Journal of Microbiology

Journal Articles

The role of Jacalin-related lectin gene AOL_s00083g511 in the development and pathogenicity of the nematophagous fungus Arthrobotrys oligospora: Xinyuan Dong , Jiali Si , Guanghui Zhang , Zhen Shen , Li Zhang , Kangliang Sheng , Jingmin Wang , Xiaowei Kong , Xiangdong Zha , Yongzhong Wang; J. Microbiol. 2021;59(8):736-745. Published online July 5, 2021; DOI: https://doi.org/10.1007/s12275-021-1029-4

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Abstract: Arthrobotrys oligospora is a model species of nematophagous fungi and has great potential for the biological control of nematode diseases. Lectin is a protein that binds to carbohydrates and their complexes with high specificity, which mediates recognition events in various physiological and pathological processes. This study aimed to investigate the role of the Jacalin-related lectin (JRL) gene, AOL_s00083g511, in A. oligospora development. Through a homology recombination approach, we obtained the AOL_s00083g511 knockout mutant strain (Δg511). Next, the biological characteristics of the Δg511 mutant strain, including growth rate, conidia germination rate, adaptation to environmental stresses, and nematocidal activity, were compared with those of the wild-type (WT) strain. The results showed that the JRL gene AOL_ s00083g511 did not affect fungal growth, conidia germination, 3D-trap formation, and the ability of A. oligospora to prey on nematodes significantly. We speculate that this phenomenon may be caused by a loss of the key β1–β2 loops in the AOL_ s00083g511-encoded JRL domain and an intrinsic genetic compensation of AOL_s00083g511 in this fungus. The growth rates of both strains on high salt or surfactant media were similar; however, in the strong oxidation medium, the growth rate of the Δg511 mutant was significantly lower than that of the WT strain, indicating that AOL_s00083g511 might play a role in oxidative stress resistance. These findings provide a basis for further analysis of the related functions of the JRL gene in A. oligospora and their potential roles in the biological control of nematodes in the future.

The hyperthermophilic α-amylase from Thermococcus sp. HJ21 does not require exogenous calcium for thermostability because of high-binding affinity to calcium: Huaixu Cheng , Zhidan Luo , Mingsheng Lu , Song Gao , Shujun Wang; J. Microbiol. 2017;55(5):379-387. Published online March 1, 2017; DOI: https://doi.org/10.1007/s12275-017-6416-5

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Abstract: The hyperthermophilic α-amylase from Thermococcus sp. HJ21 does not require exogenous calcium ions for thermo-stability, and is a promising alternative to commercially avail-able α-amylases to increase the efficiency of industrial pro-cesses like the liquefaction of starch. We analyzed the amino acid sequence of this α-amylase by sequence alignments and structural modeling, and found that this α-amylase closely resembles the α-amylase from Pyrococcus woesei. The gene of this α-amylase was cloned in Escherichia coli and the re-combinant α-amylase was overexpressed and purified with a combined renaturation-purification procedure. We con-firmed thermostability and exogenous calcium ion indepen-dency of the recombinant α-amylase and further investigated the mechanism of the independency using biochemical ap-proaches. The results suggested that the α-amylase has a high calcium ion binding affinity that traps a calcium ion that would not dissociate at high temperatures, providing a direct expla-nation as to why the addition of calcium ions is not required for thermostability. Understanding of the mechanism offers a strong base on which to further engineer properties of this α-amylase for better potential applications in industrial pro-cesses.

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

Identification of an Extracellular Thermostable Glycosyl Hydrolase Family 13 α-Amylase from Thermotoga neapolitana: Kyoung-Hwa Choi , Sungmin Hwang , Hee-Seob Lee , Jaeho Cha; J. Microbiol. 2011;49(4):628-634. Published online September 2, 2011; DOI: https://doi.org/10.1007/s12275-011-0432-7

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Abstract: We cloned the gene for an extracellular α-amylase, AmyE, from the hyperthermophilic bacterium Thermotoga neapolitana and expressed it in Escherichia coli. The molecular mass of the enzyme was 92 kDa as a monomer. Maximum activity was observed at pH 6.5 and temperature 75°C and the enzyme was highly thermostable. AmyE hydrolyzed the typical substrates for α-amylase, including soluble starch, amylopectin, and maltooligosaccharides. The hydrolytic pattern of AmyE was similar to that of a typical α-amylase; however, unlike most of the calcium (Ca2+)-dependent α-amylases, the activity of AmyE was unaffected by Ca2+. The specific activities of AmyE towards various substrates indicated that the enzyme preferred maltooligosaccharides which have more than four glucose residues. AmyE could not hydrolyze maltose and maltotriose. When maltoheptaose was incubated with AmyE at the various time courses, the products consisting of maltose through maltopentaose was evenly formed indicating that the enzyme acts in an endo-fashion. The specific activity of AmyE (7.4 U/mg at 75°C, pH 6.5, with starch as the substrate) was extremely lower than that of other extracellular α-amylases, which indicates that AmyE may cooperate with other highly active extracellular α-amylases for the breakdown of the starch or α-glucans into maltose and maltotriose before transport into the cell in the members of Thermotoga sp.

