Study On Cellulose-Decomposing Strains From Herbivorous Fish And Purification Of The Cellulase And Related Activity

Cellulose exists in the plant cell wall and is one of the most extensive carbohydrate in nature and is the largest renewable resources in the earth.It is hard to degrade to available short chain oligosaccharide such as disaccharide and monosaccharide due to its straight chain polyose throughβ-1,4- linkage that occurs in the cell wall of superior plants in strong association with hemicelluloses and lignin.So it has great realistic meaning for human being to solve environment pollution,food shortage and energy crisis that using cellulase produced by microorganism transform cellulose to energy,chemical and food.The research about cellulase has been performed for several decades.The reports showed that the fungi were the dominant contributor to producing cellulase strains both at home and abroad at present study.The cellulase is acid enzyme whose optimum pH value was 3-5 and the activity disappeared or markedly decreased in alkaline condition.The application range and yield were limited due to cellulase producing by solid state fermentation.In the research of cellulose producing condition,little attention has been put on cellulase producing by bacteria which adapt to the neutral or partial alkaline condition and its enzyme preparation has weak hydrolytic action on natural cellulose for a long time. Bacteria cellulose enzyme preparation showed good operational performance and huge economic value on the application of cotton fabrics washing processes and detergent industry over the past decade.Bacteria are also main materials of gene engineering which shows simple structure and rapid reproduction characteristic.Gene research has recently made great progress.The liquid deep layer fermentation of bacteria has the advantage of easy-controlled operating conditions,unease contaminated bacteria and higher production efficiency etc,and therefore cellulase extracted directly from bacteria has become an important and effective enzyme abstraction approach.The bacteria producing cellulose reported at present mostly were terrestrial strains such as Clostridum SP,Xanthomonas, and Acidothermus Cellulolyticus etc.Present research dealt with screen cellulose producing bacteria from water environment especially in aquatic animal is very limited.Water plants are the major source of herbivorous fish under natural condition.These fishes may have cellulose decomposed capacity in the view of physiological adaptation. Cellulose decomposing bacteria have the ability in intestine of terricolous herbivore.We presumed that some enzyme existed in the intestine of herbivorous fish have similar capacity.Grass carp(Ctenopharyngodon idellus),white bream(Parabramis pekinensis) and black bream(Megalobrama amblycephala) are common herbivorous,of which, grass carp is the quickest growth rate and the highest intensity grazing species.The study aimed to cellulose decomposing bacteria in grass carp.The activities of bacterial strains and cellulase in the intestine of grass carp were analyzed using congo red choice plate method.Filter paper and absorbent cotton as substrate and the concentration of glucose measured by the colorimetry of DNS,FPase, CMCase and absorbent cotton enzyme activity of liquid culture filtrates were measured. Based on analyses of bacterial form,physiology and biochemistry and 16S rRNA gene sequence,it was confirmed that the strain belonged to Enterobacter asburiae and bacillus subtilis.The optimal condition of strains X7 for solid state fermentation was pH=7.0-7.3, inoculation amount 5%(v/v),culture temperature37℃and the optimum medium for shoots rooting was 1%sodium carboxymethyl cellulose,1%peptone,0.1%KH₂PO₄, 0.5%NaCl.The strain was cultivated by liquid still condition in optimal growth conditions.The bacteria was smashed by ultrasonic crashing instrument,and broth was magnetic stirred and centrifugal filtered.The activity of cellulose was examined and compared.The enzyme was a kind of extracellular enzyme.It indentified preliminary multi-subunit structure by salt-out,DEAE-Sephadex A-50,Sephadex G-75 and polyacrylamide gel electrophoresis.The enzymatic properties of cellulose after purification was studied:the optimal temperature and pH were 55 and 7.0.The enzyme was rather stable in the range of pH 4.0-7.0 and temperature 45-65℃.The enzyme activity was activated by Mn2⁺,Mg2⁺ and inhibited by Fe2⁺,Ca2⁺.and Zn2⁺.Cu2⁺ and Co2⁺ had obvious toxicity to the activity. The Km value with sodium carboxymethyl cellulose as a substrate was 5.3×10^-3 g/ml.Corresponding medicine test was carried out in order to understand its influence on the growth and activity of bacteria producing cellulase.Results of sensitivity to antibiotics showed that the strains were very sensitive to norfloxacin,cefoperazone and gentamicin,but insensitive to sulfamethazine and oxytetracycline.The experiments of MIC and MBC indicated that florfenicol and norfloxacin had strong inhibitory effects on enzyme activity of four strains.The inhibition of oxytetracycline was relatively weak and that of sulfamethazine was inactive.