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한국미생물생명공학회(구 한국산업미생물학회)> Journal of Microbiology and Biotechnology> Crystal Structure and Functional Characterization of a Xylose Isomerase (PbXI) from the Psychrophilic Soil Microorganism, Paenibacillus sp.

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Crystal Structure and Functional Characterization of a Xylose Isomerase (PbXI) from the Psychrophilic Soil Microorganism, Paenibacillus sp.

Sun-ha Park , Sunghark Kwon , Chang Woo Lee , Chang Min Kim , Chang Sook Jeong , Kyung-jin Kim , Jong Wook Hong , Hak Jun Kim , Hyun Ho Park , Jun Hyuck Lee
  • : 한국미생물생명공학회
  • : Journal of Microbiology and Biotechnology 29권2호
  • : 연속간행물
  • : 2019년 02월
  • : 244-255(12pages)
Journal of Microbiology and Biotechnology

DOI


목차

Introduction
Material and Methods
Results and Discussion
Acknowledgments
Conflict of Interest
References

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Xylose isomerase (XI; E.C. 5.3.1.5) catalyzes the isomerization of xylose to xylulose, which can be used to produce bioethanol through fermentation. Therefore, XI has recently gained attention as a key catalyst in the bioenergy industry. Here, we identified, purified, and characterized a XI (PbXI) from the psychrophilic soil microorganism, Paenibacillus sp. R4. Surprisingly, activity assay results showed that PbXI is not a cold-active enzyme, but displays optimal activity at 60°C. We solved the crystal structure of PbXI at 1.94-A resolution to investigate the origin of its thermostability. The PbXI structure shows a (β/α)8-barrel fold with tight tetrameric interactions and it has three divalent metal ions (CaI, CaII, and CaIII). Two metal ions (CaI and CaII) located in the active site are known to be involved in the enzymatic reaction. The third metal ion (CaIII), located near the β4-α6 loop region, was newly identified and is thought to be important for the stability of PbXI. Compared with previously determined thermostable and mesophilic XI structures, the β1-α2 loop structures near the substrate binding pocket of PbXI were remarkably different. Site-directed mutagenesis studies suggested that the flexible β1-α2 loop region is essential for PbXI activity. Our findings provide valuable insights that can be applied in protein engineering to generate lowtemperature purpose-specific XI enzymes.

UCI(KEPA)

I410-ECN-0102-2019-400-001362999

간행물정보

  • : 자연과학분야  > 생물
  • : KCI등재
  • : SCI,SCOPUS
  • : 월간
  • : 1017-7825
  • : 1738-8872
  • : 학술지
  • : 연속간행물
  • : 1991-2021
  • : 5714


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1Two Enteropathogenic Escherichia coli Strains Representing Novel Serotypes and Investigation of Their Roles in Adhesion

저자 : Jing Wang , Hongbo Jiao , Xinfeng Zhang , Yuanqing Zhang , Na Sun , Ying Yang , Yi Wei , Bin Hu , Xi Guo

발행기관 : 한국미생물생명공학회 간행물 : Journal of Microbiology and Biotechnology 31권 9호 발행 연도 : 2021 페이지 : pp. 1191-1199 (9 pages)

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Enteropathogenic Escherichia coli (EPEC), which belongs to the attaching and effacing diarrheagenic E. coli strains, is a major causative agent of life-threatening diarrhea in infants in developing countries. Most EPEC isolates correspond to certain O serotypes; however, many strains are nontypeable. Two EPEC strains, EPEC001 and EPEC080, which could not be serotyped during routine detection, were isolated. In this study, we conducted an in-depth characterization of their putative O-antigen gene clusters (O-AGCs) and also performed constructed mutagenesis of the O-AGCs for functional analysis of O-antigen (OAg) synthesis. Sequence analysis revealed that the occurrence of O-AGCs in EPEC001 and E. coli O132 may be mediated by recombination between them, and EPEC080 and E. coli O2/O50 might acquire each O-AGC from uncommon ancestors. We also indicated that OAgknockout bacteria were highly adhesive in vitro, except for the EPEC001 wzy derivative, whose adherent capability was less than that of its wild-type strain, providing direct evidence that OAg plays a key role in EPEC pathogenesis. Together, we identified two EPEC O serotypes in silico and experimentally, and we also studied the adherent capabilities of their OAgs, which highlighted the fundamental and pathogenic role of OAg in EPEC.

