Journal of Microbiology

Research Support, Non-U.S. Gov'ts

Expression, Purification, and Biochemical Properties of Arginase from Bacillus subtilis 168: Jin-Ju Yu , Ki-Bum Park , Su-Gon Kim , Suk-Heung Oh; J. Microbiol. 2013;51(2):222-228. Published online April 27, 2013; DOI: https://doi.org/10.1007/s12275-013-2669-9

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Abstract: The arginine-degrading and ornithine-producing enzymes arginase has been used to treat arginine-dependent cancers. This study was carried out to obtain the microbial arginase from Bacillus subtilis, one of major microorganisms found in fermented foods such as Cheonggukjang. The gene encoding arginase was isolated from B. subtilis 168 and cloned into E. coli expression plasmid pET32a. The enzyme activity was detected in the supernatant of the transformed and IPTG induced cell-extract. Arginase was purified for homogeneity from the supernatant by affinity chromatography. The specific activity of the purified arginase was 150 U/mg protein. SDS-PAGE analysis revealed the molecular size to be 49 kDa (Trix·Tag, 6×His·Tag added size). The optimum pH and temperature of the purified enzyme with arginine as the substrate were pH 8.4 and 45°C, respectively. The Km and Vmax values of arginine for the enzyme were 4.6 mM and 133.0 mM/min/mg protein respectively. These findings can contribute in the development of functional fermented foods such as Cheonggukjang with an enhanced level of ornithine and pharmaceutical products by providing the key enzyme in arginine-degradation and ornithine-production.

Purification and Partial Characterization of a Detergent and Oxidizing Agent Stable Alkaline Protease from a Newly Isolated Bacillus subtilis VSG-4 of Tropical Soil: Sib Sankar Giri , V. Sukumaran , Shib Sankar Sen , M. Oviya , B. Nazeema Banu , Prasant Kumar Jena; J. Microbiol. 2011;49(3):455-461. Published online June 30, 2011; DOI: https://doi.org/10.1007/s12275-011-0427-4

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Abstract: An extracellular detergent tolerant protease producing strain VSG-4 was isolated from tropical soil sample and identified as Bacillus subtilis based on morphological, biochemical characteristics as well as 16S-rRNA gene sequencing. The VSG-4 protease was purified to homogeneity using ammonium sulphate precipitation, dialysis and sephadex G-200 gel permeation chromatography with a 17.4 purification fold. The purified enzyme was active and stable over a broad range of pH (8.0-11.0, optimum at 9.0) and temperature (40°C to 60°C, optimum at 50°C). The thermostability of the enzyme was significantly increased by the addition CaCl2. This enzyme was strongly inhibited by PMSF and DFP, suggesting that it belongs to the serine protease superfamily. The purified VSG-4 alkaline protease showed remarkable stability in anionic (5 mM SDS) and ionic (1% Trion X-100 and 1% Tween 80) detergents. It retained 97±2% and 83.6±1.1% of its initial activity after 1 h preincubation in the presence of 1% H2O2 and 1% sodium perborate, respectively. Furthermore, the purified enzyme showed excellent stability and compatibility with some commercial laundry detergents besides its stain removal capacity. Considering these promising properties, VSG-4 protease may find tremendous application in laundry detergent formulations.

Application of Terminal Restriction Fragment Length Polymorphism (T-RFLP) Analysis to Monitor Effect of Biocontrol Agents on Rhizosphere Microbial Community of Hot Pepper (Capsicum annuum L.): Young Tae Kim , Myoungho Cho , Je Yong Jeong , Hyang Burm Lee , Seung Bum Kim; J. Microbiol. 2010;48(5):566-572. Published online November 3, 2010; DOI: https://doi.org/10.1007/s12275-010-0126-6

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Abstract: Microbial communities in hot pepper (Capsicum annuum L.) cultivation fields under different cultivation methods were investigated by terminal restriction fragment length polymorphism (T-RFLP) analysis. Rhizosphere soil and leaf samples were collected from control, conventional and nature-friendly cultivation fields between May and July, 2009. Two Bacillus subtilis strains were applied to nature-friendly cultivation fields as biocontrol agents during the sampling period. Relative abundances of bacteria and plant pathogenic fungi related T-RFs were also measured to monitor the effect of biocontrol agents on potential plant pathogenic fungi. In the principal component analysis (PCA) based on T-RFLP profiles, the microbial communities from rhizosphere soil samples in July, including bacteria and fungi, showed distinct difference between nature-friendly cultivation fields and other cultivation fields. However, there was no correlation between cultivation methods and leaf microbial communities at any sampling period. Changes in the abundance of bacteria related T-RF in the rhizosphere of nature-friendly cultivation fields were observed clearly two months after application of biocontrol agent, while the abundance of plant pathogenic fungi related T-RFs significantly decreased.

