| Esterase has a wide spread applications in the field of industrial enzyme catalysis as it has the ability to catalyze esterification, transesterification, kinetic resolution of optically active compounds and interesterification. However, a large amount of enzymes used in industries are isolated from mesophile microorganisms, which are unsuitable for the environment of industrial processes, such as high temperature or organic solvents, and few literatures were reported about esterase with significant resistance came from eubacterium. Thermostable esterase has some unique advantages in regard of tolerance to organic solvents, thermostability and catalytic velocity etc, which is a hot topic in the field of biocatalysis.In this article, the dsm gene, obtained from NCBI was amplified using the genome of Clostridium Thermocellum(JCM9323) as template, then the recombinant plasmid pET-28a-dsm was constructed and transformed to BL21(DE3). The target protein was expressed by inducing with isopropyl β-d-1-thiogalactopyranoside(IPTG)and confirmed in the form of inclusion body using SDS-PAGE analysis. The target protein was purified with Ni-NTA affinity chromatography and renatured through urea concentration gradient method. The mice antiserum against recombinant protein DSM was prepared by rat immunization with renatured DSM, and detected the specificity of the antiserum by western blotting, which inferred that the antibody serum has specificity against the target protein..Enzymatic properties analysis of DSM esterase revealed that this enzyme showed highest activity with pNPB-C4 among the tested substrates, as well as it showed some activity to the long chain fatty acid. The optimal pH and temperature is7.0 and 65°C, respectively, and the residual activity of esterase was recorded as 70%and 60% after 3 hours of incubation at 65°C and 70°C, which indicated that the esterase-DSM was a thermostable enzyme. Ca2+ and Mg2+ could slightly stimulate the activity of the esterase, while Cu2+, Ni2+ inhibited it. In the presence of methanol andDMSO, the esterase-DSM could maintain some activity while other organic solvent and surfactant had an obvious inhibition to the activity of esterase. Furthermore, only in the presence of DTT(2.5 mM), the esterase could have the activity with pNPB-C4.Enzyme immobilization technology has a great potential application in the harsh conditions of the biochemical industry. The spores of Bacillus subtilis possess robust resistance, which made it an ideal carrier to immobilize thermophilic enzyme, but few research were reported in recent years. In this paper, the esterase from Clostridium thermocellium was displayed on the spore surface of Bacillus subtillis.Spore coat protein CotB was used as anchoring motif to display DSM-esterase.Firstly, overlap-PCR was used to construct fusion gene CotB-dsm, then the recombinant expression vector-pHS-CotB-dsm was constructed and transformed to Bacillus subtilis DB403 strains, and the recombinant Bacillus subtilis was induced for sporulation. But, the expression ratio of the fusion protein is low. To verify whether the DSM-esterase displayed on the surface of spore, we used western blotting and detected the esterase activity after dealing with the recombinant spore by protease,inhibitors. PNPB-C4 was used as substrate to investigate the properties of displayed esterase, results showed that its optimal temperature and pH was 60°C and 7.5,respectively. The scope of its temperature and pH adaptability had an obvious improvement compared with free esterase. The activity of recombinant spores could retain 78% and 68% after incubated for 5 hours at 60°C and 70°C, respectively, which were higher than free enzyme. In the organic reagents and surfactants, the activity of displayed esterase was also higher than free esterase, and it has an excellent DMSO tolerability, which could retain 65% activity after incubated in 20% DMSO for 7hours. Under the optimum reaction conditions(60°C, pH 7.5), its specific activity was3.0 × 10-9 U/spore. After 3 times reusing under conditions of high temperature through simple centrifugation, its relative activity retained 75%.After displayed the esterase on the surface of spore, the effect of diverse linker on the displayed esterase was also investigated. Results showed that the activities ofthe spores with different linkers were increased to some degree compared with the spores without linker at optimal temperature, and the relative activity of spores with P2(PTPTPT)2, S2(EGKSSGSGSESKST) were higher than others at pH 9.6, which were reached up to 70% and 62%, 1.75 and 1.5 times higher than the activity of the spores without linker. Under the condition of 80°C, the spores with S3(GGGGS)4 had better thermostability and could retain 55% activity after 5 hours of incubation, which was 1.2 times higher than the activity of the spores without linker. At different concentrations of DMSO(10%, 20%), activities of the spores containing different linkers were increased, activities of S2 was enhanced obviously compared with others,and at the concentration of 20%, the relative activity of S2 was 1.5 times higher than the spores without linker. At different concentration of methanol(10%, 20%), S2 significantly improve the activity of displayed esterase, which was 2.2 times higher than the activity of enzyme without linker under 20% concentration of methanol.This study revealed that, DSM esterase, isolated from Clostridium Thermocellum,has good thermostability and could tolerance organic reagents to a certain extent,having certain values for industrial applications. After displaying this esterase on the surface of spore, its thermostability, tolerance to organic solvents and reusebility showed great improvement compared with free esterase. Via fusion protein technology research, found that different linkers have a great influence on spore surface displayed esterase, such as activity, stress tolerance, etc, hint that we could find the excellent linker to the fusion enzyme displayed on the spore surface, it will benefit to abtain the recombinant spores with higher activity and stress resistance. It was concluded that Bacillus subtilis spore surface display system is a kind of effective stress-tolerance enzyme immobilization technology and has a great potential applications in hostile environment biocatalysis industry. |
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