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2Aloe-Emodin-Mediated Photodynamic Therapy Attenuates Sepsis-Associated Toxins in Selected Gram-Positive Bacteria In Vitro

저자 : Woodvine Otieno , Chengcheng Liu , Yanhong Ji

발행기관 : 한국미생물생명공학회 간행물 : Journal of Microbiology and Biotechnology 31권 9호 발행 연도 : 2021 페이지 : pp. 1200-1209 (10 pages)

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Sepsis is an acute inflammatory response that leads to life-threatening complications if not quickly and adequately treated. Cytolysin, hemolysin, and pneumolysin are toxins produced by grampositive bacteria and are responsible for resistance to antimicrobial drugs, cause virulence and lead to sepsis. This work assessed the effects of aloe-emodin (AE) and photodynamic therapy (PDT) on sepsis-associated gram-positive bacterial toxins. Standard and antibiotic-resistant Enterococcus faecalis, Staphylococcus aureus, and Streptococcus pneumonia bacterial strains were cultured in the dark with varying AE concentrations and later irradiated with 72 J/cm-2 light. Colony and biofilm formation was determined. CCK-8, Griess reagent reaction, and ELISA assays were done on bacteriainfected RAW264.7 cells to determine the cell viability, NO, and IL-1β and IL-6 pro-inflammatory cytokines responses, respectively. Hemolysis and western blot assays were done to determine the effect of treatment on hemolysis activity and sepsis-associated toxins expressions. AE-mediated PDT reduced bacterial survival in a dose-dependent manner with 32 μg/ml of AE almost eliminating their survival. Cell proliferation, NO, IL-1β, and IL-6 cytokines production were also significantly downregulated. Further, the hemolytic activities and expressions of cytolysin, hemolysin, and pneumolysin were significantly reduced following AE-mediated PDT. In conclusion, combined use of AE and light (435 ± 10 nm) inactivates MRSA, S. aureus (ATCC 29213), S. pneumoniae (ATCC 49619), MDR-S. pneumoniae, E. faecalis (ATCC 29212), and VRE (ATCC 51299) in an AE-dose dependent manner. AE and light are also effective in reducing biofilm formations, suppressing proinflammatory cytokines, hemolytic activities, and inhibiting the expressions of toxins that cause sepsis.

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3Ramlibacter terrae sp. nov. and Ramlibacter montanisoli sp. nov., Isolated from Soil

저자 : Shehzad Abid Khan , Hyung Min Kim , Ju Hye Baek , Hye Su Jung , Che Ok Jeon

발행기관 : 한국미생물생명공학회 간행물 : Journal of Microbiology and Biotechnology 31권 9호 발행 연도 : 2021 페이지 : pp. 1210-1217 (8 pages)

