Journal of Microbiology

Reviews

[Minireview]Cytoplasmic molecular chaperones in Pseudomonas species: Hyunhee Kim , Seongjoon Moon , Soojeong Ham , Kihyun Lee , Ute Römling , Changhan Lee; J. Microbiol. 2022;60(11):1049-1060. Published online November 1, 2022; DOI: https://doi.org/10.1007/s12275-022-2425-0

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Pseudomonas is widespread in various environmental and host niches. To promote rejuvenation, cellular protein homeostasis must be finely tuned in response to diverse stresses, such as extremely high and low temperatures, oxidative stress, and desiccation, which can result in protein homeostasis imbalance. Molecular chaperones function as key components that aid protein folding and prevent protein denaturation. Pseudomonas, an ecologically important bacterial genus, includes human and plant pathogens as well as growth-promoting symbionts and species useful for bioremediation. In this review, we focus on protein quality control systems, particularly molecular chaperones, in ecologically diverse species of Pseudomonas, including the opportunistic human pathogen Pseudomonas aeruginosa, the plant pathogen Pseudomonas syringae, the soil species Pseudomonas putida, and the psychrophilic Pseudomonas antarctica.

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An innovative utilization approach for by-products of biogas desulfurization：Co-hydrothermal treatment of sulfur with biogas slurry to prepare sulfur-enriched liquid fertilizer
Zhijie Xie, Fang Deng, Yuqin Wan, Yiping Luo, Qin Cao, Yichao Chen, Dong Li
Process Biochemistry.2024; 147: 522. CrossRef
Isolation of Pseudomonas oleovorans Carrying Multidrug Resistance Proteins MdtA and MdtB from Wastewater
Haifeng Wang, Chenyang Sun, Xing Chen, Kai Yan, Hongxuan He
Molecules.2023; 28(14): 5403. CrossRef
Development of heat-shock resistance in Legionella pneumophila modeled by experimental evolution
Jeffrey Liang, Gillian Cameron, Sébastien P. Faucher, Christopher A. Elkins
Applied and Environmental Microbiology.2023;[Epub] CrossRef

MINIREVIEW] Modern and Simple Construction of Plasmid: Saving Time and Cost: Hideki Nakayama , Nobuo Shimamoto; J. Microbiol. 2014;52(11):891-897. Published online October 31, 2014; DOI: https://doi.org/10.1007/s12275-014-4501-6

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Abstract

Construction of plasmids has been occupying a significant fraction of laboratory work in most fields of experimental biology. Tremendous effort was made to improve the traditional method for constructing plasmids, in which DNA fragments digested with restriction enzymes were ligated. However, the traditional method remained to be a standard protocol more than 40 years. At last, several recent inventions are rapidly and completely replacing the traditional method, because they are far quicker with less cost, and requiring less material. We here introduce three such methods that cover up most of the cases. Moreover, they are complementary with each other. Our lab protocols are provided for “no strain, no pain” construction of plasmids.

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Tumor biology experimental course design based on the integration of molecular biology and metabolomics
Xinliang Zhu, Ting Tang
Cogent Education.2024;[Epub] CrossRef
Truncation-Free Genetic Code Expansion with Tetrazine Amino Acids for Quantitative Protein Ligations
Alex J. Eddins, Riley M. Bednar, Subhashis Jana, Abigail H. Pung, Lea Mbengi, Kyle Meyer, John J. Perona, Richard B. Cooley, P. Andrew Karplus, Ryan A. Mehl
Bioconjugate Chemistry.2023; 34(12): 2243. CrossRef
Involvement of GcvB small RNA in intrinsic resistance to multiple aminoglycoside antibiotics in Escherichia coli
Akira Muto, Simon Goto, Daisuke Kurita, Chisato Ushida, Hyota Himeno
The Journal of Biochemistry.2021; 169(4): 485. CrossRef
Flagellum-mediated motility in Pelotomaculum thermopropionicum SI
Tomoyuki Kosaka, Mutsumi Goda, Manami Inoue, Toshiharu Yakushi, Mamoru Yamada
Bioscience, Biotechnology, and Biochemistry.2019; 83(7): 1362. CrossRef
Natural killer cells unleashed: Checkpoint receptor blockade and BiKE/TriKE utilization in NK-mediated anti-tumor immunotherapy
Zachary B. Davis, Daniel A. Vallera, Jeffrey S. Miller, Martin Felices
Seminars in Immunology.2017; 31: 64. CrossRef
In vivo Assembly in Escherichia coli of Transformation Vectors for Plastid Genome Engineering
Yuyong Wu, Lili You, Shengchun Li, Meiqi Ma, Mengting Wu, Lixin Ma, Ralph Bock, Ling Chang, Jiang Zhang
Frontiers in Plant Science.2017;[Epub] CrossRef
Role of 100S ribosomes in bacterial decay period
Ksenia Shcherbakova, Hideki Nakayama, Nobuo Shimamoto
Genes to Cells.2015; 20(10): 789. CrossRef