Induction of Growth Phase-Specific Autolysis in Bacillus subtilis 168 by Growth Inhibitors: Jin-Kyo Chung , Hyun Ee Yoon , Ha Chul Shin , Eun-Young Choi , Woo-Hyeon Byeon; J. Microbiol. 2009;47(1):50-59. Published online February 20, 2009; DOI: https://doi.org/10.1007/s12275-008-0256-2

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Abstract: Growth phase-specific autolysis of Bacillus subtilis by inhibitors of membrane permeability, inhibitors of macromolecule biosynthesis, inhibitors of cell wall biosynthesis and detergents were tested and characterized in glucose limited liquid medium. The minimum autolysis induction concentration (MAIC) of test compounds, which was at least 1/20th lower than the conventional autolysis induction concentration, induced autolysis only for cells at the glucose exhaustion point (diauxic point) of the growth phase, while it was not induced for cells at pre- and post-diauxic points. Inhibitors of macromolecule synthesis that are not known for inducing autolysis, such as chloramphenicol, rifampicin, nalidixic acid, and detergents, also induced specific autolysis. Two types of autolysis corresponding to the concentrations of compounds are distinguished: concentration-sensitive and concentration-insensitive types.

Cytochrome c_550 is Related to Initiation of Sporulation in Bacillus subtilis: Inji Shin , Han-Bong Ryu , Hyung-Soon Yim , Sa-Ouk Kang; J. Microbiol. 2005;43(3):244-250.; DOI: https://doi.org/2218 [pii]

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Abstract: The effect of cytochrome c_550 encoded by cccA in Bacillus subtilis during the event of sporulation was investigated. The sporulation of cccA-overexpressing mutant was significantly accelerated, while disruptant strain showed delayed sporulation in spite of the same growth rate. Activity of sporulation stage-0-specific enzyme, extracellular [alpha]-amylase of mutant strains was similar to that of the control strain, but cccA-overexpressing mutant exhibited higher activity of stage-II-specific alkaline phosphatase and stage-III-specific glucose dehydrogenase when compared to deletion mutant and control strain. Northern blot analysis also revealed that cccA-overexpressing mutant showed high level of spo0A transcripts, while the disruptant rarely expressed spo0A. These results suggested that although cytochrome c_550 is dispensable for growth and sporulation, expression of cccA may play an important role for initiation of sporulation through regulation of spo0A expression.

Expression of the Promoter for the Maltogenic Amylase Gene in Bacillus subtilis 168: Do-Yeon Kim , Choon-Hwan Cha , Wan-Seok Oh , Young-Jun Yoon , Jung-Wan Kim; J. Microbiol. 2004;42(4):319-327.; DOI: https://doi.org/2104 [pii]

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Abstract: An additional amylase, besides the typical a-amylase, was detected for the first time in the cytoplasm of B. subtilis SUH4-2, an isolate from Korean soil. The corresponding gene (bbmA) encoded a maltogenic amylase (MAase) and its sequence was almost identical to the yvdF gene of B. subtilis 168, whose function was unknown. Southern blot analysis using bbmA as the probe indicated that this gene was ubiquitous among various B. subtilis strains. In an effort to understand the physiological function of the bbmA gene in B. subtilis, the expression pattern of the gene was monitored by measuring the [beta]-galactosidase activity produced from the bbmA promoter fused to the amino terminus of the lacZ structural gene, which was then integrated into the amyE locus on the B. subtilis 168 chromosome. The promoter was induced during the mid-log phase and fully expressed at the early stationary phase in defined media containing [beta]-cyclodextrin ([beta]-CD), maltose, or starch. On the other hand, it was kept repressed in the presence of glucose, fructose, sucrose, or glycerol, suggesting that catabolite repression might be involved in the expression of the gene. Production of the [beta]-CD hydrolyzing activity was impaired by the spo0A mutation in B. subtilis 168, indicating the involvement of an additional regulatory system exerting control on the promoter. Inactivation of yvdF resulted in a significant decrease of the [beta]-CD hydrolyzing activity, if not all. This result implied the presence of an additional enzyme(s) that is capable of hydrolyzing [beta]-CD in B. subtilis 168. Based on the results, MAase encoded by bbmA is likely to be involved in maltose and [beta]-CD utilization when other sugars, which are readily usable as an energy source, are not available during the stationary phase.

Construction of secretion vectors using the α-amylase signal sequence of bacillus subtilis NA64: Kim, Sung Il , Lee, Se Yong; J. Microbiol. 1996;34(1):74-81.

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Abstract: Two secretion vectors, pUBA240 and pUB340 were constructed by using the promoter and secretory signal region of the α-amylase gene from an α-amylase hyperproducing strain, Bacillus subtilis NA64. In this secretion vector system, various restriction enzyme sites are located immediately after the proregion of the α-amylase gene for easy replacement of various foreign structural genes. To evaluate this secretion vectors, the β-lactamase gene of pBR322 was used as a reporter gene. The expressed and biologically active β-lactamase was secreted into the culture broth from B. subtilis LKS86 transformants harboring each β-lactamase secreting plasmid, pUBAbla and pUBSble. In both cases, more than 92% of expressed β- lactamases were located idn the culture medium. The amount of the secreted β-lactamase was about 80% of the total secreted proteins in the culture medium.

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