Journal of Microbiology

Journal Articles

Rhizosphere Microbial Community and Metabolites of Susceptible and Resistant Tobacco Cultivars to Bacterial Wilt: Wan Zhao , Yanyan Li , Chunlei Yang , Yong Yang , Yun Hu; J. Microbiol. 2023;61(4):389-402. Published online March 7, 2023; DOI: https://doi.org/10.1007/s12275-023-00012-0

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Abstract: Soil-borne diseases are closely related to rhizosphere microecosystem. While, plant species and genotypes are important factors affected rhizosphere microecosystem. In this study, the rhizosphere soil microbial community and metabolites of susceptible and resistant tobacco cultivars were investigated. The results showed that there were significant differences in the rhizosphere microbial community and metabolites between susceptible cultivar Yunyan87 and resistant cultivar Fandi3. Furthermore, the rhizosphere soil of Fandi3 showed a higher microbial diversity than that of Yunyan87. The abundance of R. solanacearum was much higher in the rhizosphere soil of Yunyan87 than in the rhizosphere soil of Fandi3, resulting in a higher disease incidence and index. While the abundance of beneficial bacteria in the rhizosphere soil of Fandi3 were higher than that of Yunyan87. Additionally, there were significant differences in metabolites between Yunyan87 and Fandi3 cultivars, and 4-hydroxybenzaldehyde, 3-hydroxy-4-methoxybenzoic acid, vamillic aldehyde, benzoic acid, 4-hydroxybenzyl alcohol, p-hydroxybenzoic acid and phthalic acid were notably high in Yunyan87. Redundancy analysis (RDA) indicated that the rhizosphere microbial community of Fandi3 and Yunyan87 were highly correlated with various environmental factors and metabolites. Overall, susceptible and resistant tobacco cultivars had different impact on rhizosphere microbial community and metabolites. The results expand our understanding of the roles of tobacco cultivars in plant-micro-ecosystem interactions, and provide a basis for the control of tobacco bacterial wilt.

Kinetic characterization of laccase from Bacillus atrophaeus, and its potential in juice clarification in free and immobilized forms: Lokesh Kumar Narnoliya , Neera Agarwal , Satya N. Patel , Sudhir P. Singh; J. Microbiol. 2019;57(10):900-909. Published online August 28, 2019; DOI: https://doi.org/10.1007/s12275-019-9170-z

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21 Citations

Abstract: In the present study, a laccase gene (BaLc) from a lignin degrading bacterium, Bacillus atrophaeus, has been cloned and expressed in Escherichia coli. The optimal catalytic activity of the protein was achieved at 5.5 pH and 35°C temperature, measured by oxidation of ABTS. The Km and Vmax values were determined as 1.42 mM and 4.16 μmole/min, respectively. To achieve the enzyme recovery, the biocatalyst (BaLc) was covalently attached onto the functionalized iron magnetic-nanoparticles. The nanoparticles were characterized by zeta-potential and FTIR analyses. The immobilized BaLc enzyme was physico-kinetically characterized, exhibiting retention of 60% of the residual activity after ten reaction cycles of ABTS oxidation. The immobilized biocatalyst system was tested for its biotechnological exploitability in plant juice processing, achieving 41–58% of phenol reduction, 41–58% decolorization, 50–59% turbidity reduction in the extracts of banana pseudo-stem and sweet sorghum stalk, and apple fruit juice. This is the first study to demonstrate the use of nanoparticle- laccase conjugate in juice clarification. The findings suggest that B. atrophaus laccase is a potential catalytic tool for plant juice bioprocessing activities.

