Journal of Microbiology

Journal Articles

Molecular characterization of Hsf1 as a master regulator of heat shock response in the thermotolerant methylotrophic yeast Ogataea parapolymorpha: Jin Ho Choo , Su-Bin Lee , Hye Yun Moon , Kun Hwa Lee , Su Jin Yoo , Keun Pil Kim , Hyun Ah Kang; J. Microbiol. 2021;59(2):151-163. Published online February 1, 2021; DOI: https://doi.org/10.1007/s12275-021-0646-2

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Ogataea parapolymorpha (Hansenula polymorpha DL-1) is a thermotolerant methylotrophic yeast with biotechnological applications. Here, O. parapolymorpha genes whose expression is induced in response to heat shock were identified by transcriptome analysis and shown to possess heat shock elements (HSEs) in their promoters. The function of O. parapolymorpha HSF1 encoding a putative heat shock transcription factor 1 (OpHsf1) was characterized in the context of heat stress response. Despite exhibiting low sequence identity (26%) to its Saccharomyces cerevisiae homolog, OpHsf1 harbors conserved domains including a DNA binding domain (DBD), domains involved in trimerization (TRI), transcriptional activation (AR1, AR2), transcriptional repression (CE2), and a C-terminal modulator (CTM) domain. OpHSF1 could complement the temperature sensitive (Ts) phenotype of a S. cerevisiae hsf1 mutant. An O. parapolymorpha strain with an H221R mutation in the DBD domain of OpHsf1 exhibited significantly retarded growth and a Ts phenotype. Intriguingly, the expression of heat-shock-protein‒coding genes harboring HSEs was significantly decreased in the H221R mutant strain, even under non-stress conditions, indicating the importance of the DBD for the basal growth of O. parapolymorpha. Notably, even though the deletion of C-terminal domains (ΔCE2, ΔAR2, ΔCTM) of OpHsf1 destroyed complementation of the growth defect of the S. cerevisiae hsf1 strain, the C-terminal domains were shown to be dispensable in O. parapolymorpha. Overexpression of OpHsf1 in S. cerevisiae increased resistance to transient heat shock, supporting the idea that OpHsf1 could be useful in the development of heatshock‒ resistant yeast host strains.

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A comprehensive review and comparison of L-tryptophan biosynthesis in Saccharomyces cerevisiae and Escherichia coli
Xinru Ren, Yue Wei, Honglu Zhao, Juanjuan Shao, Fanli Zeng, Zhen Wang, Li Li
Frontiers in Bioengineering and Biotechnology.2023;[Epub] CrossRef
Heat shock in Cronobacter sakazakii induces direct protection and cross-protection against simulated gastric fluid stress
Hongmei Niu, MingzheYang, Yonghua Qi, Yangtai Liu, Xiang Wang, Qingli Dong
Food Microbiology.2022; 103: 103948. CrossRef
A review of yeast: High cell-density culture, molecular mechanisms of stress response and tolerance during fermentation
Dongxu Shen, Xiaoli He, Peifang Weng, Yanan Liu, Zufang Wu
FEMS Yeast Research.2022;[Epub] CrossRef

Development of a strategy for the screening of α-glucosidase-producing microorganisms: Bo Zhou+ , Nan Huang+ , Wei Zeng+ , Hao Zhang , Guiguang Chen , Zhiqun Liang; J. Microbiol. 2020;58(2):163-172. Published online January 29, 2020; DOI: https://doi.org/10.1007/s12275-020-9267-4

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Abstract

α-Glucosidase is a crucial enzyme for the production of isomaltooligosaccharide. In this study, a novel method comprising eosin Y (EY) and α-D-methylglucoside (AMG) in glass plates was tested for the primary screening of α-glucosidaseproducing strains. First, α-glucosidase-producing Aspergillus niger strains were selected on plates containing EY and AMG based on transparent zone formation resulting from the solubilization of EY by the hydrolyzed product. Conventional
methods
that use trypan blue (TB) and p-nitrophenyl-α-Dglucopyranoside (pPNP) as indicators were then compared with the new strategy. The results showed that EY-containing plates provide the advantages of low price and higher specificity for the screening of α-glucosidase-producing strains. We then evaluated the correlation between the hydrolytic activity of α-glucosidase and diffusion distance, and found that good linearity could be established within a 6–75 U/ml enzyme concentration range. Finally, the hydrolytic and transglycosylation activities of α-glucosidase obtained from the target isolates were determined by EY plate assay and 3,5- dinitrosalicylic acid-Saccharomyces cerevisiae assay, respectively. The results showed that the diameter of the transparent zone varied among isolates was positively correlated with α-glucosidase hydrolytic activity, while good linearity could also be established between α-glucosidase transglycosylation activity and non-fermentable reducing sugars content. With this strategy, 7 Aspergillus niger mutants with high yield of α-glucosidase from 200 obvious single colonies on the primary screen plate were obtained.

