Breeding High-Production Mutant Of Neutral Cellulase And Studies On The Enzyme Production Condition

The neutral-cellulase-producing Trichoderma sp.ZC was treated with UV,NTG and N+implantation,the mutant BE40-39 was isolated with an effective screening method.The color ofc10nes 0f the mutant was slightly different from that of initial strain.The enzymatic activity of the mutant was 92 U/mL.which was 2.7 times as high as initial strain,but initial strain and mutant are still not resistant to catabolite repression.The growth of the mutant was not much different with that of initial strain when cultured in plates,while the fermentation performance was improvedconsiderably.The extended enzyme production span of the mutant leads to the improvement ofenzyme production.An effectual method of initial screening described in this paper contains ball—milled Avicelwhich is the only substrate for cellulase.The correlation between the ratio of diameter of clear zone to that of clones,namely D/d,and enzymatic activity is roughly linear. As cellulase is inducible enzyme.its production depends on effective inducers.After theeffects of several cellulosic carbon sources were studied,Avicel and paper pulp were found to be the excellent inducers for cellulase production.Besides the effects of nitrogen sources were studied.Adesired cuIture medium was established through the use of orthotropic experiment.That is Avicel·0.8g/100mL,paper pulp 0.6g/100mL,tryptone 0.45g/100mL,(NH4)2 S040.3g/100mL,bean cakeflour 0.3g/100mL,KH2P04 0.4g/100mL,wheat bran 2.5g/100mL,MgS04 O.03g/100mL and CaCl20.03g/1 00mL.The enzymatic activity could reach 1 06U/mL.The other culture conditions were alsoinvestigated.The preferred conditions:the initial pH of medium is 4.2,culture temperature is 32~C,the speed of rotary shaker is 120r/min,inoculum size is 7.5%and inoculation time is 48h.AddingO.6g/1 00mL Tween.80 when cultured 48h increases the cellulase production by 48%. Crude enzyme properties were investigated preliminarily.When the voluminal ratio ofalcohol to supernate is 3 in the alcohol precipitation,best result is obtained and cellulase recovery is 9 1%.Then the optimum pH,the stable pH range,the thermal stability,the effects of differentbuffers were investigated.Mn2+,Fe2+,Co2+ activate the crude enzyme considerably,while Cu2+inhibit the crude enzyme greatly.What role does Fe3+play depends on its concentration.Somesurfactants help to increase the enzymatic activity of the cellulase,among which PVP-K30 andsample X do the best.Sugars increase the thermal stability of the enzyme,and cellobiose has thebest resuIt.Several alcohols have a positive effect on the thermal stability of the enzyme.And Co2+,Zn2+and Ca2+improve the thermal stability of the enzyme more or less.It was noted that theseeffects Un FPase and CMCase are different.