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

Use of Denaturing High-Performance Liquid Chromatography (DHPLC) to Characterize the Bacterial and Fungal Airway Microbiota of Cystic Fibrosis Patients: Jérôme Mounier , Audrey Gouëllo , Marlène Keravec , Solène Le Gal , Grégory Pacini , Stella Debaets , Gilles Nevez , Gilles Rault , Georges Barbier , Geneviève Héry-Arnaud; J. Microbiol. 2014;52(4):307-314. Published online February 17, 2014; DOI: https://doi.org/10.1007/s12275-014-3425-5

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Abstract

The aim of this study was to evaluate the use of denaturing high-performance liquid chromatography (DHPLC) to characterize cystic fibrosis (CF) airway microbiota including both bacteria and fungi. DHPLC conditions were first optimized using a mixture of V6, V7 and V8 region 16S rRNA gene PCR amplicons from 18 bacterial species commonly found in CF patients. Then, the microbial diversity of 4 sputum samples from 4 CF patients was analyzed using cultural methods, cloning/sequencing (for bacteria only) and DHPLC peak fraction collection/sequencing. DHPLC analysis allowed identifying more bacterial and fungal species than the classical culture methods, including well-recognized pathogens such as Pseudomonas aeruginosa. Even if a lower number of bacterial Operational Taxonomic Units (OTUs) was identified by DHPLC, it allowed to find OTUs unidentified by cloning/sequencing. The combination of both techniques permitted to correlate the majority of DHPLC peaks to defined OTUs. Finally, although Aspergillus fumigatus detection using DHPLC can still be improved, this technique clearly allowed to identify a higher number of fungal species versus classical culture-based methods. To conclude, DHPLC provided meaningful additional data concerning pathogenic bacteria and fungi as well as fastidious microorganisms present within the CF respiratory tract. DHPLC can be considered as a complementary technique to culture-dependent analyses in routine microbiological laboratories.

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Chronic Diseases Associated with Malassezia Yeast
Abdourahim Abdillah, Stéphane Ranque
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The Mycobiome in Health and Disease: Emerging Concepts, Methodologies and Challenges
Pei Yee Tiew, Micheál Mac Aogain, Nur A’tikah Binte Mohamed Ali, Kai Xian Thng, Karlyn Goh, Kenny J. X. Lau, Sanjay H. Chotirmall
Mycopathologia.2020;[Epub] CrossRef
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Medical Mycology.2018; 56(suppl_1): S42. CrossRef
Aspergillus Species in Bronchiectasis: Challenges in the Cystic Fibrosis and Non-cystic Fibrosis Airways
Sanjay H. Chotirmall, Maria Teresa Martin-Gomez
Mycopathologia.2018; 183(1): 45. CrossRef
Fusarium Profiling and Barley Malt Gushing Propensity
Vertti Virkajärvi, Tuija Sarlin, Arja Laitila
Journal of the American Society of Brewing Chemists.2017; 75(3): 181. CrossRef
The lung mycobiome: an emerging field of the human respiratory microbiome
Linh D. N. Nguyen, Eric Viscogliosi, Laurence Delhaes
Frontiers in Microbiology.2015;[Epub] CrossRef
The Airway Colonization by Opportunistic Filamentous Fungi in Patients with Cystic Fibrosis: Recent Updates
Kada Touati, Do Ngoc Linh Nguyen, Laurence Delhaes
Current Fungal Infection Reports.2014; 8(4): 302. CrossRef
Characterization and quantification of the fungal microbiome in serial samples from individuals with cystic fibrosis
Sven D Willger, Sharon L Grim, Emily L Dolben, Anna Shipunova, Thomas H Hampton, Hilary G Morrison, Laura M Filkins, George A O‘Toole, Lisa A Moulton, Alix Ashare, Mitchell L Sogin, Deborah A Hogan
Microbiome.2014;[Epub] CrossRef