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Two gram-negative, catalase-positive, strictly aerobic, and white colony-forming bacteria, strains H242T and B156T, were isolated from soil in South Korea. Cells of strain H242T were oxidase-positive and non-motile short rods, while those of strain B156T were oxidase-negative and long non-motile rods. Ubiquinone-8 was identified as the sole isoprenoid quinone in both strains. C16:0, cyclo-C17:0, and summed feature 3 (C16:1 ω7c and/or C16:1 ω6c) and phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol were identified in both strains as the major cellular fatty acids and polar lipids, respectively. The DNA G+C contents of strains H242T and B156T were 69.4 mol% and 69.3 mol%, respectively. Phylogenetic analyses based on 16S rRNA and 92 concatenated core gene sequences revealed that strains H242T and B156T formed distinct phylogenic lineages from other Ramlibacter type strains. The DNA-DNA hybridization (DDH) value between strains H242T and B156T was 24.6%. Strains H242T and B156T were most closely related to Ramlibacter ginsenosidimutans BXN5-27T and Ramlibacter monticola G-3-2T with 98.4% and 98.6% 16S rRNA gene sequence similarities, respectively. Digital DDH values between strain H242T and R. ginsenosidimutans and between strain B156T and R. monticola were 23.5% and 26.1%, respectively. Phenotypic, chemotaxonomic, and molecular analyses indicated that strains H242T and B156T represent two novel species of the genus Ramlibacter, for which the names Ramlibacter terrae sp. nov. and Ramlibacter montanisoli sp. nov., respectively, are proposed. The type strains of R. terrae and R. montanisoli are H242T (=KACC 21667 T =JCM 33922T) and B156T (=KACC 21665 T =JCM 33920T), respectively.

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4Isolation and Characterization of Cold- Adapted PGPB and Their Effect on Plant Growth Promotion

저자 : Mingyuan Li , Jilian Wang , Tuo Yao , Zhenlong Wang , Huirong Zhang , Changning Li

발행기관 : 한국미생물생명공학회 간행물 : Journal of Microbiology and Biotechnology 31권 9호 발행 연도 : 2021 페이지 : pp. 1218-1230 (13 pages)

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Cold-adapted plant growth-promoting bacteria (PGPB) with multiple functions are an important resource for microbial fertilizers with low-temperature application. In this study, culturable coldadapted PGPB strains with nitrogen fixation and phosphorus solubilization abilities were isolated. They were screened from root and rhizosphere of four dominant grass species in nondegraded alpine grasslands of the Qilian Mountains, China. Their other growth-promoting characteristics, including secretion of indole-3-acetic acid (IAA), production of siderophores and ACC deaminase, and antifungal activity, were further studied by qualitative and quantitative methods. In addition, whether the PGPB strains could still exert plant growth-promoting activity at 4°C was verified. The results showed that 67 isolates could maintain one or more growth-promoting traits at 4°C, and these isolates were defined as cold-adapted PGPB. They were divided into 8 genera by 16S rRNA gene sequencing and phylogenetic analysis, of which Pseudomonas (64.2%) and Serratia (13.4%) were the common dominant genera, and a few specific genera varied among the plant species. A test-tube culture showed that inoculation of Elymus nutans seedlings with cold-adapted PGPB possessing different functional characteristics had a significant growth-promoting effect under controlled low-temperature conditions, including the development of the roots and aboveground parts. Pearson correlation analysis revealed that different growth-promoting characteristics made different contributions to the development of the roots and aboveground parts. These cold-adapted PGPB can be used as excellent strain resources suitable for the near-natural restoration of degraded alpine grasslands or agriculture stock production in cold areas.

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5Profiles of Bacillus spp. Isolated from the Rhizosphere of Suaeda glauca and Their Potential to Promote Plant Growth and Suppress Fungal Phytopathogens

저자 : Ping Lu , Ke Jiang , Ya-qiao Hao , Wan-ying Chu , Yu-dong Xu , Jia-yao Yang , Jia-le Chen , Guo-hong Zeng , Zhou-hang Gu , Hong-xin Zhao

발행기관 : 한국미생물생명공학회 간행물 : Journal of Microbiology and Biotechnology 31권 9호 발행 연도 : 2021 페이지 : pp. 1231-1240 (10 pages)

