Journal of Microbiology

Journal Article

Heterologous expression and enzymatic characterization of γ-glutamyltranspeptidase from Bacillus amyloliquefaciens: Jung-Min Lee , Jaejung Lee , Gyeong-Hwa Nam , Byung-Sam Son , Myoung-Uoon Jang , So-Won Lee , Byung-Serk Hurh , Tae-Jip Kim; J. Microbiol. 2017;55(2):147-152. Published online January 26, 2017; DOI: https://doi.org/10.1007/s12275-017-6638-6

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γ-Glutamyltranspeptidase (GGT) catalyzes the cleavage of γ- glutamyl compounds and the transfer of γ-glutamyl moiety to water or to amino acid/peptide acceptors. GGT can be utilized for the generation of γ-glutamyl peptides or glutamic acid, which are used as food taste enhancers. In the present study, Bacillus amyloliquefaciens SMB469 with high GGT activity was isolated from Doenjang, a traditional fermented soy food of Korea. The gene encoding GGT from B. amyloliquefaciens SMB469 (BaGGT469) was cloned from the isolate, and heterologously expressed in E. coli and B. subtilis. For comparison, three additional GGT genes were cloned from B. subtilis 168, B. licheniformis DSM 13, and B. amyloliquefaciens FZB42. The BaGGT469 protein was composed of 591 amino acids. The final protein comprises two separate polypeptide chains of 45.7 and 19.7 kDa, generated via autocatalytic cleavage. The specific activity of BaGGT469 was determined to be 17.8 U/mg with γ-L-glutamyl-p-nitroanilide as the substrate and diglycine as the acceptor. GGTs from B. amyloliquefaciens showed 1.4- and 1.7-fold higher transpeptidase activities than those from B. subtilis and B. licheniformis, respectively. Especially, recombinant B. subtilis expressing BaGGT469 demonstrated 11- and 23-fold higher GGT activity than recombinant E. coli and the native B. amyloliquefaciens, respectively, did. These results suggest that BaGGT469 can be utilized for the enzymatic production of various γ- glutamyl compounds.

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

Identification and Characterization of an Anti-fungi Fusarium oxysporum f. sp. cucumerium Protease from the Bacillus subtilis Strain N7: Yi Luo , Lifei Sun , Zhen Zhu , Wei Ran , Qirong Shen; J. Microbiol. 2013;51(3):359-366. Published online June 28, 2013; DOI: https://doi.org/10.1007/s12275-013-2627-6

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Abstract: A newly discovered alkaline antifungal protease named P6 from Bacillus subtilis N7 was purified and partially characterized. B. subtilis N7 culture filtrates were purified by 30–60% (NH4)2SO4 precipitation, anion-exchange chromatography and gel filtration chromatography. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) revealed a single band of 41.38 kDa. Peptide sequence of protease P6 was determined using a 4800 Plus MALDI TOF/TOFTM Analyzer System. Self-Formed Adaptor PCR (SEFA-PCR) was used to amplify the 1,149 bp open read frame of P6. Dimensional structure prediction using Automatic Modeling Mode software showed that the protease P6 consisted of two β-barrel domains. Purified P6 strongly inhibited spore and mycelium growth of Fusarium oxysporum f. sp. cucumerium (FOC) by causing hypha lysis when the concentration was 25 μg/ml. Characterization of the purified protease indicated that it had substrate specificity for gelatin and was highly active at pH 8.0–10.6 and 70°C. The P6 protease was inhibited by EDTA (2 mmol/L), phenyl methyl sulfonyl fluoride (PMSF, 1 mmol/L), Na+, Fe3+, Cu2+, Mg2+ (5 mmol/L each) and H2O2 (2%, v/v). However, protease activity was activated by Ca2+, K+, Mn2+ (5 mmol/L each), mercaptoethanol (2%, v/v) and Tween 80 (1%, v/v). In additon, activity was also affected by organic solvents such as acetone, normal butanol and ethanol, but not hexane (25%, v/v each).