Identification and Characterization of Ectoine Biosynthesis Genes and Heterologous Expression of the ectABC Gene Cluster from Halomonas sp. QHL1, a Moderately Halophilic Bacterium Isolated from Qinghai Lake: Derui Zhu , Jian Liu , Rui Han , Guoping Shen , Qifu Long , Xiaoxing Wei , Deli Liu; J. Microbiol. 2014;52(2):139-147. Published online February 1, 2014; DOI: https://doi.org/10.1007/s12275-014-3389-5

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Abstract

The moderately halophilic bacterium Halomonas sp. QHL1 was identified as a member of the genus Halomonas by 16S rRNA gene sequencing. HPLC analysis showed that strain QHL1 synthesizes ectoine in its cytoplasm. The genes involved in the ectoine biosynthesis pathway were identified on the chromosome in the order ectABC. Subsequently, the ectB gene from this strain was amplified by PCR, and the entire ectABC gene cluster (3,580 bp) was cloned using genome walking. Analysis showed that the ectA (579 bp), ectB (1269 bp), and ectC (390 bp) genes were organized in a single transcriptional unit and were predicted to encode three peptides of 21.2 kDa, 46.4 kDa, and 14.7 kDa, respectively. Two putative promoters, a δ70-dependent promoter and a δ38-controlled promoter, as well as several conserved motifs with unknown function were identified. Individual ectA, ectB, and ectC genes, and the entire ectABC gene cluster were inserted into the expression plasmid pET-28a(+) to generate the recombinant plasmids pET-28a(+)-ectA, pET-28a(+)-ectB, pET-28a(+)-ectC and pET-28a(+)-ectABC, respectively. Heterologous expression of these proteins in Escherichia coli BL21 (DE3) was confirmed by SDS-PAGE. The recombinant E. coli strain BL21 (pET-28a (+)-ectABC) displayed a higher salt tolerance than native E. coli cells but produced far less ectoine than the wild-type QHL1 strain.

