Screening,Mutation Breeding And Enzyme Production Optimization Of ?-Glycosidase-Producing Strain

?-glucosidase,hydrolyzes the?-1,4-glycoside bonds to form glucose and its corresponding ligands,which is widely found in plants,animals and microorganisms.?-glucosidase is a kind of rate-limiting enzyme in the process of cellulose degradation and plays a very important role in the development and utilization of lignocellulose.It has been widely applied to food,textile,feed,paper,medicine industries and other aspects.At present,most of the industrial using?-glucosidases were comes from microorganisms,the?-glucosidase activities are generally low,which largely limited the large-scale and efficient production and application.The effective method is developing new strains with high?-glucosidase activity by mutagenesis breeding.This study aims at screening strains with high?-glucosidase activities,and improveing the enzyme production activity by two different mutagenesis methods and optimization of enzyme production fermentation conditions.Meanwhile,cellulose enzyme activity of the related strains is detected.This study will provide an effective resource for the development and application of strains producing?-glucosidase.The main results are as follows:1.The fungi with?-glucosidase activity were isolated from the treated rotten woods,the samples were collected from Dushan Forest Park in Nanyang,Henan province.Sixteen?-glucosidase producing strains were isolated,among them,nine strains showed obviously enzyme-producing chromophores on the esculin agar plate.After shaking fermentation,the?-glucosidase activity of strain L1 was 34.85 U/mL,which was the highest among the nine strains.Strain L1 was identified as Penicillium ochrochloron by morphological observation,and molecular biology analysis of ITS and BenA gene sequence.2.The atmospheric and room temperature plasma(ARTP)mutagenesis was carried out for breeding strain L1.The mutant strain A-2 was selected by esculin agar plate screening and?-glucosidase activity re-screening,its?-glucosidase activity was 57.84U/mL,52%higher than that the original strain L1.Diethyl sulfate(DES)mutagenesis was carried out of the strain A-2,a genetically stable mutant strain D-6 with high?-glucosidase activity was finally obtained by the same screening method.The?-glucosidase activity of the strain D-6 was up to 94.74 U/mL,75.1%higher than that of the strain A-2,and 148.2%higher than that of the original strain L1.3.In order to further improve the?-glucosidase activity of the mutant strain D-6,single factor experiment,Plackett-Burman design,steepest ascent path and response surface methodology were used for studying the optimum conditions for?-glucosidase production in fermentation based on eight factors.Finally,the optimal fermentation conditions of the strain D-6 were obtained as follows:corn straw 45.74 g/L,ammonium sulfate 7.23 g/L,potassium dihydrogen phosphate 6 g/L,magnesium sulfate heptahydrate 1g/L,calcium chloride 0.5 g/L and ferrous sulfate 0.1 g/L;initial pH is 5.2,inoculation volume is 5%(spore concentration 10~8/mL),liquid loading capacity is 63 mL/250 mL,temperature is 28?,shaking speed is 160 r/min,fermentation time is 132 h.Under the above conditions,we obtained the maximum enzyme activity of 142.92 U/mL,with an increase of 54.2%compared to the original fermentation parameters.4.In order to test the cellulase activity of high-yielding?-glucosidase strains,the strain L1 was cultured on the sodium carboxymethyl cellulose plate,and the obvious transparent circle was observed by Congo red staining.The rusults showed that there were equally obvious in the transparent circles produced by the mutant strains A-2 and D-6 after Congo red staining.The filter paper enzyme activity(FPA)and internal cellulase activity(CMCase)of each strain were quantitatively measured after fermented in bottles with wild strain L1,mutant strain A-2 and D-6,respectively.The FPA of the three strains were 11.50,12.26 and 12.53 U/mL,the CMCase of the three strains were 60.09,61.55 and 61.82 U/mL.The results showed that the two enzymes activities of mutant strains A-2 and D-6 were slightly improved than that of the original strain L1.More experimental results are needed to determine the optimal enzyme-producing fermentation conditions.In this paper,the strain L1 with high?-glucosidase activity was isolated from the rotten woods.The activity of?-glucosidase of the mutant strain D-6 was 94.74 U/mL after ARTP-DES compound mutagenesis,which increased 148%compared with the original strain L1.Response surface method was used to optimize the enzyme-producing fermentation conditions of the mutant strain D-6,under the optimal conditions,the?-glucosidase activity of the mutant strain D-6 reached to 142.92 U/mL,which was 54.2%higher than that before optimization.Cellulase activity assay showed that the FPA and CMCase of mutant strains A-2 and D-6 were slightly improved than that of the original strain L1.The study aimed to obtain strains with high?-glucosidase activity and cellulose degradation abilities,the results provide a technical reference for the optimization of fermentation conditions of?-glucosidase producing strains,and also provide effective resources for the development and application of?-glucosidase strain.