Production of Saccharogenic and Dextrinogenic Amylases by Rhizomucor pusillus A 13.36: Tony M. Silva , Derlene Attili-Angelis , Ana Flavia Azevedo Carvalho , Roberto Da Silva , Mauricio Boscolo , Eleni Gomes; J. Microbiol. 2005;43(6):561-568.; DOI: https://doi.org/2289 [pii]

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Abstract: A newly-isolated thermophilic strain of the zygomycete fungus Rhizomucor pusillus 13.36 produced highly active dextrinogenic and saccharogenic enzymes. Cassava pulp was a good alternative substrate for amylase production. Dextrinogenic and saccharogenic amylases exhibited optimum activities at a pH of 4.0-4.5 and 5.0 respectively and at a temperature of 75oC. The enzymes were highly thermostable, with no detectable loss of saccharogenic or dextrinogenic activity after 1 h and 6 h at 60oC, respectively. The saccharogenic activity was inhibited by Ca2+ while the dextrinogenic was indifferent to this ion. Both activities were inhibited by Fe2+ and Cu2+ Hydrolysis of soluble starch by the crude enzyme yielded 66% glucose, 19.5% maltose, 7.7% maltotriose and 6.6% oligosaccharides.

Expression of the Promoter for the Maltogenic Amylase Gene in Bacillus subtilis 168: Do-Yeon Kim , Choon-Hwan Cha , Wan-Seok Oh , Young-Jun Yoon , Jung-Wan Kim; J. Microbiol. 2004;42(4):319-327.; DOI: https://doi.org/2104 [pii]

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Abstract: An additional amylase, besides the typical a-amylase, was detected for the first time in the cytoplasm of B. subtilis SUH4-2, an isolate from Korean soil. The corresponding gene (bbmA) encoded a maltogenic amylase (MAase) and its sequence was almost identical to the yvdF gene of B. subtilis 168, whose function was unknown. Southern blot analysis using bbmA as the probe indicated that this gene was ubiquitous among various B. subtilis strains. In an effort to understand the physiological function of the bbmA gene in B. subtilis, the expression pattern of the gene was monitored by measuring the [beta]-galactosidase activity produced from the bbmA promoter fused to the amino terminus of the lacZ structural gene, which was then integrated into the amyE locus on the B. subtilis 168 chromosome. The promoter was induced during the mid-log phase and fully expressed at the early stationary phase in defined media containing [beta]-cyclodextrin ([beta]-CD), maltose, or starch. On the other hand, it was kept repressed in the presence of glucose, fructose, sucrose, or glycerol, suggesting that catabolite repression might be involved in the expression of the gene. Production of the [beta]-CD hydrolyzing activity was impaired by the spo0A mutation in B. subtilis 168, indicating the involvement of an additional regulatory system exerting control on the promoter. Inactivation of yvdF resulted in a significant decrease of the [beta]-CD hydrolyzing activity, if not all. This result implied the presence of an additional enzyme(s) that is capable of hydrolyzing [beta]-CD in B. subtilis 168. Based on the results, MAase encoded by bbmA is likely to be involved in maltose and [beta]-CD utilization when other sugars, which are readily usable as an energy source, are not available during the stationary phase.

Purification and Characterization of α-amylase from Aspergillus sp. JP-1: Park, Hyung Nam , Yoo, Jin Cheol , Yang, Young Ki; J. Microbiol. 1995;33(1):80-84.

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Abstract: The α-amylase was purified from Aspergillus sp. JP-1 and some enzyme characteristics were studied. The enzyme waw approzimately purified 80-fold and an overall yield was 16.5% from the culture medium by ammonium sulfate fractionation, Sephadex G-150 gel permeation chromatography, and DEAE-Sephadex A-50 ion exchange column chromatography in order. The molecular weight of the purified α-amylase has been estimated to be 56 KDa on SDS-polyacrulamide gel electrophoresis and Sephadex G-150 chromatography. The purigied enzyme functions optimally at pH5.5 and 40℃, respectively. The Km value for soluble starch was 2.5 mg/ml. The enzymatic activity increased in the presence of Ca^2+, Co^2+, EDTA, Mg^2+, Mn^2+ and Zn^2+ and was inhibited by adding Cu^2+, Fe^2+, and Ni^2+.

Purification and Characteristics of Glucoamylase in Aspergillus oryzae NR 3-6 Isolated from Traditional Korean Nuruk: Yu, Tae Shick , Kim, Tae Hyoung , Joo, Chong Yoon; J. Microbiol. 1999;37(2):80-85.

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Abstract: The purification system of glucoamylase (glucan 1,4-α-glucosidase, EC 3. 2. 1. 3), some characteristics of the purified enzyme and hydrolysis rate of various raw starch were investigated through several experiments. The enzyme was produced on a solid, uncooked wheat bran medium of Aspergillus oryzae NR 3-6 isolated from traditional Korean Nuruk. The enzyme was homogeneously purified 6.8-fold with an overall yield of 28.3% by the criteria of disc- and SDS-polyacrylamide gel electrophoresis. The molecular weight was estimated to be 48 kDa by SDS-PAGE. The optimum temperature and pH were 55℃ and 4.0, respectively. The enzyme was stable at a pH range of 3.0∼10.0 and below 45℃. Enzyme activity was inhibited about 27% by 1mM Hg^2+. The hydrolysis rate of raw wheat starch was shown to be 17.5-fold faster than the hydrolysis rate of soluble starch. The purified enzyme was identified as glucoamylase because the product of soluble starch by the purified enzyme was mainly glucose by thin layer chromatography.

Monascus Red Pigment Overproduction by Coculture with Recombinant Saccharomyces cerevisiae Secreting Glucoamylase: Ho-Soo Lim , Seung-Ku Yoo , Chul-Soo Shin , Young-Min Hyun; J. Microbiol. 2000;38(1):48-51.

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Abstract: In liquid cultures using sucrose media, the coculture of Monascus with recombinant Saccharomyces cerevisiae expressing the glucoamylase gene from Aspergillus niger enhanced red pigment production by approx. 19%, compared with the coculture of wild type S. cerevisiae. Coculture with recombinant S. cerevisiae was more effective than with wild type S. cerevisiae for Monascus red pigment production. Cocultures of Monascus with commercial amylases of Aspergillus also induced high production of pigment and morphological changes in a solid culture using sucrose media.

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