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Biotechnological production of ectoine: current status and prospects
Jun Chen, Deliang Qiao, Tao Yuan, Yeyuan Feng, Pengjun Zhang, Xuejun Wang, Li Zhang
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Zhibo Wang, Yongzhen Li, Xiang Gao, Jiangwa Xing, Rong Wang, Derui Zhu, Guoping Shen
International Microbiology.2023; 26(4): 1009. CrossRef
Comparative Genome Analysis of a Novel Alkaliphilic Actinobacterial Species Nesterenkonia haasae
Shuang Wang, Lei Sun, Manik Prabhu Narsing Rao, Bao‑zhu Fang, Wen‑jun Li
Polish Journal of Microbiology.2022; 71(3): 453. CrossRef
Whole genome sequencing of the halophilic Halomonas qaidamensis XH36, a novel species strain with high ectoine production
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Antonie van Leeuwenhoek.2022; 115(4): 545. CrossRef
Identification and characterization of an ectoine biosynthesis gene cluster from Aestuariispira ectoiniformans sp. nov., isolated from seawater
Ji Young Kang, Binna Lee, Jeong Ah Kim, Min-Soo Kim, Chul Ho Kim
Microbiological Research.2022; 254: 126898. CrossRef
Genome Sequence of Streptomyces sp. Strain GQFP Isolated from Soil Near the Roots of Pharmaceutical Plant Elaeagnus pungens
Jie Zhu, David A. Baltrus
Microbiology Resource Announcements.2022;[Epub] CrossRef
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Sukhyeong Cho, Yun Seo Lee, Hanyu Chai, Sang Eun Lim, Jeong Geol Na, Jinwon Lee
Biotechnology for Biofuels and Bioproducts.2022;[Epub] CrossRef
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Applied Sciences.2021; 11(15): 6873. CrossRef
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Qi Zhao, Shannan Li, Peiwen Lv, Simian Sun, Cuiqing Ma, Ping Xu, Haijun Su, Chunyu Yang
Microbial Cell Factories.2019;[Epub] CrossRef
Analysis of Zobellella denitrificans ZD1 draft genome: Genes and gene clusters responsible for high polyhydroxybutyrate (PHB) production from glycerol under saline conditions and its CRISPR-Cas system
Yu-Wei Wu, Shih-Hung Yang, Myung Hwangbo, Kung-Hui Chu, Chih-Horng Kuo
PLOS ONE.2019; 14(9): e0222143. CrossRef
Engineering the Salt-Inducible Ectoine Promoter Region of Halomonas elongata for Protein Expression in a Unique Stabilizing Environment
Lisa Stiller, Erwin Galinski, Elisabeth Witt
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Constanze Paulus, Yuriy Rebets, Josef Zapp, Christian Rückert, Jörn Kalinowski, Andriy Luzhetskyy
Frontiers in Microbiology.2018;[Epub] CrossRef
Metabolic engineering of Escherichia coli for L-tryptophan production
V. B. Panichkin, V. A. Livshits, I. V. Biryukova, S. V. Mashko
Applied Biochemistry and Microbiology.2016; 52(9): 783. CrossRef
Draft Genome Sequence of Halomonas elongata Strain K4, an Endophytic Growth-Promoting Bacterium Enhancing Salinity Tolerance In Planta
Feras F. Lafi, Juan S. Ramirez-Prado, Intikhab Alam, Vladimir B. Bajic, Heribert Hirt, Maged M. Saad
Genome Announcements.2016;[Epub] CrossRef
High production of ectoine from aspartate and glycerol by use of whole-cell biocatalysis in recombinant Escherichia coli
Yong-Zhi He, Jiao Gong, Hai-Ying Yu, Yong Tao, Shan Zhang, Zhi-Yang Dong
Microbial Cell Factories.2015;[Epub] CrossRef
Design of an ectoine-responsive AraC mutant and its application in metabolic engineering of ectoine biosynthesis
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Review

MINIREVIEW] Development of Diagnostic and Vaccine Markers Through Cloning, Expression, and Regulation of Putative Virulence-Protein-Encoding Genes of Aeromonas hydrophila: Vijai Singh , Dharmendra Kumar Chaudhary , Indra Mani , Rohan Jain , B.N. Mishra; J. Microbiol. 2013;51(3):275-282. Published online June 28, 2013; DOI: https://doi.org/10.1007/s12275-013-2437-x

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Abstract: Aeromonas hydrophila is an opportunistic bacterial pathogen that is associated with a number of diseases in fish, amphibians, reptiles, and humans. In fish it causes several disease symptoms including tail and skin rot, and haemorrhagic septicemia; in human it causes soft-tissue wound infection and diarrhoea. The pathogenesis of A. hydrophila is multifactorial, but the mechanism is unknown so far. It is considered to be mediated by expression and secretion of extracellular proteins such as aerolysin, lipase, chitinase, amylase, gelatinase, hemolysins, and enterotoxins. A number of the putative virulence-protein-encoding genes that are present in the genome of A. hydrophila have been targeted by PCR for molecular diagnosis. These significant genes are also targeted for over-production of proteins by cloning and expression methods. In this review, we emphasize recent progress in the cloning, expression, and regulation of putative virulence-protein-encoding genes of A. hydrophila for a better understanding of the pathogenesis and also help to provide effective strategies for control of diseases.

