| There is abundant microorganisms resource in the sea. They are the valuable resources for the finding of new enzymes and new medicines, and have good potential for research and development in the future. Extremophiles are one of the research hot spot in the region of Microbiology. Cold-adapted microorganisms are one kind of extremophiles and are widely distributed in the sea. They are the good materials for isolation of cold-adapted enzymes. Cold-adapted enzymes are very useful in the trades, such as laundry, food, milk, medicine and cosmetics et al, which need enzymatic catalysis at low temperature, because they have higher catalytic efficiency at low temperature. Therefore, cold-adapted enzymes aroused wide research and development by scientists in the world. Since 1999, marine cold-adapted microorganisms and enzymes have been studied in Shandong University. A cold-adapted bacterium P. sp. SM9913 producing a cold-adapted protease was isolated from 1855 m deep-sea sediment by Xiu-Lan Chen et al (2001). Cold-adapted protease MCP-01 produced by P. sp. SM9913 was a typical cold-adapted enzyme. It had high catalytic efficiency at low temperature and high thermolability. Cold-adapted protease MCP-01 was susceptible to autolyze. Based on these researches, in this article, cold-adapted bacterium P. sp. SM9913 was identified, the gene of cold-adapted protease MCP-01 was cloned, the structure of protease MCP-01 was analyzed, the autoiysis mechanism of cold-adapted protease MCP-01 and effects of sugars on the autoiysis of cold-adapted protease MCP-01 were studied.1. Identification of cold-adapted strain SM9913According to its morphological and biochemical charateristics, cold-adapted strain SM9913 had been identified to belong to genus Pseudomonas. In this article, the gene of 16SrRNA of strain SM9913 was cloned with the primers designedaccording to the conserved sequence of 16SrDNA of ￡ coli. The homogeneity of the 16SrDNA sequence of strain SM9913 aligned with the 16SrDNA sequences of strains in GenBank database was analysed by the software Blastn. The result showed that the 16SrDNA sequence of strain SM9913 had the highest identity (98-99%) with the 16SrDNA sequences of many strains belonged to genus Pseudoalteromonas, indicating that strain SM9913 belonged to genus Pseudoalteromonas. Utilization of 95 carbon resources by strain SM9913 was analyzed with the BIOLOG microorganism identity system. The character of carbon utilization of strain SM9913 was agreed with that of genus Pseudoalteromonas. Therefore, strain SM9913 belonged to genus Pseudoalteromonas. With the phylogenetic analysis software phylip and clustalw, an unrooted phylogenetic tree was obtained by an identity analysis of the 16SrDNA sequence of strain SM9913 with the 16SrDNA sequences of 17 Pseudoalteromonas strains which had the highest identity with strain SM9913. The result indicated that cold-adapted strain SM9913 was probably a new species of genus Pseudoalteromonas and was named as Pseudoalteromonas sp. SM9913 {P. sp. SM9913).2. Gene cloning and structure analysis of cold-adapted proteaseMCP-01 produced by cold-adapted bacterium P. sp. SM9913.2.1 Gene cloning of cold-adapted protease MCP-01Previous study showed that cold-adapted protease MCP-01 produced by cold-adapted bacterium P. sp. SM9913 was a serine protease. The sequences of serine proteases in protein database were comparatively analyzed, and primers were designed according to the conserved sequences of the active centers of serine proteases. The DNA fragment of the active center of protease MCP-01 was amplified through PCR and a DNA fragment M4 of 565bp was obtained. Identity analysis of DNA fragment M4 with BLAST software showed it had identity with the sequences of many bacterial extracellular serine proteases and had the highest identity (86%) with the sequence of extracellular serine protease produced by Alteromonas sp. Strain 0-7. Therefore, DNA fragment M4 was determined as the gene fragment of the activecenter of cold-adapted protease MCP-01.The genomic DNA of bacterium P. sp. SM9913 was recovered and was partly digested by Sau 3A I. Then the digested genomic DNA was electrophorerized on agarose gel and was transferred to a nylon membrane. Southern hybridization was made with DNA fragment M4 as probe. There were two blots at about 6 kb and 3.5-4kb on the membrane respectively. DNA fragments of 3.5-4kb of the digested genomic DNA were recovered from agarose gel and cloned into puC19 which had been digested by Bam HI and transferred into E. coli DH5 a . A partial genomic DNA library was then constructed. Colony hybridization was made with DNA fragment M4 as probe and one positive colony E. coli DH5 a -MCP was obtained. The insert DNA fragment in the recombined plasmid pmT-MCP in colony E. coli DH5 a -MCP was sequenced and its length was 3.89kb. Through ORF finding and identity analysis, it was proved that only partial gene of 1689bp of cold-adapted protease MCP-01 was in the 3.89kb insert and the partial gene DNA fragment was named DNA fragment MCP. Three primers were designed according to the 5' terminal sequence of DNA fragment MCP, and a general primer was designed according to the N terminal sequences of 4 proteases which had the highest identity with protease MCP-01. A DNA fragment of 457 bp, named TAILPl, was amplified by TAIL PCR. Identity analysis of DNA fragment TAILPl with BLAST software indicated that it was a part of the gene of protease MCP-01. Three new primers were designed according to the 5' terminal sequence of TAILPl. A DNA fragment of 651 bp, named TAILP2, was amplified by another TAIL PCR with three new primers and the general primer. The full-length gene sequence encoding protease MCP-01, 2130 bp, was obtained with the join of DNA fragment TAILP1, TAILP2 and MCP, and was named as gene MCP-01. At the same time, the promotor of gene MCP-01 which was in DNA fragment TAILP2 was obtained through the second TAIL PCR . Identity analysis of sequence of gene MCP-01 with BLAST software indicated that its identity to the genes in GenBank database was very low. Therefore, tgene MCP-01 encoding protease MCP-01 is a new gene which haven't been submitted to GenBank database. 