Abstract
Paenibacillus spp. BD3526, a bacterium exhibiting a protein hydrolysis circle surrounded with an obvious precipitation zone on skim milk agar, was isolated from raw yak (Bos grunniens) milk collected in Tibet, China. Phylogenetic analysis based on 16S rRNA and whole genome sequence comparison indicated the isolate belong to the genus Paenibacillus. The strain BD3526 demonstrated strong ability to produce protease with milk clotting activity (MCA) in wheat bran broth. The protease with MCA was predominantly accumulated during the late-exponential phase of growth. The proteolytic activity (PA) of the BD3526 protease was 1.33-fold higher than that of the commercial R. miehei coagulant. A maximum MCA (6470 ± 281 SU mL−1) of the strain BD3526 was reached under optimal cultivation conditions. The protease with MCA was precipitated from the cultivated supernatant of wheat bran broth with ammonium sulfate and purified by anion-exchange chromatography. The molecular weight of the protease with MCA was determined as 35 kDa by sodium dodecyl sulfate-polyacrylamide gels electrophoresis (SDS-PAGE) and gelatin zymography. The cleavage site of the BD3526 protease with MCA in κ-casein was located at the Met106–Ala107 bond, as determined by mass spectrometry analysis.
Highlights
Used as a milk coagulant, calf rennet plays a critical role in the production of cheese.Chymosin (EC3.4.23.4), a predominant aspartic protease (AP) in calf rennet, cleaves thePhe105 –Met106 peptide bond in the κ-casein (κ-CN)
YB-3 [27] and B. subtilis natto [28], but much shorter than those required by B. amyloliquefaciens the peak milk-clotting activity (MCA) by BD3526 was longer than those required by Enterococcus faecalis TUA2495L [26], JNU002 [17], B. subtilis [22], B. licheniformis USC13 [29], and fungal milk-clotting protease producers
[34], whichhighest MCA was observed at 300 rpm (4120 ̆ 174 Soxhlet unit (SU) mL ), the upper limit of the rotary shaker indicated oxygen supply should be crucial for enzyme production by BD3526 in liquid cultures
Summary
Used as a milk coagulant, calf rennet plays a critical role in the production of cheese. Decrease in the global supply of calf rennet versus the increasing demand of coagulant in the production of cheese necessitates the exploration for potential substitutes [1]. Few commercial applications of bacterial coagulants have been reported, despite a 73 marked increase in the documentation of bacterial proteases exerting MCA. Few reports on milk-clotting enzymes expressed by members of the genus Paenibacillus. BD3526, was screened successfully applied to the preparation of cheddar and cream cheese [19,20]. The parameters of the BD3526 protease with MCA in milk curding was assessed few reports on milk-clotting enzymes expressed by members of the genus Paenibacillus. In this with study,some a potentially new coagulant producer, Paenibacillusconditions spp. The cleavage site of the BD3526 protease in κ-CN was determined by mass spectrometry analysis
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