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

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.

cDNA Cloning of Korean Human Norovirus and Nucleotidylylation of VPg by Norovirus RNA-Dependent RNA Polymerase: Byung Sup Min , Kang Rok Han , Jung Ihn Lee , Jai Myung Yang; J. Microbiol. 2012;50(4):625-630. Published online August 25, 2012; DOI: https://doi.org/10.1007/s12275-012-2087-4

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Abstract: Norovirus, a member of the Caliciviridae family, is a major causative agent of gastroenteritis worldwide. The cDNA of the entire genome of human norovirus (HuNV) was cloned using the RNA extracted from the stool sample of a Korean patient. The RNA genome consists of 7,559 nucleotides, carries 3 open reading frames (ORFs), 5' and 3' noncoding regions, and a poly(A) tail at the 3' end. Phylogenic analysis of the nucleotide sequence indicated that it belongs to GII.4, the most dominant genogroup. To analyze RNA synthesis and nucleotidylylation of VPg by RNA-dependent RNA polymerase (RdRp), recombinant RdRp and VPg were expressed in Escherichia coli as His-tagged forms. The HuNV RdRp exhibited template and divalent cation-dependent RNA synthesis in vitro. The HuNV RdRp nucleotidylylated HuNV VPg but not murine norovirus (MNV) VPg, whereas MNV RdRp nucleotidylylated both MNV and HuNV VPg more efficiently than HuNV RdRp.

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

NOTE] Fosmid Cloning, Nucleotide Sequence, and Characterization of a Beta-Lactamase Gene from Subsurface Isolates: Nurcan Vardar , Gönül Vardar-Schara; J. Microbiol. 2012;50(4):680-683. Published online July 21, 2012; DOI: https://doi.org/10.1007/s12275-012-2139-9

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Abstract: A beta-lactamase gene was isolated for the first time from a terrestrial subsurface environment using a combined cultivation and direct cloning strategy. The gene, discovered from 24 m below land surface in Hawaii, was most similar to the penicillinase from Bacillus licheniformis. The resistance gene was confirmed via subcloning and its minimum inhibitory concentration values were measured against several test betalactam antibiotics. This study extends the knowledge on resistance to antimicrobials, which may help the efforts to minimize their future threat.

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

Comparative Approach to Capture Bacterial Diversity of Coastal Waters: Hyunsoo Na , Ok-Sun Kim , Seok-Hwan Yoon , Yunmin Kim , Jongsik Chun; J. Microbiol. 2011;49(5):729-740. Published online November 9, 2011; DOI: https://doi.org/10.1007/s12275-011-1205-z

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Abstract: Despite the revolutionary advancements in DNA sequencing technology and cultivation techniques, few studies have been done to directly compare these methods. In this study, a 16S rRNA gene-based, integrative approach combining culture-independent techniques with culture-dependent methods was taken to investigate the bacterial community structure of coastal seawater collected from the Yellow Sea, Korea. For culture-independent studies, we used the latest model pyrosequencer, Roche/454 Genome Sequencer FLX Titanium. Pyrosequencing captured a total of 52 phyla including 27 candidate divisions from the water column, whereas the traditional cloning approach captured only 15 phyla including 2 candidate divisions. In addition, of 878 genera retrieved, 92.1% of the sequences were unique to pyrosequencing. For culture-dependent analysis, plate culturing, plate washing, enrichment, and high-throughput culturing (HTC) methods were applied. Phylogenetic analysis showed that the plate-washing clones formed a cluster devoid of any previously cultured representatives within the family Rhodobacteraceae. One HTC isolate (SF293) fell into the OM182 clade, which was not recovered by other culturing methods described here. By directly comparing the sequences obtained from cultures with those from culture-independent work, we found that only 33% of the culture sequences were identical to those from clone libraries and pyrosequences. This study presents a detailed comparison of common molecular and cultivation techniques available in microbial ecology. As different methods yielded different coverage, we suggest choosing the approach after carefully examining the scientific questions being asked.

