Purification, Identification And Characterization Of Neutral Cellulases From Humicola Insolens H31-3

Cellulase refers to a class of enzymes that synergistically hydrolyze the beta-1,4-D-glucosidic linkages in celluloses into oligosaccharides and cellobiose,and ultimately into glucose.There are there major types of cellulolytic enzymes produced by fungi,namely,endoglucanase,exoglucanase(cellobiohydrolase),and beta-glucosidase.The thermophilic fungus Humicola insolens H31 was isolated from humous soil by the Institute of Microbiology,Chinese Academy of Sciences,in 1970s.Humicola insolens H31-3,a mutant strain of H31,is a good producer of thermostable neutral cellulases that can work in neutral conditions and tolerate higher temperature and broad pH range.These properties determine they are particularly suitable for applications in textile industry.In this study,three extracellular neutral cellulases were purified from Humicola insolens H31-3.The main results were as follows:(1)The cellulase solution was obtained from the culture broth of liquid fermentation of H31-3,which showed high activities on CMC-Na,Avicel and salicin.Thus,it was concluded that cellulases from H31-3 have entire cellulase activities.The enzyme activities on CMC-Na,Avicel,Salicin,FP were 81.42IU/mL,0.77IU/mL,10.96IU/mL,4.27IU/mL and specific activities were 5.26IU/mg,0.05IU/mg,0.71IU/mg,0.28IU/mg respectively.(2)According to the results of gel filtration chromatography with S-100,protein SDS-PAGE and enzyme activity detected,we carded out zymogram analysis and determined that cellulases with different molecular weight may be which kind of cellulases activity and may be which type of cellulase roughtly.(3)An endoglucanase and two exoglucanases from the supematant of the culture broth of Humicola insolens H31-3 were purified by ammonium sulfatefractionation,gel filtration chromatography and ion exchange chromatography.Specific activities of purified EGI on CMC-Na was 16.49IU/mg,specific activities of purified CBHâ… ,CBHâ…¡on FP were 1.33IU/mg,1.09IU/mg respectively.(4)We identificed the purified cellulases by Protein Fingerprint Analysis,which concluded initially that EGI may be a new cellulase protein,the amino acid sequences of CBHâ… and CBHâ…¡had very high homology to Q12621_HUMGT(Swiss-Prot)and O93780_HUMGT(Swiss-Prot)from Humicola grisea var.thermoidea(also named Humicota insolens var.thermoide)respectively.Therefore,N-terminal sequences of EGI was determined and the result was T-L-P-S-E-E.(5)The molecular weight of purified EGI,CBHâ… and CBHâ…¡were 38.7547kDa(MS), 66kDa(SDS-PAGE),47.7162kDa(MS).(6)The isoelectric point of EGI and CBHâ…¡was 3.9 and 5.3 respectively detected by isoelectrofocusing,the latter was corresponded with 5.28 of O93780_HUMGT.(7)Studies on properties of EGI,CBHâ… and CBHâ…¡indicated that the optimum temperature and pH of the enzyme reaction was 60℃,60℃,65℃and 6.0,6.0～6.5,6.5 respectively.EGI and CBHâ…¡had very good thermal stability,but CBHâ… 's was relatively worse.The pH stability were 5.0～8.5,6.0～8.5 and 5.0～9.0 respectively.(8)Effect of metal ions on the activity of purified cellulases indicated:activity of EGI was stimulated by Ba²⁺,Mg²⁺ions and strongly inhibited by Zn²⁺,Fe³⁺,Cu²⁺,Ag⁺,Hg⁺, Pb²⁺,Co²⁺.Activity of CBHâ… was activated by Mg²⁺and significantly inhibited by Ag⁺,Hg⁺, Pb²⁺,Cu²⁺.Activity of CBHâ…¡was activated by Mg²⁺,K⁺ and significantly inhibited by Ag⁺, Hg⁺,Zn²⁺,Pb²⁺,Cu²⁺.However,it was unaffected by other testing ions.Some analogs of substrate or product had no effect on the activity of purified cellulases.(9)Kinetics experiment indicated Michaelis constant measured by CMC-Na of EGI and CBHâ…¡was 46.94mg/mL and 13.49mg/mL,maximum velocity was 0.64 mg/mL·min and 0.12 mg/mL·min respectively.The above results indicate that EGI,CBHâ… ,CBHâ…¡are thermostable neutral cellulases which have good thermal stability and pH stability as well as stabilized properties.These good properties will be applied in textile industry.Judging from documents and patents having been announced,EGI is extremely possible a new cellulase.Dueing to the differences of fundamental properties and characteristics between CBHâ… ,CBHâ…¡and Q12621_HUMGT,O93780_HUMGT respectively,further study still needing to identify whether they are the same proteins or not.