Screening Of Efficient Cellulase-producing Strains And The Application Of The Cellulase

The cellulases produced by most of the cellulose degrading strains of have been discovered have low enzyme activity,and the properties of the cellulases can not meet the necessity of large-scale fermentation in industry.In China,the price of cellulase is high,and most of the cellulases depend on the imports,when our own production technology and efficiency is not high.This paper have begun from the source and selected microorganisms that can efficiently produce cellulase from the cellulose-rich environment in nature.Cellulase has a good application in many fields.In this paper,the practical value of the extraction of the resveratrol and polydatin from Polygonum cuspidatum,and the utilization of water hyacinth,are analyzed.In this experiment,through Congo red staining primary screening and enzyme activity rescreening,a total of 16 cellulase-producing strains were selected from the specific environment.The maximum activity of CMCase,FPase and?Gase which were producted by numbered H-3,H-3and H-1 strains,was 1538.44 U/ml,212.87 U/ml,and 115.14 U/ml,respectively.Three strains with large hydrolysis circles and high cellulase activity?numbered H-3?Z-1 and Z-2?were analyzed by 16S rDNA gene sequence,and all of them were identified as Bacillus.The enzymatic properties of the cellulases produced by the three strains were analyzed.It was found that the optimum temperature for the enzymes produced by strains H-3?Z-1 and Z-2 were both 50°C and the optimum pH was 5,4and 5,respectively.The cellulase produced by the three strains had good heat and acid resistance,and there was more than 50%residual enzyme activity after incubation at 4°C-50°C for 4h;and the remaining enzyme activities were all above 40%after 4h of pH 3-7.The enzymatic properties of these cellulases produced by the three strains are great,and the enzymatic activity can be improved by rational use.For the efficient cellulose degradation strains of H-3,its fermentation conditions were optimized to produce more enzymes.Single-factor experiments,Plackett-Burman design and response surface design were used to optimize the cellulase-producing fermentation conditions.The experiments using CMCase as the index,evaluated the 6factors of carbon source,nitrogen source,inorganic salts,initial pH,inoculation amount and liquid volume on the influence of the strain's ability of enzyme production.The results showed yeast powder,liquid volume and initial pH is the main influence factors.Through the analysis of Design-Expert software,the optimum fermentation conditions of enzyme production is as follows:20.92 g/L of yeast strains,52.51 mL of liquid volume and initial pH 6.69;and other conditions of the fermentation were 14g/L of glucose,22g/L of peptone,6g/L of KH₂PO₄,10%of inoculation amount.In this conditions,the enzyme activity was1721.08U/mL.Compared with the original culture conditions,the enzyme activity have increased by 72.20%.The resveratrol and polydatin in Polygonum cuspidatum were extracted with cellulase.Through single-factor experiments,the effects of four factors?enzyme dosage,pH,temperature,and enzymolysis time?on the extraction rates of resveratrol and polydatin were analyzed.The results showed that the optimum conditions were as follows:8 mL cellulase,the extraction pH 5.0,55°C;but the optimal enzymolysis time for resveratrol and polydatin extraction was 4h and 8h,meanwhile the extraction rates were 6.85%and 6.95%,respectively,which were 2.70-fold and 1.46-fold than before.This experiment provides the basis for the future application of cellulase in the extraction and application of active ingredients in Chinese herbal medicines.For the utilization of water hyacinth,we have explored the hydrolysis of water hyacinth into glucose by cellulase.Through single-factor experiment,the effects of four factors?temperature,substrate amount,reaction pH and enzymolysis time?on the hydrolysis of water hyacinth by cellulase were analyzed.Using the L9?3⁴?orthogonal test,we got the optimum conditions of the hydrolysis of water hyacinth by cellulase:50°C,substrate amount 0.2g,pH 4.5,enzymolysis time 8h,which's rate was 97.16%and was 1.82-fold than before.