The ATPase Activity of The G2alt Gene Encoding an Aluminium Tolerance Protein from Anoxybacillus gonensis G2: Fatih Saban Beris , Lina De Smet , Hakan Karaoglu , Sabriye Canakci , Jozef Van Beeumen , Ali Osman Belduz; J. Microbiol. 2011;49(4):641-650. Published online September 2, 2011; DOI: https://doi.org/10.1007/s12275-011-0522-6

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Abstract: The G2ALT gene was cloned and sequenced from the thermophilic bacterium Anoxybacillus gonensis G2. The gene is 666 bp long and encodes a protein 221 amino acids in length. The gene was overexpressed in E. coli and purified to homogeneity and biochemically characterized. The enzyme has a molecular mass of 24.5 kDa and it could be classified as a member of the family of bacterial aluminium resistance proteins based on homology searches. When this fragment was expressed in E. coli, it endowed E. coli with Al tolerance to 500 μM. The purified G2ALT protein is active at a broad pH range (pH 4.0-10.0) and temperature range (25°C-80°C) with optima of 6.0 and the apparent optimal temperature of 73°C respectively. Under optimal conditions, G2ALT exhibited a low ATPase activity with Km- and Vmax- values of 10±0.55 μM and 26.81±0.13 mg Pi released/min/mg enzyme, respectively. The ATPase activity of G2ALT requires Mg2+ and Na+ ions, while Zn2+ and Al3+ stimulate the activity. Cd2+ and Ag+ reduced the activity and Li+, Cu2+, and Co2+ inhibited the activity. Known inhibitors of most ATPases, like such as β-mercaptoethanol and ouabain, also inhibited the activity of the G2ALT. These biochemical characterizations suggested that G2ALT belongs to the PP-loop ATPase superfamily and it can be responsible for aluminium tolerance in A. gonensis G2.

Identification of the Genes Involved in 1-Deoxynojirimycin Synthesis in Bacillus subtilis MORI 3K-85: Kyung-Don Kang , Yong Seok Cho , Ji Hye Song , Young Shik Park , Jae Yeon Lee , Kyo Yeol Hwang , Sang Ki Rhee , Ji Hyung Chung , Ohsuk Kwon , Su-Il Seong; J. Microbiol. 2011;49(3):431-440. Published online June 30, 2011; DOI: https://doi.org/10.1007/s12275-011-1238-3

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Abstract: 1-Deoxynojirimycin (DNJ), a D-glucose analogue with a nitrogen atom substituting for the ring oxygen, is a strong inhibitor of intestinal α-glucosidase. DNJ has several promising biological activities, including its antidiabetic, antitumor, and antiviral activities. Nevertheless, only limited amounts of DNJ are available because it can only be extracted from some higher plants, including the mulberry tree, or purified from the culture broth of several types of soil bacteria, such as Streptomyces sp. and Bacillus sp. In our previous study, a DNJ-producing bacterium, Bacillus subtilis MORI, was isolated from the traditional Korean fermented food Chungkookjang. In the present study, we report the identification of the DNJ biosynthetic genes in B. subtilis MORI 3K-85 strain, a DNJ-overproducing derivate of the B. subtilis MORI strain generated by γ-irradiation. The genomic DNA library of B. subtilis MORI 3K-85 was constructed in Escherichia coli, and clones showing α-glucosidase inhibition activity were selected. After DNA sequencing and a series of subcloning, we were able to identify a putative operon which consists of gabT1, yktc1, and gutB1 genes predicted to encode putative transaminase, phosphatase, and oxidoreductase, respectively. When a recombinant plasmid containing this operon sequence was transformed into an E. coli strain, the resulting transformant was able to produce DNJ into the culture medium. Our results indicate that the gabT1, yktc1, and gutB1 genes are involved in the DNJ biosynthetic pathway in B. subtilis MORI, suggesting the possibility of employing these genes to establish a large-scale microbial DNJ overproduction system through genetic engineering and process optimization.

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).

