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Production, purification and characterization of a milk clotting enzyme from Bacillus methanolicus LB-1.

ABSTRACT: Bacillus methanolicus LB-1 isolated from traditional rice wine was found to produce a milk clotting enzyme (MCE), and its fermentation conditions were optimized using response surface methodology. Then the MCE was produced by ethanol precipitation, and further chromatography separation resulted in a 10.46-fold purification with a 59.28% recovery. The MCA (milk clotting activity) of the purified MCE reached 597,310?±?0.13 SU/g. The optimal temperature of the MCE was determined to be 50 °C and it was stable in the low temperature range of 40-45 °C. The MCE had an optimum pH of 6.5, and it was stable under neutral conditions. Calcium chloride at the concentration of 25 mM was found to be the most effective stimulus. The MCE was identified by LC-MS to be a putative protein (ID I3EB99) containing 759 amino acids with a molecular weight of 80.37 kDa and a pI of 9.23.

SUBMITTER: Li L

PROVIDER: S-EPMC6595037 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Production, purification and characterization of a milk clotting enzyme from <i>Bacillus methanolicus</i> LB-1.

Li Liu L Zheng Zhe Z Zhao Xiao X Wu Fengyu F Zhang Jian J Yang Zhennai Z

Food science and biotechnology 20190104 4

<i>Bacillus methanolicus</i> LB-1 isolated from traditional rice wine was found to produce a milk clotting enzyme (MCE), and its fermentation conditions were optimized using response surface methodology. Then the MCE was produced by ethanol precipitation, and further chromatography separation resulted in a 10.46-fold purification with a 59.28% recovery. The MCA (milk clotting activity) of the purified MCE reached 597,310 ± 0.13 SU/g. The optimal temperature of the MCE was determined to be 50 °C ...[more]

PMID: 31275710

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Project description:Bacillus methanolicus is a Gram-positive, thermophilic, methanol-utilizing bacterium. As a facultative methylotroph, B. methanolicus is also known to utilize D-mannitol, D-glucose and, as recently discovered, sugar alcohol D-arabitol. While metabolic pathways for utilization of methanol, mannitol and glucose are known, catabolism of arabitol has not yet been characterized in B. methanolicus. In this work we present the elucidation of this hitherto uncharted pathway. In order to confirm our predictions regarding genes coding for arabitol utilization, we performed differential gene expression analysis of B. methanolicus MGA3 cells grown on arabitol as compared to mannitol via transcriptome sequencing (RNA-seq). We identified a gene cluster comprising eight genes that was up-regulated during growth with arabitol as a sole carbon source. The RNA-seq results were subsequently confirmed via qRT-PCR experiments. The transcriptional organization of the gene cluster identified via RNA-seq was analyzed and it was shown that the arabitol utilization genes are co-transcribed in an operon that spans from BMMGA3_RS07325 to BMMGA3_RS07365. Since gene deletion studies are currently not possible in B. methanolicus, two complementation experiments were performed in an arabitol negative Corynebacterium glutamicum strain using the four genes discovered via RNA-seq analysis as coding for a putative PTS for arabitol uptake (BMMGA3_RS07330, BMMGA3_RS07335, and BMMGA3_RS07340 renamed to atlABC) and a putative arabitol phosphate dehydrogenase (BMMGA3_RS07345 renamed to atlD). C. glutamicum is a natural D-arabitol utilizer that requires arabitol dehydrogenase MtlD for arabitol catabolism. The C. glutamicum mtlD deletion mutant was chosen for complementation experiments. Heterologous expression of atlABCD as well as the arabitol phosphate dehydrogenase gene atlD from B. methanolicus alone restored growth of the C. glutamicum ΔmtlD mutant with arabitol. Furthermore, D-arabitol phosphate dehydrogenase activities could be detected in crude extracts of B. methanolicus and these were higher in arabitol-grown cells than in methanol- or mannitol-grown cells. Thus, B. methanolicus possesses an arabitol inducible operon encoding, amongst others, a putative PTS system and an arabitol phosphate dehydrogenase for uptake and activation of arabitol as growth substrate.

Dataset Information

Production, purification and characterization of a milk clotting enzyme from Bacillus methanolicus LB-1.

Publications

Production, purification and characterization of a milk clotting enzyme from <i>Bacillus methanolicus</i> LB-1.

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