Characterization, Cloning, and Heterologous Expression of a Subtilisin-Like Serine Protease Gene VlPr1 from Verticillium lecanii: Gang Yu , Jin-Liang Liu , Li-Qin Xie , Xue-Liang Wang , Shi-Hong Zhang , Hong-Yu Pan; J. Microbiol. 2012;50(6):939-946. Published online December 30, 2012; DOI: https://doi.org/10.1007/s12275-012-2199-x

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Abstract: The entomopathogenic fungus Verticillium lecanii is a wellknown biocontrol agent. V. lecanii produces subtilisin-like serine protease (Pr1), which is important in the biological control activity of some insect pests by degrading insect cuticles. In this study, a subtilisin-like serine protease gene VlPr1 was cloned from the fungus and the VlPr1 protein was expressed in Escherichia coli. The VlPr1 gene contains an open reading frame (ORF) interrupted by three short introns, and encodes a protein of 379 amino acids. Protein sequence analysis revealed high homology with subtilisin serine proteases. The molecular mass of the protease was 38 kDa, and the serine protease exhibited its maximal activity at 40°C and pH 9.0. Protease activity was also affected by Mg2+ and Ca2+ concentration. The protease showed inhibitory activity against several plant pathogens, especially towards Fusarium moniliforme.

Journal Article

Chitinase Production by Bacillus thuringiensis and Bacillus licheniformis: Their Potential in Antifungal Biocontrol: Eman Zakaria Gomaa; J. Microbiol. 2012;50(1):103-111. Published online February 27, 2012; DOI: https://doi.org/10.1007/s12275-012-1343-y

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Abstract: Thirty bacterial strains were isolated from the rhizosphere of plants collected from Egypt and screened for production of chitinase enzymes. Bacillus thuringiensis NM101-19 and Bacillus licheniformis NM120-17 had the highest chitinolytic activities amongst those investigated. The production of chitinase by B. thuringiensis and B. licheniformis was optimized using colloidal chitin medium amended with 1.5% colloidal chitin, with casein as a nitrogen source, at 30°C after five days of incubation. An enhancement of chitinase production by the two species was observed by addition of sugar substances and dried fungal mats to the colloidal chitin media. The optimal conditions for chitinase activity by B. thuringiensis and B. licheniformis were at 40°C, pH 7.0 and pH 8.0, respectively. Na+, Mg2+, Cu2+, and Ca2+ caused enhancement of enzyme activities whereas they were markedly inhibited by Zn2+, Hg2+, and Ag+. In vitro, B. thuringiensis and B. licheniformis chitinases had potential for cell wall lysis of many phytopathogenic fungi tested. The addition of B. thuringiensis chitinase was more effective than that of B. licheniformis in increasing the germination of soybean seeds infected with various phytopathogenic fungi.

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

NOTE] Complete Sequence and Organization of the Sphingobium chungbukense DJ77 pSY2 Plasmid: Sun-Mi Yeon , Young-Chang Kim; J. Microbiol. 2011;49(4):684-688. Published online September 2, 2011; DOI: https://doi.org/10.1007/s12275-011-1262-3

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Abstract: Sphingobium chungbukense DJ77 is capable of metabolizing priority chemicals of human health concern such as polycyclic aromatic hydrocarbons (PAHs), extracellular polysaccharide (EPS), and antibiotics. Here, we report the complete DNA and genetic organization of the plasmid pSY2 from strain DJ77. A DNA sequence analysis revealed that pSY2 comprises 18,779 bp encoding 22 open reading frames (ORFs) with 59.5% G+C content. The ORFs on pSY2 were classified into DNA replication, conjugative function, transposition, plasmid stability/partition, and other functional groups (transport, fatty acid biosynthesis, stress, and growth rate regulation). Three ORFs on pSY2 were hypothetical proteins.

Isolation and Characterization of Ethylbenzene Degrading Pseudomonas putida E41: Lan-Hee Kim , Sang-Seob Lee; J. Microbiol. 2011;49(4):575-584. Published online September 2, 2011; DOI: https://doi.org/10.1007/s12275-011-0399-4

