Cloning, Expression And Characterization Of A Thermostable Alkaline Pullulanase And Two Thermostable Alkaline Protease From Marine Bacteria

Marine environment possesses abundant microbial resource. The hydrothermal vents provide unique environments harbor a wild range of unknown extreme microorganisms and show extremely high diversity.The thermostable alkaline pullulanase and protease can be applied in food industry, detergent industry and other industrial applications as an important industrial material. In this study, we investigated the heterologous expression of pullulanase gene of the Geobacillus kaustophilus MCCC 1A02570, the vibriolysin gene of Vibrio fluvialis MCCC 1A02761 and the Calolysin gene of Caloranaerobacter sp. TR13 in E. coli and Bacillus subtilis which could be employed in the potential industrial applications. Many method had been summed up for further investigation of the relative industrial enzymes. The mainly contribution of this paper involves the three following aspects:1. The Geobacillus kaustophilus MCCC 1A02570 were screened out from the 58 thermophiles by the pullulanase screening plate. The pullulanase gene were expressed in the E. coli BL21(DE3) and then be purified by Ni Sepharose FF resin. The pullulanase were characterized as pullulanase type I by analyzing the hydrolyzed products. The optimum p H of the pullulanase was 8.0 and the thermal stability of the pullulanase is good in p H 8.0, which indicated that it was an alkaline pullulanase. The saccharification efficiency and the saccharification rate were enhanced by 37.58% and 31.03% respectively.2. The protease of Vibrio fluvialis MCCC 1A02761 were precipitated by the adding ammonium sulphate to 70% saturation and then be purified by DEAE anion exchange. The protease was then be characterized as vibriolysin by Maldi-TOF-TOF. The protease VFP was cloned into the expression vector p ET-22 b and then transformed. The recombination protease was hard to express in the E. coli. After the optimization of the culture medium, the hosts, the induction condition and the vector, the protease was thenbe expressed as inclusion body in the E. coli, and can be renatured by further renaturing procedures.3. A novel extracellular serine protease designated Calolysin was derived from TR13 was cloned and expressed in E. coli and Bacillus subtilis. Although expression level of the protease in the Escherichia coli is extremely low, the protease was successfully overexpressed in the Bacillus subtilis as a soluble protein by recombination of a Cterminal 6×histidine tag. The molecular mass, estimated by SDS-PAGE was about 48 k Da. The phylogenetic analysis indicated that the Calolysin belongs to the pyrolysin family and it can maintain a high activity between 70-85°C with maximal activity at 80°C and was activated in a broad range of p H(6.0-10.0) in neutral and alkaline condition. The Cu2+ and Ni2+ can increase the protease activity. In the substrate specification test the casein was hydrolyzed with a high velocity. The Km value of Calolysin toward BSA was 0.1188 mg/m L which was lower than that of casein. The Suc-Ala-Ala-Pro-Leu-p NA has the lowest Km value among the synthetic peptides tested. The Calolysin is sensitive to EDTA and PMSF which indicates that it is a serine metalloprotease, which can be further applied to industrial applications.