secondary structure;and the rich AT area were removed.Primers were designed by online software DNAworks and the optimal gene of HS-BTLm was artificially synthesized and cloned.The recombinant expression vector pPICZaA-HS-btlm was constructed and successfully and secretoryly expressed in pichia GS115.The enzyme activity of the recombinant strain reached 64.97 U/mL under optimum condition induced 96h.The gene copy number is a limiting factor of recombinant protein production in pichia expression system.In most cases,the increase of copy number of gene expression box can significantly improve the expression level of recombinant proteins.In this study,the recombinant yeast expression box containing 2 and 4 copies of purpose gene were constructed in vitro,and efficienttly expressed the recombinant proteins in pichia,which the lever of the protein expression level increased by 2.6 and 3.2 times(174.93/210.83 U/mL),respectively.The relationship the copy number and the mRNA of gene was studyed by fluorescence quantitative PCR;and the relationship between gene copy number and the secretory expression of the purpose protein in pichia was initially inferred.The reason about the ratio not fully linear was analyzed.The optimum reaction conditions of the purified lipase HS-BTLm were pH 8.0,at 50?.The organic solvents Triton X-100,Tween-80 and metal ions(such as Ca2+,Mg2+,Mn2+)could promote the lipase activity of HS-BTLm.However,organic solvents Tween-20,SDS,bile salts,methanol,propylamine,acetone and Cu2+,Zn2+,as well as PMSF,EDTA could inhibit the lipase activity.The lipase gene was cloned from P.aeruginosa HS-L6 and successfully expressed in E.coli.The lipase homologous gene of the P.aeruginosa TE3285 was found in NCBI.Primers were designed according to the gene sequence and the lipase gene of P.aeruginosa HS-lip6 was cloned by PCR amplification.The length of gene fragment was 2008 bp,contains 1959bp encoding 653 amino acids and 49bp repeats.The results showed that 15 base mutations and 10 amino acid sequences were different through homologous comparison between the lipase gene of HS-lip6 and TE3285.The recombinant expression plasmid pET-HS-lip6 was constructed,and realized heterologous expression.Under the condition of 37?,recombinant protein LIP6 mainly existed in the form of inclusion body.SDS-PAGE analysis found that lipase was successfully expressed in E.coli under lower temperatures 28*C and 18? to induce expression of recombinant strain.The lipase activity of the mixture by ultrasonic was 15.65 U/mL,the lipase gene lipA and lipase fold protein gene lipB were cloned respectively from HS-L6.The recombinant expression plasmids pET-lipA and pET-lipB were constructed and expressed in E.coli.LIPA existed in the form of inclusion bodyand 8M urea treatment could refold the recombinant lipase LIPA.At the same time,the recombinant expression vector pPICZaA-lipA and pPICZ?A-/lipB were built,and expressed secretoryly in pichia.The folding renaturation of LIPA and LIPB in vitro were analyzed and the result showed that the optimal concentration of lipase folding protein LIPB promoting lipase LIPA was 0.005 mg/mL.