Cloning And Expression Of Protease And Lipase And Their Application As Cleaning Agent For Dairy Processing Equipment

Milk fouling is a kind of dairy product contaminant which deposits on the surface of processing equipment and pipes due to the adhesion between protein,fat and calcium phosphate[Ca3(PO4)2].If not complete cleaning,it is easy to cause the decreasing heat transfer efficiency of processing equipment,cross-contamination of products,affecting product quality.At present,strong acid and strong alkali combined with CIP(Clean in Place)cleaning system is widely used in dairy industry.However,strong acid and strong alkali are easy to remain and not friendly to the environment.Furthermore,HCl and NaOH have poor safety and the high energy was consumed for cleaning at high temperature.This study aims to clone and express target protease and lipase genes,detect their enzymatic properties and then screen out candidate enzymes with high activity.Our study attempt to improve the heat resistance and acid-base tolerance of enzymes through rational design and semi-rational design,and combine them with surfactants and functional additives in a proper proportion,so as to develop an enzyme preparation that is environmentally friendly,low in energy consumption and low in cost to replace part of strong acids and alkalis and be used for cleaning ultra-high temperature instant sterilization dairy processing equipment and conveying pipelines.This study is mainly divided into the following four parts:1.Take samples from a dairy co.,LTD.,and obtain milk fouling.The composition of milk fouling and commercial enzyme preparation cleaning agent was analyzed.The main components of milk fouling are protein,fat and minerals,of which Ca and P are the main minerals.2.The protease gene was amplified from Bacillus licheniformis genome and cloned and expressed in E.coli.The enzyme activity of SPr-1490 was 173.6 U/mL.The lipase gene was amplified from Nocardia sp.LMB-7 genome and cloned and expressed in E.coli.The enzyme activity of Lip-302 was 72.5 U/mL.The optimum temperature of recombinant protease was 40℃,and the optimum pH of recombinant protease was 10.0.SPr-1490 maintained a good activity in the alkaline environment(pH 9.0-11.0).Mg2+ and Al3+ activate it,while Zn2+and Mn2+inhibit it.3.Through homologous modeling and PoPMuSiC,B factor analysis,and domain analysis,mutation sites were screened to improve the heat resistance and acid-base tolerance of enzymes.The thermal stability of mutants without loss of enzyme activity(E294H,E271R,A45P,V46P,G252P)was verified.Of these mutations,the A45P mutant enhanced the half-life time at 40℃,from 43 to 50 min.4.The selected protease and lipase as well as the commercial glycosylase were combined with NaOH and enzyme in an appropriate proportion,In the experiment of single enzyme,the OD600 of protease,lipase and glucoamylase was 2.24-fold,2.05-fold and 1.93-fold of that of the control group,respectively.Protease achieved the best effect.And the cleaning effect of the enzyme on milk fouling was preliminarily verify at the laboratory level and further verified with a small UHT device.