The Journal of General and Applied Microbiology
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  • Yang-dao Wei, Ya Li, Chun Deng, Shi-hua Wu, Cui-ji Huang, Yi Yi
    Volume 63 (2017) Issue 5 Pages 259-265
    Released: November 17, 2017
    [Advance publication] Released: August 23, 2017
    JOURNALS FREE ACCESS

    A gene (gkdA) (741 bp) encoding a putative protein of 247 amino acids was cloned from the Bacillus licheniformis SR01. The protein was expressed in Escherichia coli BL21 with a molecular mass estimated by SDS-PAGE of approximately 28.03 kDa and showed a calculating isoelectric point (pI) of 6.42. Structure analysis and function identification showed that the enzyme was a multifunctional glycosidase. Its specific activity was 0.013 U/μg. The recombinant glycosidase showed a maximum activity at 50°C and pH 7.0. It was very stable below 90°C and may have heat activation at higher temperatures. The relative residual activity was still more than 80% after 120 min at pH 5.0–10.0. The enzyme activity was inhibited by Cu2+, Fe2+, Ca2+, Mg2+, Co2+, Li+, SDS and EDTA, activated by Ca2+, and not affected by Mn2+ and K+. Under simulated stomach, and in vitro intestine, conditions, the enzyme retained 80%, and more than 100%, activity, respectively, after incubation for 90 min. The excellent properties of this enzyme, specifically its thermal stability and multifunctional abilities, give it potential application in the field of feed processing and other high-temperature processing industries.

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  • Guadalupe Jiménez-Galisteo, Ester Fusté, Elisa Muñoz, Teresa Vinuesa, ...
    Volume 63 (2017) Issue 5 Pages 266-273
    Released: November 17, 2017
    [Advance publication] Released: August 23, 2017
    JOURNALS FREE ACCESS

    Gordonia jacobaea is a bacterium belonging to the mycolata group characterized by its ability to produce carotenoids. Mycolic acids in the cell wall contribute to reducing the permeability of their envelopes requiring the presence of channel-forming proteins to allow the exchange of hydrophilic molecules with the surrounding medium. Identification and purification of the channel-forming proteins was accomplished by SDS-PAGE, Mass spectrometry and Mass peptide fingerprinting and the channel-forming activity was studied by reconstitution in lipid bilayers. Here, we describe for the first time the presence of a cell-wall protein from G. jacobaea with channel-forming activity. Our results suggest that this protein bears a low similarity to other hypothetical proteins from the genus Gordonia of uncharacterized functions. The channel has an average single-channel conductance of 800 pS in 1 M KCl, is moderately anion-selective, and does not show any voltage dependence for voltages between +100 and –100 mV. The channel characteristics suggest that this protein could be of relevance in the import and export of negatively charged molecules across the cell wall. This could contribute to design treatments for mycobacterial infections, as well as being of interest in biotechnology applications.

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  • Takeshi Sato, Kazuhito Inoue, Hidehiro Sakurai, Kenji V. P. Nagashima
    Volume 63 (2017) Issue 5 Pages 274-279
    Released: November 17, 2017
    [Advance publication] Released: September 12, 2017
    JOURNALS FREE ACCESS

    The efficiency of hydrogen gas production by nitrogenase in bacteria has been improved by the inhibition of antagonistic activity by the uptake hydrogenase. In this study, a mutant lacking the gene coding for the uptake hydrogenase was generated from the photosynthetic beta-proteobacterium Rubrivivax gelatinosus IL144 to explore new ways of hydrogen gas production driven by light energy. The mutant cells produced 25–30% higher amounts of molecular hydrogen than the wild-type cells under nitrogen-deficient conditions under light. Furthermore, by the addition of 5 mM glutamate, the photosynthetic growth rate was greatly enhanced, and the hydrogen gas production activity reached 41.1 (mmol/l) in the mutant.