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Purification, characterization of a novel α-glucosidase from Debaryomyces hansenii strain MCC 0202 and chromatographic separation for high purity isomalto-oligosaccharides production
Saravanan Rengarajan, Rameshthangam Palanivel
Process Biochemistry.2024; 136: 109. CrossRef
Development of a PMA‐LAMP visual detection assay for viable Cronobacter sakazakii
Qiming Chen, Yang Yu, Xiaodi Chen, Fangming Tu, Peng Wang, Junyi Huang, Zhanmin Liu
International Journal of Dairy Technology.2024; 77(2): 427. CrossRef
Identification of chitin synthase activator in Aspergillus niger and its application in citric acid fermentation
Chunxu Jiang, Han Wang, Menghan Liu, Li Wang, Ruwen Yang, Peng Wang, Zongmei Lu, Yong Zhou, Zhiming Zheng, Genhai Zhao
Applied Microbiology and Biotechnology.2022; 106(21): 6993. CrossRef
Cloning and characterization of a recombinant α-glucosidase from Ensifer adhaerens NBRC 100388 and evaluation of its glucosyl transfer activity
Tatsuya Suzuki, Miyu Fukaya, Kazuki Takahashi, Michiki Takeuchi, Ryotaro Hara, Jun Ogawa, Makoto Ueda
Biocatalysis and Agricultural Biotechnology.2020; 30: 101837. CrossRef

Functional expression and enzymatic characterization of Lactobacillus plantarum cyclomaltodextrinase catalyzing novel acarbose hydrolysis: Myoung-Uoon Jang , Hye-Jeong Kang , Chang-Ku Jeong , Yewon Kang , Ji-Eun Park , Tae-Jip Kim; J. Microbiol. 2018;56(2):113-118. Published online February 2, 2018; DOI: https://doi.org/10.1007/s12275-018-7551-3

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Abstract

Cyclomaltodextrinases (CDases) belong to Glycoside Hydrolases (GH) family 13, which show versatile hydrolyzing and/or transglycosylation activity against cyclodextrin (CD), starch, and pullulan. Especially, some CDases have been reported to hydrolyze acarbose, a potent α-glucosidase inhibitor, and transfer the resulting acarviosine-glucose to various acceptors. In this study, a novel CDase (LPCD) gene was cloned from Lactobacillus plantarum WCFS1, which encodes 574 amino acids (64.6 kDa) and shares less than 44% of identities with the known CDase-family enzymes. Recombinant LPCD with C-terminal six-histidines was produced and purified from Escherichia coli. It showed the highest activity on β-CD at 45°C and pH 5.0, respectively. Gel permeation chromatography analysis revealed that LPCD exists as a dodecameric form (~826 kDa). Its hydrolyzing activity on β- CD is almost same as that on starch, whereas it can hardly attack pullulan. Most interestingly, LPCD catalyzed the unique modes of action in acarbose hydrolysis to produce maltose and acarviosine, as well as to glucose and acarviosineglucose.

