Screening Of Collagenase-producing Strain And Studying On The Characteristics Of Collagenase

Collagen is a kind of insoluble triple-helical structural protein that widely occurs in animals especially in skin,ligaments,bones,teeth and so on.It is very important structural substance in connective tissue to support the apparatuses and protect the economy.Nearly 30% of total protein in mammalian bodies is collagen.Due to its special configuration and function,collagen has been successfully used in food, medical treatment,health care,cosmetic and so on,and will be extensively used in future.The different use and different intents need the different size of collagen,collagen peptides have more advantages in many applications thanks to their small sizes,for example,it is easier to absorb by digestive tract while used in food,and has better effect while used in cosmetic.The traditional extraction using acid or alkali would destroy the activity of collagen,and pollute the entironment,furthermore,the general protein enzymes could not hydrolyze collagen efficiently,so we dedicate to get collagenase which could hydrolyze collagen efficiently from bacteria,and study its characteristic.The following are the contents and results of the study:1.Six strains that hydrolyze gelatin effectively were isolated from soil sample using gelatin-rich medium,the collagenase activities of strains were determined,strain JY-1 with the highest collagenase activity was selected for further study.The results showed that the collagenase activities were related to but not always consistent with gelatinase activities,the enzymes hydrolyzing gelatin efficiently did not mean they could degrade collagen effectively.2.Physiological and biochemical experiments showed that the cells of strain JY-1 were short rod about 0.9μm×1.8-3μm,gram negative,no spores,the colonies were round, gray-yellow and translucent with smooth edge,the colour of the colonies would deepen after long-time culture;The metabolism of cells was aerobic strictly,acid but no gas were produced from glucose,catalase and methyl red(M.R.) positive,amylase negative,casein and gelatin could be degraded,cells could grow in medium containing 7%NaCl or at 41℃.The total DNA of strain JY-1 was extracted using phenol chloroform extraction method,then used as template for PCR amplification of 16S rDNA.1428 bp of 16S rDNA from strain JY-1 were sequenced and submitted to Genbank(Accession number:FJ418173).Comparing with the sequences in Genbank,it showed that strain JY-1 was extremely closed to Stenotrophomonas maltophilia especially strain D1(Accession number:EU340025),the sequence identity reached 99%,so strain JY-1 was determined as S.maltophilia JY-1.3.The collagenase producing was optimized by single factor experiments and orthogonal experiments.The results showed that there was great difference among different carbon sources,glucose was optimal,and the highest activity was reached at 10 g/l;the collagenase activity was higher while using beef extract as nitrogen source,the optimal concentration was 15 g/l for the balance of cost and output.Gelatin was not suitable to be the nitrogen source,but a spot of gelatin in medium was better to induce the formation of collagenase,pH 7.5 was found to be optimum,high pH or low pH was not good for collagenase producing.The optimal cultural temperature was 33℃,and 37℃was closed to it.The effect of liquid volume in flask was negligible while less than 75 ml/250 ml,so 75 ml of liquid volume in 250 ml flasks was suitable for culture.The effects of inoculation quantities on collagenase activities were not evident from 5%to 12.5%,the highest collagenase activity appeared while inoculation quantity was 7.5%.By optimization of collagenase producing,the collagenase activity of strain JY-1 was obviously improved(from 7.6 U/ml to 13 U/ml).4.The fermentation broth of strain JY-1 which contains collagenase was purified by ion exchange chromatography(DEAE-cellulose),ammonium sulfate precipitation and gel chromatography(Sepharose CL 6B),and crude collagenase was gained.The characteristic of collagenase was determined also.The results showed that the molecular weight of strain JY-1 collagenase was less than 80 KD;the optimal pH of enzymatic reaction was 7.5,and the optimal temperature was 33℃;divalent metal ions like Mg²⁺,Ca²⁺,Zn²⁺ could promote the enzymatic reaction;this collagenase was stable while temperature was lower than 40℃,and decreased rapidly while higher than 45℃.5.Collagenase was used in extraction of collagen from bones,its characteristics were studied.Comparing the effects of collagenase and pepsin on demineralized bone powder,it showed that bone powder was not hydrolyzed completely by pepsin,only big protein chains were gained.However,collagenase could hydrolyze bone powder into different size of short peptides.The results of experiments also showed that the pretreatment of bone was very important to extraction of collagen,the smaller size of bone powder and the more completely of decalcification,the better of the collagen peptides extraction.