Cloning And Expression Of Pork Lipase Gene And Its Application In Food

Lipase can hydrolyze fats and form flavor precursors such as fatty acids.It is a widely used catalyst.Early laboratory research results show that the flavour of pork endogenous lipase for meat and dairy products were improved,in order to further verify the lipase function,in this paper,the lipase gene LPL was cloned from pork and expressed in E.coli.The effect of recombinant lipase on pork,dairy products and starch was investigated.The mechanism of the effect of pork lipase on food flavor was analyzed from the molecular level and enzyme level,to provide new ideas for the industrial production of lipase.The research results are as follows:1.According to the LPL gene sequence contained in the GenBank database,the LPL gene was amplified by RT-PCR.The sequencing result are aligned by BLAST,the homology is 99%,and the LPL gene is 1756 bp,encoding 478 amino acids.The constructed recombinant plasmid pET30a-LPL is successfully expressed in E.coli.The recombinant strain reach the maximum expression level when the concentration of IPTG is 0.4 mM,the induction time is 5 h and the effect of temperature on expression is small.Purification by nickel column affinity chromatography gave a single target band,which is consistent with the theoretical molecular weight of the predicted recombinant protein of 53.58 kD.The recombinant protein concentration is 189 ?g/mL and the enzyme activity is 0.932 U/mL.The recombinant enzyme pH stability is good,the optimal pH and optimum temperature at pH 8.0 and 40? respectively,and the low concentration of methanol and glycerol,recombinant enzyme has good tolerance.Electronic nose combined with GC-MS to detect pork flavor,a total of 36 volatile flavor substances are detected,mainly hydrocarbons,heterocyclic and aromatic compounds,organic acids and esters,aldehydes,ketones,alcohols,etc.The relative content of the recombinase and the endogenous enzyme treated pork aldehyde flavor component increased,and the effect of the recombinase is better than the endogenous enzyme.2.Recombinant lipase and endogenous pork lipase are respectively applied to pork,and the fat hydrolysis oxidation index is measured.The results show: the peroxide value,thiobarbituric acid value(TBA),carbonyl value and diene value of the lipase treatment are higher than those of the blank control group,indicating that the recombinant enzyme has a similar effect to endogenous lipase.Hydrolysis oxidation has a positive effect.It is further indicated that the recombinant enzyme can promote the increase of pork flavor and enhance the flavor of pork.3.Electronic nose detection adds significant changes to the flavor of dairy products after adding endogenous enzymes and recombinase.The volatile components in dairy samples by GC-MS are mainly organic acids and esters,hydrocarbons,aldehydes and ketones,and heterocyclic and aromatic substances.The relative content of volatile substances such as 2-undecone,2-heptanone and 2-anthrone in milk samples is significantly improved.It indicates that the recombinant enzyme can enzymatically hydrolyze milk fat to produce flavor substances,and effectively improve the flavor of dairy products.4.Taking starch as an example,the effects of recombinant lipase and endogenous lipase on the hydrolysis of polysaccharides are investigated.When the enzyme dosage of endogenous enzyme is 2.0 mL,the starch concentration is 6%,the reaction temperature is 50°C,and the pH value of the system is 6,the hydrolysis degree of starch is the highest,and the glucose content is 1.01 mg.When the enzyme addition amount of the recombinase is 2.0 mL,the starch concentration is 6%,the reaction temperature is 55°C,and the pH value of the system is 8,the hydrolysis degree of the starch reached the highest,and the hydrolysis of the glucose content is 1.32 mg.