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Genetic and enzymatic characterization of Amy13E from Cellvibrio japonicus reclassifies it as a cyclodextrinase also capable of α-diglucoside degradation
Giulia M. Mascelli, Cecelia A. Garcia, Jeffrey G. Gardner, Isaac Cann
Applied and Environmental Microbiology.2024;[Epub] CrossRef
Food-grade expression and characterization of cyclomaltodextrinase from B. sphaericus E−244 in Bacillus subtilis
Ruiqi Zhou, Luhua Zheng, Bo Jiang, Weiwei He, Ran Zhang, Jingjing Chen, Assam Bin Tahir
Food Bioscience.2024; 61: 104726. CrossRef
Enhancement of the structure and biochemical function of cyclomaltodextrinase from the Anoxybacillus flavithermus ZNU-NGA with site-directed mutagenesis
Ziba Mirzaee, Vahab Jafarian, Khosrow Khalifeh
International Microbiology.2024;[Epub] CrossRef
A Single Strain of Lactobacillus (CGMCC 21661) Exhibits Stable Glucose- and Lipid-Lowering Effects by Regulating Gut Microbiota
Yuying Wang, Xiaozhong Wang, Xinzhu Xiao, Shufang Yu, Wennan Huang, Benqiang Rao, Fenglin Chen
Nutrients.2023; 15(3): 670. CrossRef
Enzymatic Approaches for Structuring Starch to Improve Functionality
Ming Miao, James N. BeMiller
Annual Review of Food Science and Technology.2023; 14(1): 271. CrossRef
Gut bacteria thwart the blood sugar-lowering effect of acarbose
Melanie M. Brauny, Lisa Maier
Nature Metabolism.2023; 5(5): 732. CrossRef
Investigating the role of carbohydrate-binding module 34 in cyclomaltodextrinase from Geobacillus thermopakistaniensis: structural and functional analyses
Iqra Aroob, Maryam Javed, Nasir Ahmad, Mehwish Aslam, Naeem Rashid
3 Biotech.2022;[Epub] CrossRef
Cyclodextrin-preferring glycoside hydrolases: properties and applications
Iqra Aroob, Nasir Ahmad, Naeem Rashid
Amylase.2021; 5(1): 23. CrossRef
A highly active α-cyclodextrin preferring cyclomaltodextrinase from Geobacillus thermopakistaniensis
Iqra Aroob, Nasir Ahmad, Mehwish Aslam, Abeera Shaeer, Naeem Rashid
Carbohydrate Research.2019; 481: 1. CrossRef

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

Detailed Modes of Action and Biochemical Characterization of endo-Arabinanase from Bacillus licheniformis DSM13: Jung-Mi Park , Myoung-Uoon Jang , Jung-Hyun Kang , Min-Jeong Kim , So-Won Lee , Yeong Bok Song , Chul-Soo Shin , Nam Soo Han , Tae-Jip Kim; J. Microbiol. 2012;50(6):1041-1046. Published online December 30, 2012; DOI: https://doi.org/10.1007/s12275-012-2489-3

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Abstract: An endo-arabinanase (BLABNase) gene from Bacillus licheniformis DSM13 was cloned and expressed in Escherichia coli, and the biochemical properties of its encoded enzyme were characterized. The BLABNase gene consists of a single open reading frame of 987 nucleotides that encodes 328 amino acids with a predicted molecular mass of about 36 kDa. BLABNase exhibited the highest activity against debranched α-(1,5)-arabinan in 50 mM sodium acetate buffer (pH 6.0) at 55°C. Enzymatic characterization revealed that BLABNase hydrolyzes debranched or linear arabinans with a much higher activity than branched arabinan from sugar beet. Enzymatic hydrolysis pattern analyses demonstrated BLABNase to be a typical endo-(1,5)-α-L-arabinanase (EC 3.2.1.99) that randomly cleaves the internal α-(1,5)-linked L-arabinofuranosyl residues of a branchless arabinan backbone to release arabinotriose mainly, although a small amount of arabino-oligosaccharide intermediates is also liberated. Our results indicated that BLABNase acts preferentially along with the oligosaccharides longer than arabinopentaose, thus enabling the enzymatic production of various arabinooligosaccharides.

Identification and Characterization of a Novel β-Galactosidase from Victivallis vadensis ATCC BAA-548, an Anaerobic Fecal Bacterium: Uyangaa Temuujin , Won-Jae Chi , Jae-Sun Park , Yong-Keun Chang , Jae Yang Song , Soon-Kwang Hong; J. Microbiol. 2012;50(6):1034-1040. Published online December 30, 2012; DOI: https://doi.org/10.1007/s12275-012-2478-6

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Abstract: Victivallis vadensis ATCC BAA-548 is a Gram-negative, anaerobic bacterium that was isolated from a human fecal sample. From the genomic sequence of V. vadensis, one gene was found to encode agarase; however, its enzymatic properties have never been characterized. The gene encoding the putative agarase (NCBI reference number ZP_01923925) was cloned by PCR and expressed in E. coli Rosetta-gami by using the inducible T7 promoter of pET28a(+). The expressed protein with a 6×His tag at the N-terminus was named His6-VadG925 and purified as a soluble protein by Ni2+-NTA agarose affinity column chromatography. The purification of the enzyme was 26.8-fold, with a yield of 73.2% and a specific activity of 1.02 U/mg of protein. The purified His6-VadG925 produced a single band with an approximate MW of 155 kDa, which is consistent with the calculated value (154,660 Da) including the 6×His tag. Although VadG925 and many of its homologs were annotated as agarases, it did not hydrolyze agarose. Instead, purified His6-VadG925 hydrolyzed an artificial chromogenic substrate, p-nitrophenyl-β-D-galactopyranoside, but not p-nitrophenyl-α-D-galactopyranoside. The optimum pH and temperature for this β-galactosidase activity were pH 7.0 and 40°C, respectively. The Km and Vmax of His6-VadG925 towards p-nitrophenyl-β-D-galactopyranoside were 1.69 mg/ml (0.0056 M) and 30.3 U/mg, respectively. His6-VadG925 efficiently hydrolyzed lactose into glucose and galactose, which was demonstrated by TLC and mass spectroscopy. These results clearly demonstrated that VadG925 is a novel β-galactosidase that can hydrolyze lactose, which is unusual because of its low homology to validated β-galactosidases.

