Characterization, Cloning And Expression Of A Cold-active Protease From Psychrophilic Bacterium Pseudoalteromonas Sp. QI-1

There exist huge permanent cold ecosystems in polar regions, of which the biodiversity and exploitation on biological resources are attracting more and more attentions. Many novel species of marine organisms, enzymes and potential drugs are being studied and exploited. The microorganisms living in the extreme environment have their own special characters and adaptation mechaisms, compared with those of common microorganisms, to survive and adapt in the harsh environment. Therefore, polar microorganisms provide a useful model system for studying cold adaptation, and also are one of the optimal sources of cold-active enzymes. It is of not only greatly biological value to study on these microorganisms and their cold-active enzymes, but also important practical value due to their potential for biotechnological applications.106 strains of polar bacteria producing extracellular protease were screened from the polar microbial resources of the laboratory by method of casein plate. The phylogenetic analysis and the enzymatic activity of the strains with lager casein hydrolyzation halos were studied. The phylogenetic analysis showed that 8 isolates with higher protease activity at low temperature belonged to genus of Pseudoalteromonas, Colwelli, Shewanella and Psychrobacter respectively. Using casein as the substrate, the characteristics of the crude protease produced by QI-1 which had higher activity at low temperature than other strains were preliminarily studied. The results showed that the optimal temperature and pH values of the protease were 40℃and 10.0, respectively. The enzymatic activity was independent of divalent cation and was very susceptible to heat treatment, as the activity would be completely lost after incubation at 60℃for 15 min. The enzyme activity retained more than 90% relative activity when it was treated in 0.05mol/L pH 9.0 Tris-HCl buffer for 36h, and retained more than 80% relative activity when the protease was treated in 0.05mol/L pH 7.0～pH 9.0 Tris-HCl buffer and pH 9.0～pH 11.0 Glycine-NaOH buffer for 36h. Zymogram analysis of crude proteases from Pseudoalteromonas sp. QI-1 showed that it might secret four different kinds of proteases.Individual factorial design was optimized to select the important components in the medium for the production of cold-adapted protease of Pseudoalteromonas sp. QI-1. The results showed that both of the optimum temperature for microoganism growth and protease production were 5℃. The optimum inoculation density, air content and initial pH of the medium were 1%,10% and pH 5.0～pH 10.0, respectively, and 20‰NaCl salinity had favorable effect on the growth and protease production. Bran and CH3COONa were selected as the optimal source of nitrogen and carbon. Mg2+, K+ and 0.5% tween-80 had favorable effect on protease production. Base on the above results, a L16(45) orthogonal experiment was designed to determine the maximum protease production at optimum levels for bran, yeast extract, casein, Mg2+ and K+. The optimal combinations of media constituents for maximum protease production were determined as bran 5g/L, yeast extract 2.5g/L, casein 0.3%, MgCl2·6H2O 3g/L, KCl 1.5g/L,5℃,150 r/min for 96 h, and the maximum protease activity was 166.20U/ml.The cold-active protease secreted by Pseudoalteromonas sp. QI-1 will have great industrial prospect if manipulated by genetic engineering. The chromosomal DNA was extracted from strain Pseudoalteromonas sp. QI-1, and then partially digested with Sau3AI. The DNA fragments of 2-9kb were isolated and then ligated with pUC118 vector that had been previously digested with BamUI and dephosphorylated with bacterial alkaline phosphatase. The resultant plasmids were introduced into E. coli DH5a and consequently a genomic library containing about 20000 recombinant clones was produced. The clones producing protease were detected based on the formation of clear halos around the colonies on LB agar plates supplemented with ampicillin, IPTG and casein. After incubation at 37℃until colonies became visible, the plates were incubated further at 4℃for 4 days.1 clone with clear halos was selected for DNA sequence analysis. We determined its nucleotide sequence and found an open reading frame comprising 2127bp(GenBank accession No: HM047289), which encodes a putative protein of 708 amino acids. Alignment of amino acid sequences of PRO-2127 suggested it might have 4 structual domains:a signal peptide sequence, an N-t prosequence, a catalytic domain and a C-t extension. Amino acid residues Asp185, His244 and Ser425 likely formed the catalytic triad of the protease. Analysis of PRO-2127 amino acid composition showed some structural commom in most of psychrophilic enzymes. For example, there was a decrease of the Arg/Arg+Lys ratio, a reduction of the Pro content and a increase in the Gly content. The study on heterologous expression of pro-2127 revealed that the specific activity of the expression systems of pColdⅢ-pro-2127 and pMAL-c4x-pro-2127 were 4.44 U/ml and 3.65 U/ml culture, respectively. The optimum temperature and pH for protease PRO-2127 of recombinant E. coli pColdⅢ-pro-2127 were 40℃and pH 10.0～pH 11.0, respectively. The optimum temperature and induction time for protease expression of recombinant strain E. coli pColdⅢ-pro-2127 and pMAL-c4x-pro-2127 were 10℃and 22h, and the optimum IPTG concentration for protease expression of recombinant strain E. coli pColdⅢ-pro-2127 and pMAL-c4x-pro-2127 were 0.5 mmol/L～1.0 mmol/L and 0.5 mmol/L, respectively.