Molecular Modification Of Acidic Protease Based On Visualized Transformation System Of Trichoderma Reesei

The demand and quota for proteases are the highest in the global enzyme preparation market compared to other enzyme preparation products.Acid protease is a kind of protease preparation,which is very important for industrial processing.In order to obtain a large number of acid proteases and modify their properties,a system for the expression of exogenous genes in Trichoderma reesei was established.In order to rapidly and efficiently screen the transformation of Trichoderma reesei,a visual filamentous fungal expression vector was constructed based on the plant expression vector pCAMBIA2301 and labelled with hygromycin and Green fluorescent protein gene（GFP）.In order to improve the transformation efficiency,the neural network was used to optimize the transformation conditions of Exogenous genes mediated by Agrobacterium tumefaciens-mediated transformation（ATMT）.Finally,the expression of the proA acid protease gene was realized.In order to make proA acidic protease better meet the application of relevant industrial production,the rational design was carried out based on the existing properties and industrial production requirements,and finally obtained proA heat-resistant mutants and proA enzyme activity enhancing mutants.Specific research results are as follows:（1）Hygromycin B was selected as the resistance marker,and 200 mg/mL was selected as the antibiotic concentration through the tolerance test of Trichoderma reesei CICC40932.A visual screening vector with hygromycin resistance marker and green fluorescent protein marker was constructed based on plant expression vector pCAMBIA2301,and fluorescence excitation of positive transformants was successfully achieved under the excitation light field.The transformation conditions of Trichoderma reesei mediated by ATMT method were classified and optimized.The species of agrobacterium used in ATMT method and co-culture temperature were taken as a single factor optimization group.The concentration of acetosyringone,co-culture time,proportion of agrobacterium,pre-induction time of agrobacterium and initial p H of co-culture medium were optimized in the multi-factor optimization group.BP neural network and GA genetic algorithm were used to fit and optimize the transformation conditions.The agrobacterium species was AGL1,the co-culture temperature was 24.5 ℃,the concentration of acetosyringone was 225 μmol/L,the co-culture time was 52 h,the ratio of agrobacterium to liquid was 55%,and the pre-induction time of agrobacterium was six h.The initial p H of the medium was 5.4.After optimization,the maximum number of positive converters reached118 /106,and the lowest number was 96 /106.Compared with before optimization,the average increase was 30%.（2）Visual screening expression vector TR2301-CBH was constructed by expression box of endogenous gene Cbh1 of Trichoderma reesei,and acid protease gene proA was expressed in CICC40932.Trichoderma reesei was fermented,and the fermentation process was detected.Finally,under the condition of 27 ℃ and 150 r/min,the protease activity in fermentation broth reached the highest level after 144 h of fermentation.The enzyme activity reached 344.12±11.182 U/mL.（3）The thermal stability and enzyme activity of proA acid protease were modified based on the spatial structure analysis.In order to improve the thermal stability of proA,artificial disulfide bonds were introduced into THE G220-S290 region of proA.By software CAVER analysis,protein substrate channels were obtained to form residues.In order to improve proA enzyme activity,residues in protein channels were modified.The thermal stability of the mutant with disulfide bond was measured,and the residual enzyme activity of mutant S294C-S296 C increased by 15.6% after 30 min tolerance at 55 ℃compared with wild proA.The D77 G enzyme activity enhancing mutant was obtained by modifying the internal residues.Compared with the wild-type proA,the enzyme activity of the mutant was increased by 24% at 50 ℃ and p H 3.0.The enzyme activity of the D77 G mutant was 428.4±12.671U/mL.In summary,this study successfully constructed the visual screening vector for Trichoderma reesei.The expression of the acidic protease gene in Trichoderma reesei expression system was completed based on this vector.At the same time,the molecular transformation of acidic protease was carried out,which provided a reference for the study of expression systems and rational design of filamentous fungi.