| The kitchen waste ethanol fermentation mash is one of the wastes derived from the process of ethanol fermentation of kitchen waste,which contains a large amount of protein.If it is directly discharged,the kitchen waste fermentation mash is easy to cause environmental pollution.If the protein in the mash is converted into an amino acid feed,it can solve the pollution and generate economic benefits.Therefore,this study focused on the construction of a recombinant Candida utilis with the abilities to secreting exogenous proteases,as well as the optimization of the usage amount ratios of proteases for fermentation mash hydrolyzing,which will provide a reference for future work applications.Comparing the growth conditions of Pichia pastoris,Saccharomyces cerevisiae and C.utilis in a low-pH and high-concentration of protease environment,we chose C.utilis as the microbial model to express the exogenous protease.The protease genes from different sources were integrated into C.utilis.After comparing the activities of recombinant protease,the carboxypeptidase pepF,endoprotease pepA and aminopeptidase YPDF genes with the highest enzyme activity were selected.After optimizing the promoter,the promoter was transferred.Summarily the recombinant enzyme activities of YPDF,pepF and pepA were 8.48 U/mL,12.58U/mL and 17.14 U/mL,respectively.After being co-expressed in C.utilis,the obtained recombinant enzyme activity was 14.84 U/mL.Proven its optimum pH value was between 3-4,the recombinant enzyme could adapt to the acidic environment.To further explore the processing of fermentation mash hydrolysis,the optimum synergistic ratio of the three recombinant enzymes was tested.It was found that the combination of endoproteinase pepA and carboxypeptidase pepF had the highest hydrolysis efficiency,and the amino acid yield at 60 h was 23.85 mg/ml.The rate of proteolysis was 51.2%,which was equivalent to 82.2% when using 0.2 g/g commercial protease. |
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