| Glucoamylase, an extracellular enzyme with exoglucohydrolase activity,catalyzes the hydrolysis of ?-1,4 glycosidic bond from the non-reducing terminal of starch successively. Glucoamylase has been widely utilized on industry, such as the fermentation of glucose, amino acids, antibiotic, etc. Most glucoamylase from Aspergillus niger is able to hydrolyze over 80% of starch considering the characteristics of safety, high activity and high-yield, hence Aspergillus niger has been chosen as the starting strain in this study. On the other hand, ?-glucosidase was generated in the same process accordingly. It may hydrolyzes terminal non-reducing?-1,4 glycosidic bond, breaks down starch to ?-glucose, or transfer glucose residues to other sugar substrates for forming ?-1,6-glucosidic bond, thus nonfermented isomalto-oligosaccharide, glycolipids and glycopeptides were produced. Competition with ?-glucosidase means less substrates and limited hydrolysis efficiency for glucoamylase. Deletion is a method which makes ?-glucosidase no longer to be a concomitant product, and industrial costs on removing ?-glucosidase will be reduced with deletion of ?-glucosidase gene. It may contribute to design new research methods on the modification of Aspergillus niger for better efficiency and high-yielding glucoamylase.Split-Marker method was used in this paper for gene deletion. In the case of?-glucosidase gene, a nourseothricin resistance cassette was used. In the first round of polymerase chain reaction(PCR), upstream segment ?-gluE1(995 bp) and downstream segment ?-gluE2(1 013 bp) were amplified from ?-glucosidase gene of Aspergillus niger HE01, while the selective marker gene of nourseothricin resistance(NR, 1 776 bp) was amplified from the vector of pBC-Nours.R similarly. Then,deletion cassettes of ?-glucosidase gene were constructed through each segments that from ?-glucosidase gene fused to the marker gene by overlap PCR. Aspergillus nigerHE01 protoplasts were transformed by the resulting PCR products with nourseothricin cassettes. As a result of homologous recombination, ?-glucosidase gene has been replaced by selective gene. Transformants were selected on medium containing 125?g mL-1 nourseothricin. Mutants were selected by PCR. The PCR amplified the junctions specific to the replaced locus, using two primers that annealed at an end of the selective resistance gene and upstream of the proximal flank used in the deletion cassette. Homologous recombination of the deletion cassette allowed amplification of a predictable fragment on each side of the selective resistance gene. After that,phenotypic analyses including determination of the growth situations of mycelium,the activities of glucoamylase and ?-glucosidase, and the expression quantities of glucoamylase gene on mutant strain(??-glu) of Aspergillus niger, has been investigated.15 transformants were obtained from the selective medium plates, and 10 of them had been verified to be positive with 66.67% conversion rate. Difference was found in ??-glu, mutants developed a mycelium that extended longer on sucrose medium plates than starting strain. But there was no significant difference of starting strain and ??-glu in the ability to degrade soluble starch on soluble starch medium plates. Fungi biomass of starting strain was heavier than ??-glu in sucrose and soluble starch medium of liquid, respectively, which indicates the hypha of starting strain grew more quickly than ??-glu on soluble starch medium of liquid.Shake-flask fermentation was conducted in this paper. Highest activity of glucoamylase in ??-glu was 21.06% higher than in starting strain, with number of7 437.73 U mL-1 and 6 143.42 U mL-1 respectively. In contrast with these results, the activity of ?-glucosidase in above-mentioned two strains were distinguished, the former one is 72.31% lower than the latter, with number of 573.54 U mL-1 and2 068.59 U mL-1 respectively. Additionally, we developed a PCR-based detection system to measure the expression quantity of glucoamylase. And these results suggest that ?-glucosidase gene deletion is conducive to increase not only the expression quantity level of glucoamylase but the activity of its.In conclusion, ?-glucosidase gene was deleted from Aspergillus niger HE01 successfully in this study, while the mutant strain of Aspergillus niger HE01 was obtained. Deletion of ?-glucosidase gene eliminates the process of removing?-glucosidase in the production of glucoamylase in Aspergillus niger, and saves the cost of glucoamylase purification. Moreover, the deletion of ?-glucosidase gene in Aspergillus niger HE01 provided an opportunity for strain producing more pure and effective glucoamylase in factories. |
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