Producing Alkaline Cellulase Producing Strain IS-B4 Breeding, Optimization Of Fermentation Conditions And Enzymatic Properties

Alkaline Cellulase(CMCase)is a part of alkaline cellulase system.Alkaline cellulase reacts mainly with cellulose in uncrystalloid section(unformed section, accounts for 10 percent of cotton fibre)of cotton fibre and it has little effect on hydrolyzing the cellulose in the crystalloid section so it can't reduce the fastness of the cotton fibre and can be used widly in the detergent.It comes from the nature and is easy to decompose.Bewsides,it can get good results with little dose and has no harmful effect on the environment,so it is the trend of the world detergent industry.In addition,it can be uesd in the fields of food,chemistry,paper making,textile,environment protecting and so on,which make it very important to study the production of CMC.In this study,a higher CMCase production strain IS-B4 was obtained,and an emphasis was placed on optimization of fermentation conditions for production of the CMCase by the strain IS-B4 by flask culture and in 5 L-fermentor;also some enzymatic characteristics were studied.The results are summarized as follows:1.At first,The morphological physiological and biochemical characteristics of the original strain Bacillus.macerans IS-B was studied.Then it was mutated by UV,DES,He-Ne laser and UV+ DES.The optimal mutation effect were obtained by UV 40 s,DES 10min,He-Ne laser 20 rain and 40s UV+20min DES;we use a rapid method for screening the higher production strain,also the stability of the mutants were studied,finally the mutant IS-B4 was obtained and its enzyme productivity was 52.3 U/mL,and it is 5.13 fold higher than that of original strain.2.The optimal fermentation conditions for CMCase production were determined by a single-factorial experiment and orthogonal experiment.The compositions of fermentation medium were,saccharose 2%,yeast extract 2%,NaCL 0.5%, KH₂PO₄ 0.1%.The incubation temperature 36℃;amount of inoculum is 5%; pH 9.0.The load of 250 mL-flask was 60 mL,and the enzyme activity could reach as high as 56.0 U/mL. 3.In 5 L-fermentor,by increasing the dissolution oxygen of medium at the exponential phase,the time to reach enzyme activity peak was shortened in 5 L-fermentor,the strain could produce as much as 59.8 U/mL.4.The optimal conditions for reaction of the CMCase produced by IS-B4 was pH 9.0～10.0at 36℃.The enzyme was in a range of pH 7.0～10.0,at the temperature beteewn 30～70℃.The enzyme was inactivated at the presence of Cu²⁺,Mn⁺,Zn²⁺,whereas it was activated at the presence of Na⁺,Ca²⁺;However, Tween80 had insignificant effect on the activity of the CMCase concerned.