| Protease is a kind of biological catalyst can catalyze the hydrolysis of peptide bonds,often used as additives are used in washing products.In this paper,the DNA fragment was found in yeast strain yeryeyeh yeast strain(W29),and the cDNA sequence of two possible proteases was cloned from yeasts by reverse transcriptional PCR.After joining the pPICZα vector,it was injected into Pichia pastoris GS115 and X33 to express the activity,and a total of four recombinant proteins were synthesized and the protease producing strain GS115/pPICZ a-AXP was obtained.The fermentation conditions were optimized from culture time,carbon source,culture environment pH and inoculation quantity.The fermentation conditions were optimized from culture time,carbon source,culture environment and inoculation quantity.The recombinant Pichia pastoris cells were expanded in 3L triangle bottle,the fermentation liquid was concentrated by ultrafiltration,and the enzyme solution was granulation by fluidized bed granulation technology.The influence factors of granulation were investigated,and the properties of the particles were investigated.Compare the test and explore the best means of granulation.The main contents and results are as follows:(1)the recombinant Pichia pastoris type GS115/pPICZα-AXP was synthesized by exogenous vector,and the strain of recombinant Pichia pastoris type GS115/pPICZα-AXP strain was prominent on the plate culture medium,and the multiple copy transformants were screened by high concentration Zeocin(1000 g/ mL),and the enzyme activity produced by the initial fermentation of recombinant Pichia pastoris was 18.9U/mL.(2)P.pastoris GS115/pPICZα-AXP was fermented by shaking flask culture,and methanol was used as carbon source and inducer.The recombinant strain was 2% in methanol concentration,6.5 in the initial environment,6.5 in the initial environment and 6% when the inoculation amount was 6%,and the maximum enzyme activity was 39.87 U/mL after 120 h.With glycerol as carbon source,the recombinant strain was 1.8% in glycerol concentration,7 in the initial environment pH and 4% when the inoculation amount was 4%.The fermentation broth of 120 h reached the highest enzyme activity 110.67U/mL.The decontamination effect of protease produced by the recombinant Pichia pastoris cell fermentation was preliminarily studied.The decontamination ability of the fermentation liquid was determined from the whiteness difference before and after washing,the greater the mass fraction of the protease,the better the decontamination effect.(3)the recombinant Pichia pastoris cells were cultured in the 3L triangle bottle,and the fermentation liquid was concentrated by ultrafiltration,and the enzyme activity of the concentrated liquid was 366.67U/mL.The influence of some factors on protease granulation was explored from prescription and technology.On the prescription,we choose three kinds of commonly used granulating agents to study.The results showed that when the corn starch was dextrin: polyethylene glycol was 1:1:1,the quality of extruded granules was the best.The coating experiment was carried out with three factors: the dosage of adhesive,the content of the filler and the content of protease.The orthogonal results showed that the capacity of the three factors on the enzyme activity retention rate was: the content of protease > filler content > adhesive content,the best proportioning ratio was binder: filling agent: protease =7:12:14.The fluidized bed coating experiment showed that the coating selection was titanium dioxide and Magnesium Sulfate,Arabia gum,dextrin and polyethylene glycol from inside to outside,and the particle properties were the best.The influence of extrusion speed and fluidization temperature on granulation was explored.The optimum technological parameters were obtained: extrusion speed 30 Hz and fluidization temperature 75 degrees.(4)the fermentation broth was treated with fluidized bed granulation.Compared with the original enzyme solution,the granulation treatment of protease reduced the loss of enzyme activity and greatly enhanced the thermal stability.The qualified particle 20-40 mesh(0.425-0.85mm)accounted for 97.29%,and the qualified rate of the product was high. |
