| α-Glucosidase(α-D-glucoside glucohydrolase,EC 3.2.1.20),which belongs to the exoglycosidase,is capable of catalyzing the cleavage of the non-reducing terminal of substrates to liberates α-D-glucose,and supposed to play a important role in the processes of hydrolyzing starch to produce non-fermentable sugars.In addition,α-glucosidase can transfer free glucose residues to another saccharide substrate,thereby producing oligosaccharides.The transglycosylation activity from α-glucosidase is being used to produce glucosylate compounds or to prebiotic isomaltooligosaccharides.The production of α-glucosidase is usually accompanied by the amylases as glucoamylase from Aspergillus niger.Glucoamylase,which reduce the amount of starch that can be used by α-glucosidase,thereby inhibiting the ability of α-glucosidase to hydrolyze and convert substrates.Glucoamylase is an exoglucohydrolase that basic catalyzes the hydrolysis of α-1,4-glycosidic linkages in starch,glycogen and Oligosaccharides,it also hydrolyse α-1,6-glycosidic linkages at lower rate.And glucosamylase from Aspergillus niger in industrial production is often affected by transglucosidation of the α-glucosidase,resulting in a significant reduction in glucose production.Since the same substrate can be hydrolyzed,α-glucosidase and glucoamylase may be competitively inhibited from each other.In this study,a glucoamylase gene was investigated by targeted gene deletion and aimed to construct mutant strains of Aspergillus niger with higher α-glucosidase activity and the effect of glucoamylase on α-glucosidase activity was studied.In this paper,homologous recombination method was used to delete the glucoamylase gene and obtain the mutant.Two 1000 bp regions located upstream and downstream of the glucoamylase sequence were amplified from Aspergillus niger genomic DNA.The nourseothricin resistant marker was amplified from the p BC-Nours.R vector.Deletion cassette of α-glucosidase gene was constructed by overlap PCR,and the transformant with nourseothricin cassettes was obtained by protoplast transformation and selected on mediumcontaining 125 μg/m L nourseothricin.The positive transformants were further screened by PCR.And the phenotypic analysis was carried out,including determination of the growth situations of mycelium,the activities of α-glucosidase and glucoamylase,the relative expression quantities of α-glucosidase gene on mutant strain of Aspergillus niger,and the transcosylation of α-glucosidase.Based on the analysis of the mutant strain ΔGA,the glucoamylase was inactivated,and the rate of growth of the ΔGA on the culture medium was slowed down in a certain extent.But there were no apparent defects in the growth development,such as colony phenotype,morphology and pigmentation.In addition,enzymatic assays on the original strain and the mutant suggest that α-glucosidase activity of the ΔGA was increased by 63.08% compared with original strain.The ΔGA mutant lost partly glucoamylase activity,glucoamylase activity decreased by 21.14%.The expression level of α-glucosidase gene in ΔGA was 2.53 flod higher than that of the original strain by RT-q PCR.And these results suggest that glucoamylase gene deletion is conducive to increase not only the activity of α-glucosidase but the expression quantity level of its.Determination of transcosylation of ΔGA mutant and original strain by high-performance liquid chromatography.The concentration of isomaltose increased by 2.00 flod and the concentration of panose increased by 1.59 flod,indicating that the deletion of the glucoamylase gene beneficial to increase of the concentration of the transglycoside products.In conclusion,glucoamylase gene was deleted in this study,while the mutant strain ΔGA was obtained.The results indicated that glucoamylase had a negative effect on the activity ofα-glucosidase.The ΔGA can be better to gain higher α-glucosidase activity by deleting the glucoamylase gene in original Aspergillus niger,and provide a α-glucosidase producing strain with a useful value in industry. |
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