Transcriptome analysis of differential gene expression in Dichomitus squalens during interspecific mycelial interactions and the potential link with laccase induction: Zixuan Zhong , Nannan Li , Binghui He , Yasuo Igarashi , Feng Luo; J. Microbiol. 2019;57(2):127-137. Published online September 13, 2018; DOI: https://doi.org/10.1007/s12275-019-8398-y

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11 Citations

Abstract: Interspecific mycelial interactions between white rot fungi are always accompanied by an increased production of laccase. In this study, the potential of the white rot fungus Dichomitus squalens to enhance laccase production during interactions with two other white rot fungi, Trametes versicolor or Pleurotus ostreatus, was assessed. To probe the mechanism of laccase induction and the role that laccase plays during combative interaction, we analyzed the differential gene expression profile of the laccase induction response to stressful conditions during fungal interaction. We further confirmed the expression patterns of 16 selected genes by qRT-PCR analysis. We noted that many differentially expressed genes (DEGs) encoded proteins that were involved in xenobiotic detoxification and reactive oxygen species (ROS) generation or reduction, including aldo/keto reductase, glutathione S-transferases, cytochrome P450 enzymes, alcohol oxidases and dehydrogenase, manganese peroxidase and laccase. Furthermore, many DEG-encoded proteins were involved in antagonistic mechanisms of nutrient acquisition and antifungal properties, including glycoside hydrolase, glucanase, chitinase and terpenoid synthases. DEG analyses effectively revealed that laccase induction was likely caused by protective responses to oxidative stress and nutrient competition during interspecific fungal interactions.

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

Transcriptional profiles of laccase genes in the brown rot fungus Postia placenta MAD-R-698: Hongde An , Dongsheng Wei , Tingting Xiao; J. Microbiol. 2015;53(9):606-615. Published online August 1, 2015; DOI: https://doi.org/10.1007/s12275-015-4705-4

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11 Citations

Abstract: One of the laccase isoforms in the brown rot fungus Postia placenta is thought to contribute to the production of hydroxyl radicals, which play an important role in lignocellulose degradation. However, the presence of at least two laccase isoforms in this fungus makes it difficult to understand the details of this mechanism. In this study, we systematically investigated the transcriptional patterns of two laccase genes, Pplcc1 and Pplcc2, by quantitative PCR (qPCR) to better understand the mechanism. The qPCR results showed that neither of the two genes was expressed constitutively throughout growth in liquid culture or during the degradation of a woody substrate. Transcription of Pplcc1 was upregulated under nitrogen depletion and in response to a high concentration of copper in liquid culture, and during the initial colonization of intact aspen wafer. However, it was subject to catabolite repression by a high concentration of glucose. Transcription of Pplcc2 was upregulated by stresses caused by ferulic acid, 2, 6-dimethylbenzoic acid, and ethanol, and under osmotic stress in liquid culture. However, the transcription of Pplcc2 was downregulated upon contact with the woody substrate in solid culture. These results indicate that Pplcc1 and Pplcc2 are differentially regulated in liquid and solid cultures. Pplcc1 seems to play the major role in producing hydroxyl radicals and Pplcc2 in the stress response during the degradation of a woody substrate.

Effect of Natural Mediators on the Stability of Trametes trogii Laccase during the Decolourization of Textile Wastewaters: Rim Khlifi-Slama , Tahar Mechichi , Sami Sayadi , Abdelhafidh Dhouib; J. Microbiol. 2012;50(2):226-234. Published online April 27, 2012; DOI: https://doi.org/10.1007/s12275-012-1421-1

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26 Citations

Abstract: The purpose of the present study was to determine the effect of natural mediators on the stability of the Trametes trogii crude laccase in the process of decolourization of textile effluents. Acetosyringone allowed the highest wastewaters decolourization rate of 25%. At higher concentrations of acetosyringone, the relative activity of laccase decreased approximately by between 38% and 88% after 5 days of incubation. T. trogii laccase was strongly inactivated at 3 mM syringaldehyde, after 3 days of incubation. However, laccase activity is more stable in the presence of the vanillin and m-coumarate. The T. trogii growth on solid effluentbased- medium was examined and evaluated by measuring the colony diameter in cm. T. trogii was completely inhibited on 100:0 and 80:20 effluent:water solid medium, however, colony diameter reached 5 cm on 60:40 effluent:water solid medium after 13–14 days incubation. When the textile effluent was pre-treated with laccase and laccase-acetosyringone system, the colony diameter of 2 cm of T. trogii on 80:20 effluent:water solid medium was reached after 14 and 10 days of incubation respectively.