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  • Meiru Si, Yanyan Feng, Keqi Chen, Yiwen Kang, Can Chen, Yao Wang, Xihu ...
    Volume 63 (2017) Issue 5 Pages 280-286
    Released: November 17, 2017
    [Advance publication] Released: September 12, 2017
    JOURNALS FREE ACCESS

    Methionine sulphoxide reductases (Msr) are able to reduce methionine sulfoxide to methionine and protect bacteria against reactive oxygen species (ROS). Many organisms express both methionine sulphoxide reductase A (MsrA), specific for methionine-S-sulfoxide and methionine sulphoxide reductase B (MsrB), active against methionine-R-sulfoxide. Corynebacterium glutamicum expresses MsrA, the function of which has been well defined; however, the function of MsrB has not been studied. Whether MsrB and MsrA play an equally important role in the antioxidant process is also poorly understood. In this study, we identified MsrB encoded by ncgl1823 in C. glutamicum, investigated its function and made a comparison with MsrA. The msrB gene showed a slight effect on utilizing methionine sulfoxide (MetO) as the sole Met source; however, the survival rates showed no sensitivity to oxidants. MsrB showed catalytic activity using thioredoxin/thioredoxin reductase (Trx/TrxR) reducing system as electron donors, but independent from the mycoredoxin 1/mycothione reductase/mycothiol (Mrx1/Mtr/MSH) system. Therefore, MsrB plays a limited role in resisting oxidative stress and it could reduce MetO to Met by the Trx/TrxR reducing system, which is useful for expanding the understanding of the functions of Msr in this important industrial microbe.

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  • Miki Tashiro, Akira Fujii, Shigeko Kawai-Noma, Kyoichi Saito, Daisuke ...
    Volume 63 (2017) Issue 5 Pages 287-295
    Released: November 17, 2017
    [Advance publication] Released: September 26, 2017
    JOURNALS FREE ACCESS

    To achieve an efficient production of geraniol and its derivatives in Escherichia coli, we aimed to improve the activity of geraniol synthase (GES) through a single round of mutagenesis and screening for higher substrate consumption. We isolated GES variants that outperform their parent in geraniol production. The analysis of GES variants indicated that the expression level of GES was the bottleneck for geraniol synthesis. Over-expression of the mutant GESM53 with a 5ʹ-untranslated sequence designed for high translational efficiency, along with the additional expression of mevalonate pathway enzymes, isopentenyl pyrophosphate isomerase, and geranyl pyrophosphate synthase, yielded 300 mg/L/12 h geraniol and its derivatives (>1000 mg/L/42 h in total) in a shaking flask.

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  • Wasana Suyotha, Hidehisa Fujiki, Vipavee Cherdvorapong, Kazuyoshi Taka ...
    Volume 63 (2017) Issue 5 Pages 296-304
    Released: November 17, 2017
    [Advance publication] Released: September 26, 2017
    JOURNALS FREE ACCESS

    Thermally stable α-1,3-glucanase HF65 was purified from culture filtrate of Streptomyces thermodiastaticus HF3-3. The molecular mass of this enzyme was estimated to be 65 kDa and 45.7 kDa by using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion chromatography, respectively. The purified enzyme retained more than 50% of maximum activity even after incubation at 65°C more than 2 h. Moreover, α-1,3-glucanase HF65 was stable in the presence of chemicals like SDS, benzethonium chloride, and sodium fluoride at a concentration of 1%. The enzyme also exhibited salt tolerance at a concentration up to 20%. The observed stability of α-1,3-glucanase HF65 to salt and surfactants is a great advantage for its addition to commercial oral care products. Interestingly, the N-terminal amino acid sequence did not show any similarity to those of known α-1,3-glucanases, while the sequence of internal eight amino acid residues of this enzyme was homologous with those of mycodextranases. Nevertheless, the enzyme exhibited high specificity against α-1,3-glucan. According to these results, the enzyme purified from S. thermodiastaticus HF3-3 was classified as α-1,3-glucanase which was highly homologous to mycodextranase in amino acid sequence.

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