Expression Of Lipase Gene From Aspergillus Oryzae In Aspergillus Niger

Filamentous fungi are widely used for the efficient expression of exogenous proteins for its unique advantages which is different from bacteria and yeast:it has a strong exocrine ability of protein,which is similar to that of higher eukaryotes,and can carry out various post-translational processing correctly.Among them the Aspergillus niger become a hotspot in the research for it have high security and can be applied in the field of food and drug.One of the important sources of the Industrial lipase is the lipase from microbial,the lipase gene aol?Gen Bank:KP975533?of one strain of Aspergillus oryzae strain WZ007 screened by our laboratory has been successfully expressed in the expression system of escherichia coli and Pasteur pichia pastoris.To further improve the expression of the lipase gene of Aspergillus oryzae,this subject attempted to use agrobacterium-mediated fungal expression system to achieve exocrine expression in Aspergillus niger and its Enzymology properties were studied,the research results are as follows:1.Construction of Aspergillus niger engineering bacteria expressing Aspergillus oryzae lipase gene.Firstly,the aol target gene of Aspergillus oryzae lipase was connected with the p CAMBIA-Pgpd A-tcbh1-hph-ptrpc vector and transformed into E.coli DH5?,and positive transformant was screened.The transformant was plasmid extracted and transformed into agrobacterium AGL-1 competent cells after verification by sequencing,and the positive transformant was identified by PCR.The suspension of Aspergillus niger spores was cultured with agrobacterium for 48 h at22?,then transferred to the hydomycin B resistance screening medium to acquire the Aspergillus niger whose genome was extracted and identified with PCR.Through the enzyme activity assay after the fermentation of Aspergillus niger engineering bacteria,the result shows that Aspergillus niger engineering bacteria AN-aol been constructed successfully.2.The optimization of fermentation conditions for Aspergillus niger engineering bacteria.First,single factor optimization was carried out on the fermentation conditions for the expression of lipase in Aspergillus niger.With the shaking flask fermentation conditions for 31?as the fermentation temperature,inoculation quantity of 7%,initial p H 7.0,shaking flask fluid volume is 110 m L?500 m L flask with damper?,shaking table speed of 180 r/min,with glucose as the carbon source whose content is 40 g/L,peptone and yeast powder compound as the nitrogen source,no addition of inorganic salt,induced using olive oil for fermentation,the enzyme activity reached 217.6 U/m L which is 1.1 higher than the results without optimization before.3.The research of isolation and purification of recombinant lipase AOL.Using highspeed freezing centrifugation to acquire the supernate from Aspergillus niger fermentation broth,then 10 k Da ultrafiltration concentration was performed,and purification was performed by anion exchange chromatography and hydrophobic chromatography,the volume of enzyme liquid was 100?L,the protein concentration was 13 mg/m L,the total protein content was 1.3 mg,the total enzyme activity was163.42 U,and the specific enzyme activity was 125.71 U/mg.The purification factor was 65.7 times and the recovery rate of enzyme activity was 10.32%.A single stripe could be seen in the SDS-PAGE which have lipase activity proved by the reactive staining.It indicated that the target protein was separated and purified successfully to electrophoretic purity.4.The research of enzymatic properties of recombinant lipase AOL.The optimum temperature and p H of lipase were studied.The results show that the optimum temperature of lipase was 40?,and it has better thermo stability within30-45?;the optimum p H for the enzyme was 8.0.AOL substrate specificity:The p-nitrophenyl?p NP?acyl esters with different acyl chain lengths from C2 to C16 all had certain hydrolysis activity,and the substrate specific hydrolysis activity of p NP-butyric acid?C4?was the highest.It can be seen from the results of influence experiment of metal ions that they have no obvious activation on the activity of AOL,among them the Cu²⁺and Zn²⁺have distinct inhibitory effect on AOL,while it has better tolerance of Ca²⁺?Mg²⁺and Mn²⁺which almost have no inhibitory effect on AOL.The results from influence experiment of organic solvent shows that AOL can be activated during the existence of both Isooctane and n-hexane.The dynamic parameters of AOL were measured and the K_mand V_max value from Lineweaver-Burk figure were 3.03 m M and 11.40 m M/min/mg separately.This paper increases the fermentation enzyme activity unit of the strain and the enzyme activity reached 217.6 U/m L through the successful expression of Aspergillus oryzae lipase achieved by Aspergillus niger expression system and the optimization of fermentation conditions.And the isolation and purification as well as the enzymatic properties of recombinant Aspergillus oryzae lipase were also carried out.The research of this paper provides some technical guidance to the industrial manufacture of recombinant Aspergillus oryzae lipase.