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Abstract: Pseudomonas putida E41 was isolated from oil-contaminated soil and showed its ability to grow on ethylbenzene as the sole carbon and energy source. Moreover, P. putida E41 show the activity of biodegradation of ethylbenzene in the batch culture. E41 showed high efficiency of biodegradation of ethylbenzene with the optimum conditions (a cell concentration of 0.1 g wet cell weight/L, pH 7.0, 25°C, and ethylbenzene concentration of 50 mg/L) from the results of the batch culture. The maximum degradation rate and specific growth rate (μmax) under the optimum conditions were 0.19±0.03 mg/mg-DCW (Dry Cell Weight)/h and 0.87±0.13 h-1, respectively. Benzene, toluene and ethylbenzene were degraded when these compounds were provided together; however, xylene isomers persisted during degradation by P. putida E41. When using a bioreactor batch system with a binary culture with P. putida BJ10, which was isolated previously in our lab, the degradation rate for benzene and toluene was improved in BTE mixed medium (each initial concentration: 50 mg/L). Almost all of the BTE was degraded within 4 h and 70-80% of m-, p-, and o-xylenes within 11 h in a BTEX mixture (initial concentration: 50 mg/L each). In summary, we found a valuable new strain of P. putida, determined the optimal degradation conditions for this isolate and tested a mixed culture of E41 and BJ10 for its ability to degrade a common sample of mixed contaminants containing benzene, toluene, and xylene.

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.

A Thermostable Phytase from Neosartorya spinosa BCC 41923 and Its Expression in Pichia pastoris: Patcharaporn Pandee , Pijug Summpunn , Suthep Wiyakrutta , Duangnate Isarangkul , Vithaya Meevootisom; J. Microbiol. 2011;49(2):257-264. Published online May 3, 2011; DOI: https://doi.org/10.1007/s12275-011-0369-x

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Abstract: A phytase gene was cloned from Neosartorya spinosa BCC 41923. The gene was 1,455 bp in size, and the mature protein contained a polypeptide of 439 amino acids. The deduced amino acid sequence contains the consensus motif (RHGXRXP) which is conserved among phytases and acid phosphatases. Five possible disulfide bonds and seven potential N-glycosylation sites have been predicted. The gene was expressed in Pichia pastoris KM71 as an extracellular enzyme. The purified enzyme had specific activity of 30.95 U/mg at 37°C and 38.62 U/mg at 42°C. Molecular weight of the deglycosylated recombinant phytase, determined by SDS-PAGE, was approximately 52 kDa. The optimum pH and temperature for activity were pH 5.5 and 50°C. The residual phytase activity remained over 80% of initial activity after the enzyme was stored in pH 3.0 to 7.0 for 1 h, and at 60% of initial activity after heating at 90°C for 20 min. The enzyme exhibited broad substrate specificity, with phytic acid as the most preferred substrate. Its Km and Vmax for sodium phytate were 1.39 mM and 434.78 U/mg, respectively. The enzyme was highly resistant to most metal ions tested, including Fe2+, Fe3+, and Al3+. When incubated with pepsin at a pepsin/phytase ratio of 0.02 (U/U) at 37°C for 2 h, 92% of its initial activity was retained. However, the enzyme was very sensitive to trypsin, as 5% of its initial activity was recovered after treating with trypsin at a trypsin/phytase ratio of 0.01 (U/U).

Characterization, Gene Cloning, and Heterologous Expression of β-Mannanase from a Thermophilic Bacillus subtilis: Pijug Summpunn , Suttidarak Chaijan , Duangnate Isarangkul , Suthep Wiyakrutta , Vithaya Meevootisom; J. Microbiol. 2011;49(1):86-93. Published online March 3, 2011; DOI: https://doi.org/10.1007/s12275-011-0357-1

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Abstract: Bacillus subtilis BCC41051 producing a thermostable β-mannanase was isolated from soybean meal-enriched soil and was unexpectedly found to be thermophilic in nature. The extracellular β-mannanase (ManA) produced was hydrophilic, as it was not precipitated even with ammonium sulfate at 80% saturation. The estimated molecular weight of ManA was 38.0 kDa by SDS-PAGE with a pI value of 5.3. Optimal pH and temperature for mannan-hydrolyzing activity was 7.0 and 60°C, respectively. The enzyme was stable over a pH range of 5.0-11.5, and at temperatures of up to 60°C for 30 min, with more than 80% of its activity retained. ManA was strongly inhibited by Hg2+ (1 mM), but was sensitive to other divalent ions to a lesser degree. The gene of ManA encoded a protein of 362 amino acid residues, with the first 26 residues identified as a signal peptide. High expression of recombinant ManA was achieved in both Escherichia coli BL21 (DE3) (415.18 U/ml) and B. megaterium UNcat (359 U/ml).