Journal Article

Cloning, Expression, and Characterization of Serine Protease from Thermophilic Fungus Thermoascus aurantiacus var. levisporus: An-Na Li , Chen Xie , Jie Zhang , Jia Zhang , Duo-Chuan Li; J. Microbiol. 2011;49(1):121-129. Published online March 3, 2011; DOI: https://doi.org/10.1007/s12275-011-9355-6

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Abstract: The serine protease gene from a thermophilic fungus Thermoascus aurantiacus var. levisporus, was cloned, sequenced, and expressed in Pichia pastoris and the recombinant protein was characterized. The full-length cDNA of 2,592 bp contains an ORF of 1,482 bp encoding 494 amino acids. Sequence analysis of the deduced amino acid sequence revealed high homology with subtilisin serine proteases. The putative enzyme contained catalytic domain with active sites formed by three residues of Asp183, His215, and Ser384. The molecular mass of the recombinant enzyme was estimated to be 59.1 kDa after overexpression in P. pastoris. The activity of recombinant protein was 115.58 U/mg. The protease exhibited its maximal activity at 50°C and pH 8.0 and kept thermostable at 60°C, and retained 60% activity after 60 min at 70°C. The protease activity was found to be inhibited by PMSF, but not by DTT or EDTA. The enzyme has broad substrate specificity such as gelatin, casein and pure milk, and exhibiting highest activity towards casein.

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

Cloning, Expression, and Characterization of Thermostable Manganese Superoxide Dismutase from Thermoascus aurantiacus var. levisporus: Ning-Ning Song , Yan Zheng , Shi-Jin E , Duo-Chuan Li; J. Microbiol. 2009;47(1):123-130. Published online February 20, 2009; DOI: https://doi.org/10.1007/s12275-008-0217-9

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Abstract: A superoxide dismutase (SOD) gene of Thermoascus aurantiacus var. levisporus, a thermophilic fungus, was cloned, sequenced, and expressed in Pichia pastoris and its gene product was characterized. The coding sequence predicted a 231 residues protein with a unique 35 amino acids extension at the N-terminus indicating a mitochondrial-targeting sequence. The content of Mn was 2.46 ug/mg of protein and Fe was not detected in the purified enzyme. The enzyme was found to be inhibited by NaN3, but not by KCN or H2O2. These results suggested that the SOD in Thermoascus aurantiacus var. levisporus was the manganese superoxide dismutase type. In comparison with other MnSODs, all manganese-binding sites were also conserved in the sequence (H88, H136, D222, H226). The molecular mass of a single band of the enzyme was estimated to be 21.7 kDa. The protein was expressed in tetramer form with molecular weight of 68.0 kDa. The activity of purified protein was 2,324 U/mg. The optimum temperature of the enzyme was 55oC and it exhibited maximal activity at pH 7.5. The enzyme was thermostable at 50 and 60oC and the half-life at 80oC was approximately 40 min.

Molecular Cloning of the Phospholipase D Gene from Streptomyces sp. YU100 and Its Expression in Escherichia coli: Ji-Seon Lee , Munkhtsetseg Bat-Ochir , Atanas V. Demirev , Doo Hyun Nam; J. Microbiol. 2009;47(1):116-122. Published online February 20, 2009; DOI: https://doi.org/10.1007/s12275-008-0161-8

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Abstract: The gene for phospholipase D (PLD) of Streptomyces sp. YU100 was cloned from λ phage library and heterologously expressed in Escherichia coli. Using an amplified gene fragment based on the consensus sequences of streptomycetes PLDs, λ phage library of Streptomyces sp. YU100 chromosomal DNA was screened. The sequencing result of BamHI-digested 3.8 kb fragment in a positive phage clone revealed the presence of an open reading frame of a full sequence of PLD gene encoding a 540-amino acid protein including 33-amino acid signal peptide. The deduced amino acid sequence showed a high homology with other Streptomyces PLDs, having the highly conserved ‘HKD’ motifs. The PLD gene excluding signal peptide sequence was amplified and subcloned into a pET-32b(+) expression vector in E. coli BL21(DE3). The recombinant PLD was purified by nickel affinity chromatography and compared the enzyme activity with wild-type PLD. The results imply that the recombinant PLD produced by E. coli had the nearly same enzyme activity as PLD from Streptomyces sp. YU100.

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