Journal Article

Symbiotic Relationship between Microbacterium sp. SK0812 and Candida tropicalis SK090404: Seung Won Kang , Bo Young Jeon , Tae Sik Hwang , Doo Hyun Park; J. Microbiol. 2009;47(6):721-727. Published online February 4, 2010; DOI: https://doi.org/10.1007/s12275-009-0146-2

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Abstract: A bacterium growing inside yeast cytoplasm was observed by light microscope without staining. The bacterium was separately stained from yeast cell by a fluorescent dye, 4′,6-diamidino-2-phenylindole (DAPI). The bacterium actively moved inside yeast cytoplasm and propagated in company with the yeast growth. The bacterium was separated from the yeast cytoplasm by selective disruption of yeast cells and the yeast without the intracellular bacterium (YWOB) was obtained by selective inactivation of bacterial cells. The yeast and the intracellular bacterium were identified as Candida tropicalis and Microbacterium sp., respectively. The length of Microbacterium sp. and C. tropicalis measured with SEM image was smaller than 0.5 μm and was larger than 5 μm, respectively. The yeast with the intracellular bacterium (YWIB) grew in a starch-based medium but the YWOB was not C. tropicalis has neither extracellular nor intracellular saccharification enzyme. Glucose was produced from starch by the extracellular crude enzyme (culture fluid) of Microbacterium sp. YWIB produced significantly more ethanol from glucose than YWOB but did not from starch. Conclusively, C. tropicalis is thought to catabolize starch dependent upon Microbacterium sp. growing in its cytoplasm and furnish stable habitat for the Microbacterium sp.

Research Support, Non-U.S. Gov't

Analysis of Double Stranded DNA-dependent Activities of Deinococcus radiodurans RecA Protein: Jong-Il Kim; J. Microbiol. 2006;44(5):508-514.; DOI: https://doi.org/2447 [pii]

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Abstract: In this study, the double-stranded DNA-dependent activities of Deinococcus radiodurans RecA protein (Dr RecA) were characterized. The interactions of the Dr RecA protein with double-stranded DNA were determined, especially dsDNA-dependent ATP hydrolysis by the Dr RecA protein and the DNA strand exchange reaction, in which multiple branch points exist on a single RecA protein-DNA complex. A nucleotide cofactor (ATP or dATP ) was required for the Dr RecA protein binding to duplex DNA. In the presence of dATP, the nucleation step in the binding process occurred more rapidly than in the presence of ATP. Salts inhibited the binding of the Dr RecA protein to double-stranded DNA. Doublestranded DNA-dependent ATPase activities showed a different sensitivity to anion species. Glutamate had only a minimal effect on the double-stranded DNA-dependent ATPase activities, up to a concentration of 0.7 M. In the competition experiment for Dr RecA protein binding, the Dr RecA protein manifested a higher affinity to double-stranded DNA than was observed for single-stranded DNA.

Physiological Relevance of Salt Environment for in vitro recA System: Kim , Jong Il; J. Microbiol. 1999;37(2):59-65.

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Abstract: RecA protein can promote strand assimilation, homologous pairing, and strand exchange. All these reactions require DNA-dependent ATP hydrolysis by recA protein, and the activities of recA protein are affected by the ionic environment. In this experiment, DNA-dependent ATPase activity showed different sensitivity to anionic species. ATP hydrolysis and strand exchange were relatively sensitive to salt in the reactions with NaCl, strongly inhibited at 100 mM NaCl. However, the inhibition by sodium acetate or sodium glutamate was not observed at 50∼100 mM concentration. Addition of sodium glutamate to the standard reaction condition increased the apparent efficiency of ATP hydrolysis during strand exchange. The condition including 50∼100 mM sodium-glutamate might be similar to the physiological condition.

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