Characterization of Plasmid pSY3 in Sphingobium chungbukense DJ77: Sun-Mi Yeon , Young-Chang Kim; J. Microbiol. 2009;47(6):796-800. Published online February 4, 2010; DOI: https://doi.org/10.1007/s12275-009-0329-x

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Abstract: This study determined the complete nucleotide sequence of the plasmid pSY3 from Sphingobium chungbukense DJ77. It was 35,735 bp long with a G+C content of 61.9%. Forty open reading frames (ORFs) were found. We predicted these ORFs would encode proteins associated with plasmid replication, conjugative transfer, transposition of genes, plasmid stability/partition, hypothetical protein, and some other functions. Genes for biodegradation were not found. No other plasmid homologous to pSY3 in the overall nucleotide sequence or gene organization could be found in the NCBI database.

Journal Article

Characterization of the Bacillus subtilis WL-3 Mannanase from a Recombinant Escherichia coli: Ki-Hong Yoon , Seesub Chung , Byung-Lak Lim; J. Microbiol. 2008;46(3):344-349. Published online July 5, 2008; DOI: https://doi.org/10.1007/s12275-008-0045-y

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Abstract: A mannanase was purified from a cell-free extract of the recombinant Escherichia coli carrying a Bacillus subtilis WL-3 mannanase gene. The molecular mass of the purified mannanase was 38 kDa as estimated by SDS-PAGE. Optimal conditions for the purified enzyme occurred at pH 6.0 and 60°C. The specific activity of the purified mannanase was 5,900 U/mg on locust bean gum (LBG) galactomannan at pH 6.0 and 50°C. The activity of the enzyme was slightly inhibited by Mg2+, Ca2+, EDTA and SDS, and noticeably enhanced by Fe2+. When the enzyme was incubated at 4°C for one day in the presence of 3 mM Fe2+, no residual activity of the mannanase was observed. The enzyme showed higher activity on LBG and konjac glucomannan than on guar gum galactomannan. Furthermore, it could hydrolyze xylans such as arabinoxylan, birchwood xylan and oat spelt xylan, while it did not exhibit any activities towards carboxymethylcellulose and para-nitrophenyl-β-mannopyranoside. The predominant products resulting from the mannanase hydrolysis were mannose, mannobiose and mannotriose for LBG or mannooligosaccharides including mannotriose, mannotetraose, mannopentaose and mannohexaose. The enzyme could hydrolyze mannooligosaccharides larger than mannobiose.

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Strain Improvement of Candida tropicalis for the Production of Xylitol:Biochemical and Physiological Characterization of Wild-type and Mutant Strain CT-OMV5: Ravella Sreenivas Rao , Cherukuri Pavana Jyothi , Reddy Shetty Prakasham , Chaganti Subba Rao , Ponnupalli Nageshwara Sarma , Linga Venkateswar Rao; J. Microbiol. 2006;44(1):113-120.; DOI: https://doi.org/2328 [pii]

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Abstract: Candida tropicalis was treated with ultraviolet (UV) rays, and the mutants obtained were screened for xylitol production. One of the mutants, the UV1 produced 0.81g of xylitol per gram of xylose. This was further mutated with N-methyl-N’-nitro-N-nitrosoguanidine (MNNG), and the mutants obtained were screened for xylitol production. One of the mutants (CT-OMV5) produced 0.85g/g of xylitol from xylose. Xylitol production improved to 0.87 g/g of xylose with this strain when the production medium was supplemented with urea. The CT-OMV5 mutant strain differs by 12 tests when compared to the wild-type Candida tropicalis strain. The XR activity was higher in mutant CT-OMV5. The distinct difference between the mutant and wild-type strain is the presence of numerous chlamydospores in the mutant. In this investigation, we have demonstrated that mutagenesis was successful in generating a superior xylitol-producing strain, CT-OMV5, and uncovered distinctive biochemical and physiological characteristics of the wild-type and mutant strain, CT-OMV5.

Purification and characterization of a xylanase from alkalophilic cephalosporium sp. RYM-202: Kang, Myoung Kyu , Kwon, Tae Ik , Yang, Young Ki , Rhee, Young Ha; J. Microbiol. 1995;33(2):109-114.