2.2 Structure analysis of cold-adapted protease MCP-01The amino acid sequence of protease MCP-01 was deduced according to its gene sequence. There were 709 amino acid residues in the primary structure of protease MCP-01. Analysis of its amino acid composition showed that it had typical characteristics of cold-adapted enzyme molecular. Identity analysis of the amino acid sequence of protease MCP-01 with BLAST software showed that it had identity with the amino acid sequences of many bacterial extracellular serine proteases and had the highest identity (67%) with the sequence of extracellular alkaline serine protease produced by Alteromonas sp. Strain 0-7. Identity analysis also showed that cold-adapted protease MCP-01 belonged to thermitase family in subtilase superfamilay in serine proteases and it was a new member of thermitase family. Protease MCP-01 was composed of three domains. Its catalytic domain Peptidase_S8, composed of 291 amino acid residues, was from Glyl67 to K458, which had the character of catalytic center of proteases in subtilase family--Asp/Ser/His. The three amino acids which took part in catalyzing were Aspl88, Ser428 and His247. Two similar domains PPC1 and PPC2, all composed of 85 amino acid residues were from A622 to Q706 and from Q514 to Gly598, respectively. This domain was usually found at the C-terminal region of peptidases secreted by many bacteria. Its function probably related to the peptidase secretion toward the cellular outside. The 3D strcture model of the catalytic domain was constructed by an automated homology modeling server. According to the 3D strcture model, the catalytic cavity of protease MCP-01 was surrounded by a -helixes and P -folds. The three amino acids asp 152, ser392, his211 which took part in catalyzing were all in the catalytic cavity.3. The autolysis mechanism of cold-adapted protease MCP-01Previous study showed cold-adapted protease MCP-01 was susceptible to autolyze and capillary electrophoresis was a sensitive, efficient and rapid method to study protease autolysis. In this article, effect of temperature on the autolysis and thermostability of cold-adapted protease MCP-01 in Tris buffer of pH6.5, pH7.5 and pH8.5 was studied respectively. The autolysis rates and thermostabilities of cold-adapted protease MCP-01 at different pH and different temperatures weredifferent. At each pH and temperature, the thermostability of cold-adapted protease MCP-01 decreased as its autolysis rate increased. This result further proved that the autolysis of protease MCP-01 was closely related to its thermostability and its autolysis was the main reason why it was thermolabile at moderate temperature (<40 °C). In addition, the concentration of protease MCP-01 also affected its autolysis. The autolysis rate of protease MCP-01 decreased as its concentration increased at the range of 0.2-1.Omg/ml. High concentration of protease MCP-01 actually could inhibit its autolysis and enhanced its thermostability, although the concentration 1 .Omg/ml of protease MCP-01 couldn't completely inhibit its autolysis. The effect of pH on the autolysis of protease MCP-01 in Tris and borate buffer was also studied in this article. In tris buffer, protease MCP-01 at pH7.5~9.0 was relatively stable and had a slow autolysis rate at 35 °C. On the other hand, when protease MCP-01 was at pH6.0~7.0 in tris buffer, it was not stable and autolyzed rapidly. However, in borate buffer, when protease MCP-01 was at pH7.0~8.0, it was relatively stable and had a slow autolysis rate at 35*C. when protease MCP-01 was at pH8.5~9.0 in borate buffer, it was not stable and autolyzed rapidly. Therefore, pH could affected the autolysis and stability of protease MCP-01. Moreover, in different buffer, the pH range for protease MCP-01 stability was different. Because the structure of protease MCP-01 was flexible and loose, changes of elements, such as ion strength and electric charge et al, in the buffer could directly affected the autolysis rate and structure stability of protease MCP-01.4. Effect of sugars on the autolysis of cold-adapted proteaseTrehalose is a nonreducing disaccharide, which exists extensively in the body of bacteria, yeasts, fungi, plants, insects and mammals. Trehalose played an important role in the process of these creatures to resist stress. Trehalose could stabilize the structure of macromoleculars when they were at extreme environments such as dehydration and high temperature et al. Trehalose could keep, even activate enzyme activity at high temperature. Effect of trehalose on the autolysis of protease MCP-01 in different concentration and at different pH was studied in this article. Trehalose had preventing effect on the autolysis of protease MCP-01 both in different concentrationand at different pH. The preventing effect of trehalose on the autolysis of protease MCP-01 increased as the trehalose concentration increased. Studies showed that many kind sugars had protective effect on protein activity and thermostability. Besides trehalose, effects of sucrose and three monosaccharides—mannose, glucose and galactose, on the autolysis of cold-adapted protease MCP-01 was also studied in this article. These sugars all could prevent the autolysis of protease MCP-01. Among three monosaccharides, the preventing effect of mannose was inferior to that of glucose and galactose. At the same concentration, the preventing effect of trehalose was the best among the 5 sugars. Previous studies showed that the effect of trehalose on stabilizing protein was the best among all the sugars. The same result on protease autolysis was obtained at this article. However, the results in this article also showed that, for all the sugars used, the preventing effect on protease autolysis was affected by sugar concentration. The preventing effect of 1.5 M glucose and galactose was better than that of 1 M trehalose.
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