Cloning, Expression And Enzymatic Activity Detection Of Lipase Gene From Bacillus Amyloliquefaciens SWB16

Bombyx mori is the model insect of Lepidopteran species, which has created excellent income in sericulture industry as an important economic insect. However, the infectious disease of silkworm seriously restricted the development of sericulture industry, especially a viral disease named ‘nuclear polyhedrosis’ caused by Bombyx mori nucleopolyhedrovirus(BmNPV). In the process of natural evolution, silkworm developed three defensive lines to fight against pathogen infection which including physicalbarrier, cellular immunity and humoral immunity. The idea that Bmlipase as an antiviral protein in silkworm intestine which plays a role in humoral immunity against BmNPV has been already confirmed by Japanese researchers. Domestic scholars revealed that the intestinal lipase from Bombyx mandarina Moore showed inhibitory effect against BmNPV as well, and the bioactive silkworm lipase were purified in vitro. In addition, a research team established a transgenic silkworm line which highly resistant to BmNPV by overexpressing Bmlipase-1 gene. Our previous studies found that the viral susceptibility between silkworms feeding with mulberry leaves and Tricuspid cudrania leaves were significantly different, and the later was much more susceptible to BmNPV. Subsequently, we found that both microbial community and dominant microorganisms were dramatically changed along with different forages. In addition, we noticed that not only total bacterial abundance but also lipase-producing bacteria in silkworm intestine were significantly reduced while feed with Tricuspid cudrania leaves, and the total activity of lipase and trypsin in digestive juice were also decreased. The above results suggested that exogenous lipase produced by silkworm enterobacteria or other microbes might participate in host immunization to prevent BmNPV infection.In order to obtain a vast amount of high-purity microbial lipase, the lipase gene of exogenous bacterial strain Bacillus amyloliquefaciens SWB16 were cloned, prokaryotically expressed and purified by using Ni-NTA column. The enzymatic activity and amount of recombination protein were then detected. The primary findings are shown as below:(1) Cloning of SWB16 lipase gene and bioinformatics analysisThe genomic DNA of strain SWB16 were extracted as template, and the lipase gene was successfully cloned by using the primer pair LIP-F and LIP-R. The complete open reading frame(ORF) of this gene is 645 bp long which encoding a polypeptide of 214 amino acids. The computed molecular mass of this lipase is about 22.73 kDa, and the predicted isoelectric point is 9.82 which is an alkaline protein. In the N-terminal region, it is predicted that there is a signal peptide within the first 32 amino acidesand two transmembrane domains which are located at amino acid 7-29 and 39-61 respectively and no N glycosylation sites exist. The predicted secondary structure of SWB16 lipase consists of several random coils and extend strands with a few α-helixes and β-sheets, and the amino acid residues 33-134 constitute the functional domain. By BLAST searching in NCBI datebase, a conserved domain Lipase-2 which located from amino acid 36-180 of this enzyme is determined, and also shows a conserved sequence of α/β hydrolase family from amino acid 39-138. All these results indicated that SWB16 lipase which belongs to α/β hydrolase family possesses specific enzymatic structure and function as fatty acid or lipid hydrolase.(2) Prokaryotic expression and solubility analysis of SWB16 lipaseThe pET28-lipase recombinant expression plasmid is constructed successfully by sub-cloning lipase gene into pET28 vectors. The recombinant plasmid was then transformed into E. coli BL21(DE3)and expressed under IPTG induction with the optimal conditions of 0.1 mmol/L IPTG, 100 r/min shaking for 12 h at 20 °C. SDS-PAGE analysis showed that the protein is mainly expressed as inclusion bodies in bacterial cells at 37 °C, 28 °C and 20 °C, however, the soluble form of this protein was also detected.(3) Purification of fusion protein and enzymatic activity detectionThe fusion protein HIS-LIPASE was separated based on Ni-NTA chromatography column, and the protein samples were purified by ultrafiltration. The enzymatic activity of the recombination protein was detected by using rhodamine B method, and the paper disks contained lipase emitted orange fluorescence under the ultraviolet. The results suggested that the purified lipase samples showed a certain lipolytic activity. The content of recombination protein was determined by BCA method which showed a high concentration of 1.019 mg/m L that could be used in the further studies.