Characterization Of The Milk-clotting Enzyme From Paenibacillus Bovis Sp. Nov.

Calf rennet, as a traditionally used milk coagulant, plays a critical role in the production of cheese. The use of calf rennet in cheese-making dates back to approximately 6000 BC, and it is one of the earliest exogenous enzymes applied in food processing for human beings. Chymosin(EC3.4.23.4), a predominant aspartic proteinase(AP) in calf rennet, specifically cleaves the Phe105–Met106 peptide bond in the κ-casein(κ-CN). After cleaving κ-CN, the destabilized casein micelles consequently aggregate to form cheese curd. The increment in the worldwide production of cheese, concomitant with a reduced supply of calf rennet, led to the scarcity and high price of calf rennet. In addition, religious concerns(e.g., Islam and Judaism), diet(vegetarianism) and ban on recombinant chymosin have encouraged the research on rennet substitute. Microorganism proteinases, especially the aspartic proteinases from fungi exerting the same or similar mechanism as chymosin, have been produced in large sacle and applied in cheese-making. However, reports on bacterial proteinases with milk clotting activity(MCA) are sporadic. In this study, a novel candidate of coagulant producer, BD3526 was screened, identified and named. The cultivation parameters for milk clotting enzyme(MCE) production by the strain in wheat bran both were optimized. After purification, the characteristics of the enzyme were studied. The amino acid sequence and encoding gene of its zymogen were determined by peptide mass fingerprinting(PMF) and whole-genome sequencing. The main results are described as follows:(1) Based on the casein precipitate zone and hydrolysis circle around the colony on the skim milk agar, a potential MCE producing strain BD3526 was isolated from the raw yak milk collected in Tibet, China. The MCA and proteolytic activity(PA) in the supernatant reached 2422.09±68.35 SU/mL and 0.40±0.01U/mL, when BD3526 was cultivated in the 30 g/L wheat bran broth at 30°C and 180 r/min for 48 h. At the same level of MCA, the PA of the BD3526 MCE was comparable to that of Rhizomucor miehei coagulant, and was mildly stronger than that of calf rennet and recombinant chymosin, which indicating the BD3526 MCE was able to serve as a coagulant without excessive proteolysis. BD3526 was identified as a novel species belonging to the genus Paenibacillus and named as Paenibacillus bovis sp. nov.(CGMCC No.8333 = DSM No.28815 = ATCC No.00318), based on the results of biochemical analysis, 16 S rRNA sequencing, total cellular fatty acid content, mean DNA G+C content, homologous hybridization and whole genome sequencing.(2) The cultivation conditions for MCE production by BD3526 were optimized using the single-factor experiment and response surface methodology(RSM). The optimal conditions for BD3526 to produce MCE in shaking-flasks were 30 mL of 30 g/L wheat bran broth with an initial pH 6.0 in a 250 mL Erlenmeyer flask, inoculated with 5%(v/v) seed, cultivated at 300 r/min(the upper limit of the rotate shaker) and 30°C. Three factors(temperature, dissolved oxygen and time) had significantly influences on the enzyme production. Maximum MCA was achieved to 6733.81±663.73 SU/mL, and the time of which was shortened to 18 h by RSM. In 7-liter scale fermentor, the MCA were enhanced as the agitation speeds(200–400 r/min) and aeration rates(3.5–5.5 L/min) increased. Maximum MCA(14777.78±1099.94 SU/mL) and MCA/PA(32999.34±918.48) were achieved when cultivation was carried out at agitation speed of 400 r/min, aeration rate of 5.5 L/min and flowed with 500 mmol/L hydrogen peroxide(H2O2) after 24 h cultivation. The average flow rate of H2O2 was 2.5 mL/h.(3) The obstruction of the enzyme salting out from culture supernatant with exopolysaccharide(EPS) was overcome. The synthesis of EPS by BD3526 had a positive correlation to the dissolved oxygen, and the EPS was synthesized first and then depleted. The enzyme was successfully precipitated with 60% saturated ammonium sulfate when the concentration of EPS decreased to a much lower level. The BD3526 MCE was a 35 kDa neutral zinc metalloproteinase, and had a tendency to cannibalistic autolysis, which could be inhibited by calcium. The optimal p H of the enzyme was 7.0, and the optimal temperature for MCA and PA was 65°C and 60°C, respectively. The MCA was completely inactivated by heating at 60°C for 30 min, and the PA was totally inactivated for 20 min at 55°C or 10 min at 60°C. The Km and Vmax of the enzyme for casein was 1.36 g/L and 0.017 μmol/min at pH 6.0 and 35°C. The BD3526 MCE was preferentially active towards κ-casein and β-casein, analyzing by sodium dodecyl sulphate-polyacrylamide gels(SDS-PAGE), whereas the hydrolysis of αs-casein was slow and comparable to recombinant chymosin and asparatic acid proteinase from R. miehei. The cleavage site of the enzyme in κ-CN was at Met106–Ala107 bond, which was different from that of chymosin(Phe105–Met106).(4) The amino acid sequence and encoding gene of the BD3526 MCE zymogen were determined by PFM and whole-genome sequencing. The zymogen of the enzyme was encoded by a gene named zinc metalloproteinase precursor 1(zmp1)with 1671 bp. N-terminal amino acid sequencing of the mature enzyme showed that the cleavage at His252–Ala253 peptide bond was responsible for converting zymogen into active enzyme. Sequence alignment revealed the presence of two highly conserved motifs, HEXXH and GXXNEXXSD, indicating that the enzyme is a new member belongs to metalloprotease family M4(thermolysin-like proteinases, TLPs). Interestingly, the three residues to form calcium binding site 3(Ca3) in the BD3526 TLP were obviously different from the aligned TLPs, i.e. the Asp57 and Asp59 both mutated to Ser, and V61 or Q61 changed to Thr, which might be the implication for the weak calcium affinity and calcium-dependent stability of the enzyme. The three-dimensional structure of the BD3526 MCE was predicted to mainly contain two domains. The N-terminal domain contains a series of β-strands that cradle one long α-helix, whereas the C-terminal domain is mainly α-helical and carries the terminal four-helix bundle. The C-terminal and N-terminal domains are connected by a central α-helix containing several catalytically crucial residues.