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Abstract: Alkalophilic Cephalosporium sp. RYM-202 produced multiple xylanases extracellularly. One of these xylanases was purified to electrophoretical homogeneity by chromatography with DEAE-Sephadex A-50, Sephacryl S-200 HR and Superose 12 HR. The purified xylanase differed from most other microbial xylanases in that it had low-molecular weight and acidic isoelectric point. The molecular weight of the xylanase in that it had low-molecular weight and acidic isoelectric point. The molecular weight of the xylanase was 23 kDa by SDS-polyacrylamide electrophoresis and 24 kDa by gel permeation chromatography, and the isoelectric point was 4.3. The xylanase had the highest activity permentation chromatography, and the isoelectric point was 4.3. The xylanase had the highest activity permeation chromatography, and the isoelectric point was 4.3. The xylanase had the highest activity at pH 8.0 and 50℃. It was stable over a wide range of pH and retained more than 80% of its original activity after 24 h of incubation even at pH 12. The Km values of this enzyme on birchwood xylan and oat spelts xylan were 2.33 and 3.45 mg/ml, respectively. The complete inhibition of the enzyme of n-bromosuccinimide suggests the involvement of tryptophan in the active site. The sylanase lacked activity towards crystalline cellulose and carboxymethyl cellulose.

Purification and Characterization of an Extracellular Protease from Culture Filtrate of Salmonella schttmulleri: Na, Byoung Kuk , Song, Chul Yong; J. Microbiol. 1995;33(3):244-251.

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Abstract: An extracellular protease of Salmonella schottmulleri was purified from culture filtrate by using 0-75% ammonium sulfate precipitation, DEAE Sepharose Fast Flow ion exchange chromatography, Ultrogel HA chromatography and Sephacryl S-200 HR molecular sieve chromatography. To measure enzyme activity, synthetic dipeptide substrate (CBZ-arg-arg-AFC) with low molecular weight was employed as substrate. The molecular weight of the purified enzyme was approximately 80 kDa when determined by gel filtration on Sephacryl S-200 HR and 73 kDa when estimated by SDS-PAGE. The isoelectric point was 5.45. The activity of the purified enzyme was inhibited by metal chelating agesnts such as EDTA and 1.10-phenanthroline. The divalent cations, such as Ca^2+, Zn^2+, Fe^2+, Mg^2+ enhanced its activity. These results suggested that it was a metalloprotease. It had a narrow pH optimum of 6.5-7.5 with a maximum at pH 7.0 and a temperature optimum of 40℃. It was stable at least for 1 week at 40℃ and maintained its activity for 24 hours at 50℃, but it was rapidly inactivated at 65℃. This protease was shown to be sensitive to sodium 50℃, but it was rapidly inactivated at 65℃. This protease was shown to be sensitive to sodium 50℃, but it was rapidly inactivated at 65℃. This protease was shown to be sensitive to sodium 50℃, but it was rapidly inactivated at 65℃. This protease was shown to be sensitive to sodium dodecyl sulfate (SDS) and was inactivated in a dose-dependent manner. However, it was resistant to Triton X-100 and the activity was enhanced to 32.3% with treatment of 0.025% Triton X-100.

Partial characterization of proteases from culture filtrate of mycobacterium tuberculosis: Na, Byoung Kuk , Song, Chul Yong , Park, Young Kil , Bai, Gill Han , Ki, Sang Jae; J. Microbiol. 1996;34(2):198-205.

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Abstract: Two proteases were partially characterized from culture filtrate of Mycobacterium, tuberculosis KIT110. Their molecular weights were approximately 200 and 180 kDa, respectively and they exhibited similar enzymatic characteristics. These enzymes were inhibited significantly by EDTA and to some extent by EGTA. Their activity was enhanced by Ca^2+ and Mg^2+ to some degree. However, Cu^2+ and Ag^2+ completely inhibited the enzyme activity at the concentration of 2.5 and 5 mM, respectively. The optimal pH was 7.0 and optimal temperature was around 40℃. These enzymes were rapidly inactivated at 80℃. Therefore, they were heat-labile, neutral metalloproteases. These enzymes exhibited antigenicity shown by their reacting with sera from the partients with pulmonary tuberculosis. These enzymes were able to degrade serum proteins including hemoglobin, bovine serum albumin, lysozyme and immunoglobulin G and structural matrix protein such as type I collagen. Therefore, these enzymes may be thought to contribute to tissue necrosis and pathogenesis during infection.

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