Homologous Expression Of Asparaginase Gene In Aspergillus Niger

Asparaginase (asparaginase), also known as L-asparaginase, asparaginase, L-asparaginase, asparaginase, the enzyme can catalyze hydrolysis of aspartic acid and ammonia, widely exist in the animal, plant tissues and microorganisms. Animals asparaginase mainly exists in mammals and birds, pancreas, liver, kidney, spleen, and stomach.Asparaginase from Aspergillus niger is light yellow to brown liquid, or pale yellow to deep yellow granules or powder. Soluble in water, But in organic solvents which the chloroform, acetone is he insoluble. In solution at20℃storage for7days or14days of storage at5℃enzyme has activity.In this study, we put the Aspergillus niger CICC2462as t receptor material, the strain has top levels of protein expression, the ability of secretion is very strong, the activity of extracellular protease is low, sensitive to selectable marker of hygromycin etc. With gene replacement skill to be integrated into the gene specified high expression,which the endogenous gene glucoamylase (glaA) locus, the locus which is the active transcription, It is very simple to gene regulation, in this way, Not only can undock the endogenous expression of Glucoamylase Gene competition impact which the exogenous gene, but also can gain the efficient expression of exogenous gene, simplified engineering bacteria fermentation conditions, structure the gene replacement vector pSZHG-Asp and transformed the gene applying Agrobacterium-mediated transformation(ATMT).The method has high conversion efficiency, homologous recombination rate tall, good genetic stability etc. And through project forwardrepeat sequences on both sides of the hygromycin gene expression frame,so that dispel hygromycin marker gene transformants posterity,Achieve food-grade Asparaginase engineering strain. This research thinking for the domestic structre of asparaginase engineering strain,which offers a new way to afford a new expression system for the efficient and safe production of enzymes.The main results are as follows:1. Asparaginase gene cloning and amplificationusing CTAB to extract Aspergillus niger genome, then in the PCR clone the asparaginase gene, linked to T-vector, sequenced correctly.2. Construction expression vector of AsparaginaseMaking use of he same enzyme to deal with the correct sequence T-Asp carriers, pSZHG vector, then connect the construction of expression vector pSZHG-Asp.3. Agrobacterium mediated genetic transformation system of Aspergillus niger Making use of Agrobacterium-mediated genetic transformation of Aspergillus niger, were screened by colony PCR, positive conversion rate of56.14%.Then with Agrobacterium co-cultivation of Aspergillus niger,Theextracted genomic DNA as template with5’Gla upstream and3’Gla downstream primers for PCR detection, filtrating of positive transformation, the positive was30.16%. Homologous recombination with the design of primers for identification of transformants which the correct transformants of PCR confirmed, whether the identified homologous recombinants, obtained the homologous recombinants was92.45%. four strains in the PDA liquid or solid medium containing200mmol/L hygromycinB continuous passage, screening, using the primers of glucoamylase gene,which20colonies were identified by PCR.results showed three colonies with homozygous recombinants.4.Asparaginase gene expression in Aspergillus nigerAsparaginase with the fermentation medium for fermentation, using Berthelot color reaction to measure asparaginase enzyme activity, the activity up to20000U fermentation time21days. By SDS-PAGE electrophoresis, at approximately40kD has aim protein band, thus,asparaginase in Aspergillus niger has been secreted expression. Protein expression of recombinant strains of185μg/mL-417μg/mL.