Breeding And Characterization Of Milk Clotting Enzyme From Bacteria

Milk clotting enzyme（MCE）is a key enzyme that curdles milk to make cheese and plays an important role in food and dairy processing.The properties of MCE can affect the efficiency of cheese making and the quality of cheese（such as texture,flavor,etc.）.Traditional MCE,such as calf rennet,lamb rennet and camel rennet,mainly come from animals.They are restricted by human ethics,growth cycle and production cost,which are not good for expanding production.Therefore,it is becoming a focus of attention to find alternatives to traditional MCE.The purpose of this study is to isolate and identify a wild strain with the potential of curding,optimize the fermentation conditions for MCE producing,explore the physicochemical properties of MCE and the differences of hydrolyzed casein.Finally,amino acid sequence of MCE was obtained by N-terminal sequencing.The specific research content and results are as follows:（1）From the soil samples of Northeast dairy farm,a strain that could form a larger sedimentation circle and a small hydrolysis circle on casein plates was screened.Identified by physiological and biochemical and 16S r DNA,it was determined to be Bacillus velezensis and named DB219.Fermented in bran basal medium for 36 h,the milk clotting activity（MCA）of DB219 MCE was 754±13 SU/m L and the proteolytic activity（PA）was 82±6 U/m L.Therefore,DB219 is a strain with curd potential.（2）The enzyme production of Bacillus Velezensis DB219 MCE in 250 m L shake flask was optimized.Firstly,the single factor method was used to optimize the fermentation medium（bran concentration,type and concentration of carbon and nitrogen sources）and fermentation parameters（inoculation amount,liquid loading amount and initial p H）.Three factors that had the greatest influence on DB219 MCE yield were selected by Plackett-Burman design,and their ranges were determined using the steepest ascent/descent path.The Box-Behnken design was used to perform a response surface analysis experiment with three components and three levels.The following were the theoretical ideal fermentation conditions for DB219 MCE:wheat bran concentration 60.14 g/L,soluble starch 12.5 g/L,corn steep liquor 3 g/L,inoculum size 5%,volume 40.08 m L and initial p H 6.15.DB219 MCE achieved the maximal MCA（3164.84 SU/m L）that was 101.9%of the predicted value（3104.49SU/m L）and 4.3-fold higher than the control.（3）DB219 MCE exhibited a notable specific activity of 6110 SU/mg and3.16-fold purification yield with 28.87%recovery through ammonium sulfate fractionation and DEAE-Sepharose Fast Flow.The purified DB219 MCE was metalloproteinase and about 36 k Da.DB219 MCE was weak acid resistance and stable at p H 6.0–10.0 and temperature<45°C.The highest MCA appeared at substrate p H 5.5 and 20–30 m M of Ca Cl₂.The K_mand V_mfor casein were 0.31 g/L and 14.22μmol/min.DB219 MCE preferred to hydrolyzeβ-casein（β-CN）compared toα-casein（α-CN）.DB219 MCE hydrolyzedα-CN,β-CN andκ-casein（κ-CN）to generate significantly different peptides in comparison with calf rennet and ES6023MCE through SDS-PAGE and RP-HPLC analysis.DB219 MCE mainly cleaved Thr124-Ile125 and Ser104-Phe105 bonds inκ-CN and had unique casein cleavage sites and peptide composition through LC-MS/MS analysis.After sequencing and comparison,DB219 MCE has 521 amino acid residues and a mature peptide molecular weight of about 33 k Da,which is a novel M4 metalloproteinase.