Screening And Preliminary Fermentation Optimization Of Neutral Cellulase Producing Bacteria

As a kind of cellulase with optimal pH value at6.0to8.0, neutral cellulase hassignificant application prospect and market potential in textile, dyeing and other fields. Asincreasingly broad application of neutral cellulase in textile industry, the market demand forneutral cellulose is increasing rapidly, especially in China, which has a big textile industry.While, the neutral cellulase used in China was mainly imported, therefore the research aboutneutral cellulase has great significance.In this paper, a strain belong to bacterium which can produce neutral cellulase wasscreened and isolated from soil and used two methods to improve the production of enzyme.One was optimized the fermentation conditions, and another was screened the positive mutantstrains through traditional mutation breeding means.A neutral cellulase-producing bacterium, JN-D2, was screened via5steps from the soilsamples around the campus, like enrichment culture, pre-screening with hydrolytic circlemethod on congo-red plate, screening by hydrolytic circle of oxford-cup, re-screening byenzyme activity in shaking-flasks and measurement of enzyme activity under different pH.And the strain JN-D2was proved to be a G+bacterium and can formed spores by gramstaining, spore staining and colony morphology observation. Finally this strain was identifiedas Bacillus through16S rDNA sequence analysis.The composition of fermentation medium was optimized for neutral cellulase productionin shaking flasks. The result of single factor experiments showed that: maltose was the bestcarbon source, and the best nitrogen source was a mixed nitrogen source of yeast extract andpeptone, and the mixed mass ratio was1:4. At the same time, maltose and mixed nitrogenwere identified as main factors for enzyme production than other factors, like MgSO4·7H2O,CaCl2, FeSO4·7H2O, K2HPO4·3H2O, through factional factorial experiment, and theirconcentrations were optimized by response surface methodology (RSM) and artificial neuralnetwork-genetic algorithm (ANN-GA). The optimal concentrations obtained by RSM weremaltose1.97%and mixed nitrogen sources2.03%, and the maximum enzyme activity was194.23U·mL-1. Meanwhile, the optimal concentrations obtained through ANN-GA weremaltose1.94%and mixed nitrogen sources2.02%with a maximum enzyme activity of193.40U·mL-1, which were highly similar with RSM results.The cultivation conditions were optimized for neutral cellulase production in shakingflasks. The single factor experimental results showed that, the initial pH of medium andinoculum volume slightly affects enzyme activity when they were in the range of6.0~8.0and0.4%~10%respectively. Besides, the optimum culture temperature, culture time and were30℃,48h and20mL respectively. With JN-D2as a starting strain, traditional mutation breeding was used to improve itsenzyme production capacity. A positive mutant strain, JN-D2AU18, was obtained byhigh-throughput screening of porous culture plate after atmospheric-room temperature plasma(ARTP) and ultraviolet (UV) mutagenesis. Its enzyme activity increased about20%comparedwith JN-D2.