Journal Article

Purification and Characterization of a Novel Laccase from the Edible Mushroom Hericium coralloides: Ya-Jie Zou , He-Xiang Wang , Tzi-Bun Ng , Chen-Yang Huang , Jin-Xia Zhang; J. Microbiol. 2012;50(1):72-78. Published online February 27, 2012; DOI: https://doi.org/10.1007/s12275-012-1372-6

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40 Citations

Abstract: A novel laccase from the edible mushroom Hericium coralloides was purified by ion exchange chromatography on diethylaminoethyl (DEAE) cellulose, carboxymethyl (CM) cellulose, and Q-Sepharose columns followed by fast protein liquid chromatography gel filtration on a Superdex 75 column. Analysis by gel filtration and SDS-PAGE indicated that the protein is a monomer in solution with a molecular mass of 65 kDa. Its N-terminal amino acid sequence was AVGDDTPQLY, which exhibits partial sequence homology to previously isolated laccases. Optimum activity was observed at pH 2.2 and at 40°C. The enzyme showed activity toward a variety of substrates, the most sensitive of which was 2,2􍿁-azinobis [3-ethylbenzothiazolone-6-sulfonic acid] diammonium salt (ABTS). The degradation activity toward substrates was ABTS > N,N-dimethyl-1,4-phenylenediamine > catechol > 2-methylcatechol > pyrogallol. The laccase did not exert any antiproliferative activity against Hep G2 or MCF 7 tumor cell lines at a concentration of 60 μM, unlike some previously reported mushroom proteins, but showed significant activity toward human immunodeficiency virus-1 (HIV-1) reverse transcriptase with an IC50 of 0.06 μM.

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

Effect of Fungal Pellet Morphology on Enzyme Activities Involved in Phthalate Degradation: Young-Mi Kim , Hong-Gyu Song; J. Microbiol. 2009;47(4):420-424. Published online September 9, 2009; DOI: https://doi.org/10.1007/s12275-009-0051-8

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27 Citations

Abstract: Pellet size of white rot fungus, Pleurotus ostreatus may affect the secretion of its degradative enzymes and accompanying biodegrading capability, but could be controlled by several physical culture conditions in liquid culture. The pellet size of P. ostreatus was affected by the volume of inoculum, flask, and medium, but the agitation speed was the most important control factor. At the lower agitation speed of 100 rpm, the large pellets were formed and the laccase activity was higher than that of small pelleted culture at 150 rpm, which might be due to loose intrapellet structure. However, the biodegradation rates of benzylbutylphthalate and dimethylphthalate were higher in the small pelleted culture, which indicated the involvement of other degradative enzyme rather than laccase. The activity of esterase which catalyzes the nonphenolic compounds before the reaction of ligninolytic enzymes was higher in the small pelleted culture, and coincided with the degradation pattern of phthalates. This study suggests the optimization of pellet morphology and subsequent secretion of degradative enzymes is necessary for the efficient removal of recalcitrants by white rot fungi.

Enhanced Expression of Laccase during the Degradation of Endocrine Disrupting Chemicals in Trametes versicolor: Yunjung Kim , Sumin Yeo , Hong-Gyu Song , Hyoung T. Choi; J. Microbiol. 2008;46(4):402-407. Published online August 31, 2008; DOI: https://doi.org/10.1007/s12275-007-0236-y

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24 Citations

Abstract: A putative laccase cDNA from a white-rot basidiomycete, Trametes versicolor, that consisted of 1,769 nucleotides was cloned using the rapid amplification of cDNA ends (RACE)-PCR method. The deduced amino acid sequence had 4 putative copper binding regions, which are common to fungal laccases. In addition, the sequence was 57~97% homologous to sequences of other T. versicolor laccases. Additionally, the expression of laccase and manganese peroxidase in this fungus were both greatly increased under degrading conditions for bisphenol A, nonylphenol and two phthalic esters (benzylbutylphthalate and diethylphthalate), all of which are reportedly endocrine disrupting chemicals (EDCs). Furthermore, the estrogenic activities of the EDCs also decreased rapidly during incubation when examined in a two-hybrid yeast system. Finally, kojic acid inhibited the removal of estrogenic activities generated by bisphenol A and nonylphenol, which confirmed that laccase was involved in the degradation of EDCs in T. versicolor.

