Studies On Purification, Gene Cloning And Expression Of Collagenolytic Enzymes

Collagenolytic proteases are defined as proteases with the ability to decompose native collagen, which are not only important for cell dispersion and for structural studies of collagen and collagen-like proteins but also clinically significant, because they are effective in the treatment of various pathological conditions involving collagen-rich structures. Collagenolytic proteases from both microbes and mammalian tissues have been widely studied. There are more than 40 different collagenolytic proteases isolated from bacteria and fungi but few bacterial ones are commercially prepared.By using gelatin plate screening, fresh-hides decomposing test and collagenolytic assay, twelve bacterial stains with strongly collagenolytic activity were selected and identified as Pseudomonas aeraginosa and Photobacterium logei, respectively.The optimal fermentation media for P.aeraginosa MBL13 and Ph.Logei MBL59 producing collagenolytic enzyme were determined as follows. The compositions of fermentation medium for the former one are: 1% gelatin,4% saccharu, 0.15% yeast; 0.02% CaCl2,0.25% K2HPO4 ·3H2O and 0.05%NaH2PO4 ·2H2O, pH 6.0, and for the latter one are: 0.5% beef extract, 0.5% NaCl and 1% tryptone. Many nitrogen and carbon compounds, as well as metalions were shown to inhibit the collagenolyticenzyme production of Ph. logei MBL59.A collagenolytic enzyme from P.aeraginosa MBL13 was purified by ammonium sulfate precipitation, SP sepharose fast flow, Sephadex G-200 gel filtration and native SDS-PAGE cutting method. The final yield was about 0.95% and the specific enzyme activity was increased 36.1-fold, with 5.77U/mg protein.The purified enzyme called PAC has an apparent molecular weight of about 110kD by SDS PAGE without β-mercaptoethanol. Treatment with β-Me suggested that PAC was dissociated into three subunits approximately 33,25 and 20kD with a ratio of 2:1:1, designed as subA, subB and subC, repectively.The optimal temperature and pH for PAC collagenolytic activity is 37℃ and 7.5 respectively. EDTA and EGTA displayed a significant inhibitory effect on the enzyme activity while PMSF, leupeptin and pepstain did not appreciably inhibit it. The first 15 amino acid residues of the major subunit (subA) were determined and the sequence was Ala-Glu-Ala-Gly-Gly-Pro-Gly-Gly-Asn-Gln-Lys-Ile-Gly-Lys-Tyr. This sequence was identical to that of elastase of P. aeruginosa PA01. Since elastase from P.aeruginosa has been reported as no collagenolytic activity, these results demonstrated that PAC is a novel collagenolytic protease different from those detected in other P.aeruginosa strains before.A collagenolytic enzyme from Pklogei was purified by ammonium sulfate precipitation, ion-exchange chromatography on DEAE-sepharose fast flow, hydrophobic interaction chromatography on phenyl sepharose 6 fast flow and gel filtration on sephadex G50. This protein was purified 15.7-fold with a 1.1% yield by these steps. The specific activity of the finial enzyme was estimated to be 2.2U/mg protein.The enzyme called PLC has a molecular weight of 35kD with or without β-Me treatment. The optimal temperature and pH for PLC collagenolytic activity is 37℃ and 8.5. The enzyme activity could be completely inhibited by EDTA and EGTA while PMSF and Leupeptin and pepstain had no influence on it. These results suggested that PLC also is a metalloprotease with collagenolytic activity. Pklogeiwas firstly found to produce a coUagenolytic enzyme so that PLC also is a novel coUagenolytic protease.Based on the high homologous N-terminal amino acid sequence between SubA and elastase, the intact gene of SubA was amplified from total DNA of P. aeruginosa MBL13 by polymerase chain reaction. After cloned into pMD18-T vector, the gene sequence was determined and its size is 2011bp. The coding region of the SubA mature peptide has 1494bp in length and encods 498 amino acid residues. The deduced amino acid sequence contained a putative signal sequence consisting of 23 residues followed by a propeptide containing 174 residues. Homologous analysis shows there are 99% an...