Study On The Development And Utilization Of Collagenase And The Function Of Collagen Polypeptide

With the development of modern productivity,people's living standards have been steadily improved,but there are also many hidden problems.On the one hand,due to the meat consumption,million tons of underutilized animal skeleton,innards and skin which are rich in collagen are produced,and have not been fully utilized;on the other hand,the market looks forward to developing more healthy food products to further provide endogenous sources for improving the quality of life power.It was reported that the market share of health care products in the United States reached 42 billion dollars in 2018,and according to the reports of the China Business Industry Research Insititute,the market share of health care products in China exceeded 350 billion yuan in 2019 as well.The use of collagen-rich "trimmings" to prepare functional collagen peptide products not only improves resource utilization,but is also an effective measure to turn waste into treasure and meet market demand.In this paper,the enzyme decomposition method was used to hydrolyze the collagen,the tool enzyme was cloned from Bacillus cereus and expressed successfully,and the prepared functional peptides were evaluated for efficiency,separation,purification and structure identification.,the main research results were as follows:(1)The level of collagenase secretion of Bacillus cereus was investigated,Three pairs of primers were designed using NCBI GenBank homology analysis and two sets of different collagenase genes were cloned successfully:LYCol-1 and LYCol-2.Next,in order to investigate the compatibility of the plasmid with the engineering bacteria system,four sets of different recombinant plasmids were constructed by combining the pET-28a and pETDuet-1 plasmids which were stored in the laboratory,and combined with the rare codon analysis of the target gene,it was decided to take the E.coli Rosetta(DE3)as chassis cell.Through SDS-PAGE analysis showed that pETDuet-1 and E.coli Rosetta(DE3)had good adaptability.(2)In order to further screen for better collagenase,by measuring the Mie constant and other kinetic parameters,it was found that the comprehensive performance of LYCol-2 was better,its Km=40.34 mg/mL,Vm=2.36 U,kcat/Km=0.46 mL/(mg·s)and kcat=18.63/s;the optimal temperature of the collagenase was 37? and the optimal pH was 7.0,it had good tolerance to alkali solution and Co2+,Ba2+,Na+,K+,Mg2+Ca2+,non-ionic surfactants and serine protease inhibitors,but the addition of metal chelating agents had a negative effect on their activity,indicating that they belong to a class of neutral metal protease.(3)The properties of the tested collagen substrates were investigated,and it was found that the N content accounted for 1/6,the substrates were aggregated,had the corresponding characteristic absorption peaks of collagen,and the molecular weight was above 12 kDa.the collaegnase concentration,reaction time,pH and temperature were used as independent variables,and ABTS·,DPPH·radical scavenging activities and reducing power were used as response values to design RSM experiments to optimize collagen-soluble polypeptide(CPP)preparation conditions.The optimal preparation conditions of antioxidative peptides were determined as follows:collagenase concentration(X1)was 110.0 ?g/mL,reaction temperature(X2)was 35.0? pH(X3)was 8.0 and reaction time(X4)was 6.0 h.At the concentration of 40 mg/mL,the CPP sample prepared under this condition,ABTS·radical scavenging activity of 99.21±0.35%,DPPH· radical scavenging activity of 18.98±0.20%,and reducing power of 0.41±0.03 were obtained.(4)Using FPLC tandem superdex 75 10/300 GL gel chromatography column,successfully separating CPP-1(1.5 kDa?6.5 kDa),CPP-2(0.5 kDa?1.5 kDa)and CPP-3(?500 Da)three components,combined with radical scavenging activity index and in vitro cell antioxidative experiment(CAA),comprehensively screened the best antioxidative CPP-3 for peptide structure identification.Through RP-HPLC-MS/MS measurement,five antioxidative peptides not reported were found,which were rich in typical collagen "Gly-Xaa-Yaa(X,Y can be any amino acid)" structural unit,indicating the structure of the polypeptide has universal antioxidative capacity and has the potential for functional food development.