Increase of Yeast Survival under Oxidative Stress by the Expression of the Laccase Gene from Coprinellus congregatus: Dongsik Kim , Eunju Kwak , Hyoung T. Choi; J. Microbiol. 2006;44(6):617-621.; DOI: https://doi.org/2466 [pii]

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Abstract: Coprinellus congregatus secreted a laccase isozyme when the culture was transferred to an acidic liquid medium (pH 4.1). The laccase cDNA gene (clac2) was used as a probe for cloning of the genomic laccase gene (lac2) including the promoter (Plac2). The open reading frame (ORF) of lac2 had 526 deduced amino acids and four conserved copper binding domains as other fungal laccases. Recombinant plasmid (pRSlac2p-cDNA) of lac2 cDNA with its own promoter was transformed in Saccharomyces cerevisiae. Expression of the transformed lac2 gene was induced by oxidative stress (H2O2) in yeast and the survival rate of the transformed yeast strain was greatly increased when compared with that of the control strain transformed with pRS316 yeast vector.

Purification and Characterization of Laccase from the White Rot Fungus Trametes versicolor: Moon-Jeong Han , Hyoung-Tae Choi , Hong-Gyu Song; J. Microbiol. 2005;43(6):555-560.; DOI: https://doi.org/2290 [pii]

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Abstract: Laccase is one of the ligninolytic enzymes of white rot fungus Trametes versicolor 951022, a strain first isolated in Korea. This laccase was purified 209-fold from culture fluid with a yield of 6.2% using ethanol precipitation, DEAE-Sepharose, Phenyl-Sepharose, and Sephadex G-100 chromatography. T. versicolor 951022 excretes a single monomeric laccase showing a high specific activity of 91,443 U/mg for 2,2''-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) as a substrate. The enzyme has a molecular mass of approximately 97 kDa as determined by SDS-PAGE, which is larger than those of other laccases reported. It exhibits high enzyme activity over broad pH and temperature ranges with optimum activity at pH 3.0 and a temperature of 50oC. The Km value of the enzyme for substrate ABTS is 12.8 M and its corresponding Vmax value is 8125.4 U/mg. The specific activity and substrate affinity of this laccase are higher than those of other white rot fungi, therefore, it may be potentially useful for industrial purposes.

Journal Article

Laccase Production Using Pleurotus ostreatus 1804 Immobilized on PUF Cubes in Batch and Packed Bed Reactors: Influence of Culture Conditions: K. Krishna Prasad , S. Venkata Mohan , Y. Vijaya Bhaskar , S. V. Ramanaiah , V. Lalit Babu , B. R. Pati , P. N. Sarma; J. Microbiol. 2005;43(3):301-307.; DOI: https://doi.org/2209 [pii]

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Abstract: The feasibility of laccase production by immobilization of Pleurotus ostreatus 1804 on polyurethane foam (PUF) cubes with respect to media composition was studied in both batch and reactor systems. Enhanced laccase yield was evidenced due to immobilization. A relatively high maximum laccase activity of 312.6 U was observed with immobilized mycelia in shake flasks compared to the maximum laccase activity of free mycelia (272.2 U). It is evident from this study that the culture conditions studied, i.e. biomass level, pH, substrate concentration, yeast extract concentration, Cu^2^+ concentration, and alcohol nature, showed significant influence on the laccase yield. Gel electrophoretic analysis showed the molecular weight of the laccase produced by immobilized P. ostreatus to be 66 kDa. The laccase yield was significantly higher and more rapid in the packed bed reactor than in the shake flask experiments. A maximum laccase yield of 392.9 U was observed within 144 h of the fermentation period with complete glucose depletion.

