Optimization Of Solid Fermentation Conditions By A Marine Aspergillus Niger And The Preparation Of Crude Cellulase

Cellulase was compound.enzyme,which was consisted of endoglucanase,exoglucanase and β-glucosidase.By the synergy of these three enzymes,cellulase can produced economic and abundant raw materials,which was expected to solve the natural problems of solid waste.In order to develop and apply cellulase,the optimization of solid fermentation,cellulase extraction process in solid and liquid fermentation,and the vacuum freeze-drying protective agent of cellulase has been studied in this paper.The main contents and results were as follows:Single-factor was adopted to determine the optimal carbon source and nitrohen source.Carbon source was consisted of corn stalk powder and wheat bran(1:1),nitrogen source was NH4C1,water content 70%.Water content and initial pH value were identified by Plackett-Burman(PB)as two main factors on FPA,and their concentration were ascertained by steepest ascent path.Finally,by response surface analysis,the optimal fermentation medium of 30 g was determined as follows:the percentage of wheat bran and corn stalk powder was 48.53%,the percentage of NH4C1 was 1.94%,the percentage of KH2PO4 was 0.2%,the percentage of Fe(OH)3 was 0.16%,the ratio of material and water was 1:2.2,initial pH value 4.28,inoculum concentration of 2%,and incubated in an incubator at 28 ℃ for 7 days.The sample was dissolved in 10 volumes of tap water,which was extracted for 1 hour at 20 ℃,after optimization,the activity of cellulase reached 14.826 U/g,which was increased by 75.12%。The crude enzyme solution was preliminary concentrated and purified.The process of tannin-PEG in the solid fermentation was investigated,the results showd that the optimum flocculating agent concentration was 12 mg/mL,precipation time was 70 min,the extraction effect of PEG was better than PVP,the recovery rate of cellulase reached 96.97%,when the PEG concentration was 30 mg/mL,extraction time was 15 min.The concentration effect increased 10 folds.The comparion of tannin-PEG,ammonium sulfate precipitation and ultrafiltration was researched,we found that ammonium sulfate,ultrafiltration and tannin-PEG all had a condensing effect,but they were different:the usage of ammonium sulfate was very big and it reduced the activity of cellulase;ultrafiltration costed much time,and the membrane was easy to pollution;the process of tannin-PEG was very convenient,fast,and easy to operate,but the cost will also be a prolem that can not be ignored.The process of concentrating liquid fermentation by tannin-PEG was studied,it was found that the method was suitable for it.The results showed that the recovery rate of total enzyme activity was up to 93.5%,when the concentration of tannin was 18mg/mL,the sedimentation time was 70 min,the concentration of PEG was 36 mg/mL and the extraction time was 10min.The vacuum freeze-drying protective agent of cellulase was screened.Bran,corn stover,straw powder,sucrose,trehalose,maltose,lactose and starch as the protective agent were tested,the results showed that trehalose was the best,and the residual enzyme activity of cellulase filter paper after vacuum freeze-drying was 78.80%.