title: High Milk-Clotting Activity Expressed by the Newly Isolated Paenibacillus spp. Strain BD3526.
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.
authors.affiliation: State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, China. fhang0427@126.com.|State Key Laboratory of Dairy Biotechnology, Technology Center and Dairy Research Institute of Bright Dairy &amp; Food Co. Ltd., Shanghai 200436, China. fhang0427@126.com.|Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi 214122, China. fhang0427@126.com.
authors.forename: Feng
authors.initials: F
authors.lastname: Hang
authors.affiliation: State Key Laboratory of Dairy Biotechnology, Technology Center and Dairy Research Institute of Bright Dairy &amp; Food Co. Ltd., Shanghai 200436, China. liupeiyi@brightdairy.com.
authors.forename: Peiyi
authors.initials: P
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Dairy Biotechnology, Technology Center and Dairy Research Institute of Bright Dairy &amp; Food Co. Ltd., Shanghai 200436, China. wangqinbo@brightdairy.com.
authors.forename: Qinbo
authors.initials: Q
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Dairy Biotechnology, Technology Center and Dairy Research Institute of Bright Dairy &amp; Food Co. Ltd., Shanghai 200436, China. hanjin@brightdairy.com.
authors.forename: Jin
authors.initials: J
authors.lastname: Han
authors.affiliation: State Key Laboratory of Dairy Biotechnology, Technology Center and Dairy Research Institute of Bright Dairy &amp; Food Co. Ltd., Shanghai 200436, China. wuzhengjun@brightdairy.com.
authors.forename: Zhengjun
authors.initials: Z
authors.lastname: Wu
authors.affiliation: State Key Laboratory of Dairy Biotechnology, Technology Center and Dairy Research Institute of Bright Dairy &amp; Food Co. Ltd., Shanghai 200436, China. gaocaixia@brightdairy.com.
authors.forename: Caixia
authors.initials: C
authors.lastname: Gao
authors.affiliation: State Key Laboratory of Dairy Biotechnology, Technology Center and Dairy Research Institute of Bright Dairy &amp; Food Co. Ltd., Shanghai 200436, China. liuzhenmin@brightdairy.com.
authors.forename: Zhenmin
authors.initials: Z
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, China. zhanghao@jiangnan.edu.cn.|Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi 214122, China. zhanghao@jiangnan.edu.cn.
authors.forename: Hao
authors.initials: H
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, China. chenwei66@jiangnan.edu.cn.|Beijing Innovation Centre of Food Nutrition and Human Health, Beijing Technology &amp; Business University, Beijing 100048, China. chenwei66@jiangnan.edu.cn.
authors.forename: Wei
authors.initials: W
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D000645:Ammonium Sulfate / Q000737:chemistry; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D002364:Caseins / Q000737:chemistry; D002417:Cattle; D002852:Chromatography, Ion Exchange; D004043:Dietary Fiber; D005260:Female; D005427:Flocculation; D015870:Gene Expression; D016680:Genome, Bacterial; D008892:Milk / Q000737:chemistry* / Q000382:microbiology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D056507:Paenibacillus / Q000737:chemistry* / Q000201:enzymology / Q000235:genetics; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D059748:Proteolysis; D012336:RNA, Ribosomal, 16S / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D002364:Caseins; D004043:Dietary Fiber; D012336:RNA, Ribosomal, 16S; D010447:Peptide Hydrolases; D000645:Ammonium Sulfate
keywords: Paenibacillus; coagulant; milk clotting activity; proteolytic activity