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

Degradation of Phenanthrene by Trametes versicolor and Its Laccase: Mun-Jung Han , Hyoung-Tae Choi , Hong-Gyu Song; J. Microbiol. 2004;42(2):94-98.; DOI: https://doi.org/2039 [pii]

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Abstract: Phenanthrene is a three-ring polycyclic aromatic hydrocarbon and commonly found as a pollutant in various environments. Degradation of phenanthrene by white rot fungus Trametes versicolor 951022 and its laccase, isolated in Korea, was investigated. After 36 h of incubation, about 46% and 65% of 100 mg/l of phenanthrene added in shaken and static fungal cultures were removed, respectively. Phenanthrene degradation was maximal at pH 6 and the optimal temperature for phenanthrene removal was 30^oC. Although the removal percentage of phenanthrene was highest (76.7%) at 10 mg/ l of phenanthrene concentration, the transformation rate was maximal (0.82 mg/h) at 100 mg/L of phenanthrene concentration in the fungal culture. When the purified laccase of T. versicolor 951022 reacted with phenanthrene, phenanthrene was not transformed. The addition of redox mediator, 2,2'- azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) or 1-hydroxybenzotriazole (HBT) to the reaction mixture increased oxidation of phenanthrene by laccase about 40% and 30%, respectively.

Journal Article

Molecular Characteristics of Two Laccase from the Basidiomycete Fungus Polyporus brumalis: Sun-Hwa Ryu , A-Young Lee , Myungkil Kim; J. Microbiol. 2008;46(1):62-69.; DOI: https://doi.org/10.1007/s12275-007-0110-y

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14 Citations

Abstract: Two laccase cDNAs, pblac1 and pblac2, were cloned from a white-rot fungus strain, Polyporus brumalis (KFRI 20912). The cloned cDNAs consisted of 1,829 bp and 1,804 bp, and their open reading frames encoded proteins of 520 and 524 amino acids, with calculated molecular masses of approximately 55.9 kDa and 56 kDa, respectively. The deduced amino acid sequences of each protein showed 70% similarity. The copper binding regions were conserved in both proteins, as in other fungal laccases. RT-PCR analysis revealed that the transcript levels of the two laccases increased progressively in shallow stationary culture liquid medium. The transcript level of each laccase was induced when the fungus was exposed to di-butyl phthalate (DBP), suggesting that the two laccases are involved in DBP degradation. The overexpression of the pblac1 gene was derived by the promoter of a gene for glyceraldehyde-3-phosphate dehydrogenase, using a homologous system. The activity of laccase in the transformants was significantly higher than that of the wild type. The identification of these laccase cDNAs was a first step to characterize the molecular events related to the lignin degradation ability of this basidiomycetous fungus, as well as the degradation of many recalcitrant xenobiotics.

Purification and Properties of Laccase of the White-rot Basidiomycete Coriolus hirsutus: Lee, Yeo Jin , Shin, Kwnag Soo; J. Microbiol. 1999;37(3):148-153.

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Abstract: Laccase produced by Coriolus hirsutus was purified to electrophoretic homogeneity by acetone precipitation, Sephacryl S-2000 HR chromatography, DEAE Sepharose CL-6B chromatography, and Mono Q HR 5/5 chromatography. The purification of laccase was 46.6-fold with an overall yield of 23.7%. Laccase from this fungus was a monomeric glycoprotein with 16% carbohydrate content, and has an isoelectric point of 4.2, and molecular mass of 78 kDa, respectively. The N-terminal amino acid sequence of the enzyme showed significant homology to hoste of laccases from Coriolus versicolor, Pycnoporus cinnabarius, and an unidentified basidiomycete, PM1. The highest rate of 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) oxidation by laccase was reached at 45℃, and the pH optima of the enzyme varied depending on the substrate in the range of 2.0 to 4.5. The enzyme was stable at 60℃ for 5 h and lost 80% activity at 80℃ in 30 min. The enzyme oxidized a variety of usual laccase substrates including lignin-related phenol, and had the highest affinity toward ABTS. Under standard assay conditions, the apparent Km value of the enzyme toward ABTS was 8.1 μM. The enzyme was completely inhibited by L-cysteine and sodium azide, but not by potassium cyanide, SDS, ad thiourea.

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