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- Tubulysin Production by the Dead Cells of Archangium gephyra KYC5002.
- Seohui Park, Chaehyeon Park, Yujin Ka, Kyungyun Cho
- J. Microbiol. 2024;62(6):463-471. Published online June 13, 2024
- DOI: https://doi.org/10.1007/s12275-024-00130-3
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Abstract
- Archangium gephyra KYC5002 produces tubulysins during the death phase. In this study, we aimed to determine whether dead cells produce tubulysins. Cells were cultured for three days until the verge of the death phase, disrupted via ultrasonication, incubated for 2 h, and examined for tubulysin production. Non-disrupted cells produced 0.14 mg/L of tubulysin A and 0.11 mg/L of tubulysin B. Notably, tubulysin A production was increased by 4.4-fold to 0.62 mg/L and that of tubulysin B was increased by 6.7-fold to 0.74 mg/L in the disrupted cells. The same increase in tubulysin production was observed when the cells were killed by adding hydrogen peroxide. However, when the enzymes were inactivated via heat treatment of the cultures at 65 °C for 30 min, no significant increase in tubulysin production due to cell death was observed. Reverse transcription-quantitative polymerase chain reaction analysis of tubB mRNA revealed that the expression levels of tubulysin biosynthetic enzyme genes increased during the death phase compared to those during the vegetative growth phase. Our findings suggest that A. gephyra produces biosynthetic enzymes and subsequently uses them for tubulysin production in the cell death phase or during cell lysis by predators.
- Saxibacter everestensis gen. nov., sp. nov., A Novel Member of the Family Brevibacteriaceae, Isolated from the North Slope of Mount Everest.
- Mao Tian, Shiyu Wu, Wei Zhang, Gaosen Zhang, Xue Yu, Yujie Wu, Puchao Jia, Binglin Zhang, Tuo Chen, Guangxiu Liu
- J. Microbiol. 2024;62(4):277-284. Published online March 6, 2024
- DOI: https://doi.org/10.1007/s12275-024-00108-1
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Abstract
- We isolated and analyzed a novel, Gram-stain-positive, aerobic, rod-shaped, non-motile actinobacterium, designated as strain ZFBP1038(T), from rock sampled on the north slope of Mount Everest. The growth requirements of this strain were 10-37 °C, pH 4-10, and 0-6% (w/v) NaCl. The sole respiratory quinone was MK-9, and the major fatty acids were anteiso-C(15:0) and iso-C(17:0). Peptidoglycan containing meso-diaminopimelic acid, ribose, and glucose were the major cell wall sugars, while polar lipids included diphosphatidyl glycerol, phosphatidyl glycerol, an unidentified phospholipid, and an unidentified glycolipid. A phylogenetic analysis based on 16S rRNA gene sequences showed that strain ZFBP1038(T) has the highest similarity with Spelaeicoccus albus DSM 26341( T) (96.02%). ZFBP1038(T) formed a distinct monophyletic clade within the family Brevibacteriaceae and was distantly related to the genus Spelaeicoccus. The G + C content of strain ZFBP1038(T) was 63.65 mol% and the genome size was 4.05 Mb. Digital DNA-DNA hybridization, average nucleotide identity, and average amino acid identity values between the genomes of strain ZFBP1038(T) and representative reference strains were 19.3-25.2, 68.0-71.0, and 52.8-60.1%, respectively. Phylogenetic, phenotypic, and chemotaxonomic characteristics as well as comparative genome analyses suggested that strain ZFBP1038(T) represents a novel species of a new genus, for which the name Saxibacter gen. nov., sp. nov. was assigned with the type strain Saxibacter everestensis ZFBP1038(T) (= EE 014( T) = GDMCC 1.3024( T) = JCM 35335( T)).
- Characterization of Trichoderma reesei Endoglucanase II Expressed Heterologously in Pichia pastoris for Better Biofinishing and Biostoning
- Sutanu Samanta , Asitava Basu , Umesh Chandra Halder , Soumitra Kumar Sen
- J. Microbiol. 2012;50(3):518-525. Published online June 30, 2012
- DOI: https://doi.org/10.1007/s12275-012-1207-5
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Abstract
- The endoglucanase II of Trichoderma reesei is considered the most effective enzyme for biofinishing cotton fabrics and biostoning denim garments. However, the commercially available preparation of endoglucanase II is usually mixed with other cellulase components, especially endoglucanase I, resulting in hydrolysis and weight loss of garments during biofinishing and biostoning. We thus isolated the endoglucanase II gene from T. reesei to express this in Pichia pastoris, under the control of a methanol-inducible AOX1 promoter, to avoid the presence of other cellulase components. A highly expressible Mut+ transformant was selected and its expression in BMMH medium was found most suitable for the production of large amounts of the recombinant protein. Recombinant endoglucanase II was purified to electrophoretic homogeneity, and functionally characterized by activity staining. The specific activity of recombinant endoglucanase II was found to be 220.57 EU/mg of protein. Purified recombinant endoglucanase II was estimated to have a molecular mass of 52.8 kDa. The increase in molecular mass was likely due to hyperglycosylation. Hyperglycosylation of recombinant endoglucanase II secreted by P. pastoris did not change the temperature or pH optima as compared to the native protein, but did result in increased thermostability. Kinetic analysis showed that recombinant endoglucanase was most active against amorphous cellulose, such as carboxymethyl cellulose, for which it also had a high affinity.
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