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Members of the genus Bacillus are known to play an important role in promoting plant growth and protecting plants against phytopathogenic microorganisms. In this study, 21 isolates of Bacillus spp. were obtained from the root micro-ecosystem of Suaeda glauca. Analysis of the 16S rRNA genes indicated that the isolates belong to the species Bacillus amyloliquefaciens, Bacillus velezensis, Bacillus subtilis, Bacillus pumilus, Bacillus aryabhattai and Brevibacterium frigoritolerans. One of the interesting findings of this study is that the four strains B1, B5, B16 and B21 are dominant in rhizosphere soil. Based on gyrA, gyrB, and rpoB gene analyses, B1, B5, and B21 were identified as B. amyloliquefaciens and B16 was identified as B. velezensis. Estimation of antifungal activity showed that the isolate B1 had a significant inhibitory effect on Fusarium verticillioides, B5 and B16 on Colletotrichum capsici (syd.) Butl, and B21 on Rhizoctonia cerealis van der Hoeven. The four strains grew well in medium with 1-10% NaCl, a pH value of 5-8, and promoted the growth of Arabidopsis thaliana. Our results indicate that these strains may be promising agents for the biocontrol and promotion of plant growth and further study of the relevant bacteria will provide a useful reference for the development of microbial resources.

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6Suppression of Fusarium Wilt Caused by Fusarium oxysporum f. sp. lactucae and Growth Promotion on Lettuce Using Bacterial Isolates

저자 : Dil Raj Yadav , Mahesh Adhikari , Sang Woo Kim , Hyun Seung Kim , Youn Su Lee

발행기관 : 한국미생물생명공학회 간행물 : Journal of Microbiology and Biotechnology 31권 9호 발행 연도 : 2021 페이지 : pp. 1241-1255 (15 pages)

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This study was carried out to explore a non-chemical strategy for enhancing productivity by employing some antagonistic rhizobacteria. One hundred eighteen bacterial isolates were obtained from the rhizospheric zone of various crop fields of Gangwon-do, Korea, and screened for antifungal activity against Fusarium wilt (Fusarium oxysporum f. sp. lactucae) in lettuce crop under in vitro and in vivo conditions. In broth-based dual culture assay, fourteen bacterial isolates showed significant inhibition of mycelial growth of F. oxysporium f. sp. lactucae. All of the antagonistic isolates were further characterized for the antagonistic traits under in vitro conditions. The isolates were identified on the basis of biochemical characteristics and confirmed at their species level by 16S rRNA gene sequencing analysis. Arthrobacter sulfonivorans, Bacillus siamensis, Bacillus amyloliquefaciens, Pseudomonas proteolytica, four Paenibacillus peoriae strains, and Bacillus subtilis were identified from the biochemical characterization and 16S rRNA gene sequencing analysis. The isolates EN21 and EN23 showed significant decrease in disease severity on lettuce compared to infected control and other bacterial treatments under greenhouse conditions. Two bacterial isolates, EN4 and EN21, were evaluated to assess their disease reduction and growth promotion in lettuce in field conditions. The consortium of EN4 and EN21 showed significant enhancement of growth on lettuce by suppressing disease caused by F. oxysporum f. sp. lactucae respectively. This study clearly indicates that the promising isolates, EN4 (P. proteolytica) and EN21 (Bacillus siamensis), can be commercialized and used as biofertilizer and/or biopesticide for sustainable crop production.

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7Freeze-Dried Powder of Rubus coreanus Miquel Ameliorates Isoproterenol-Induced Oxidative Stress and Tissue Damage in Rats

저자 : Jin Tae Kim , Shuai Qiu , Yimeng Zhou , Ji Hyun Moon , Seung Beom Lee , Ho Jin Park , Hong Jin Lee

발행기관 : 한국미생물생명공학회 간행물 : Journal of Microbiology and Biotechnology 31권 9호 발행 연도 : 2021 페이지 : pp. 1256-1261 (6 pages)

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Rubus coreanus Miquel (bokbunja), Korean black raspberry, is known to possess various phytochemicals that exert antioxidative, anti-inflammatory, and anti-cancer effects. However, most studies on Rubus coreanus Miquel have been performed with the solvent extracts and/or a single component to demonstrate the efficacy, while studies evaluating the effect of the whole fructus of Rubus coreanus Miquel are limited. In this study, therefore, we employed the isoproterenol (IPN)- induced myocardial infarction model and investigated the effect of freeze-dried powder of Rubus coreanus Miquel (RCP) on oxidative stress and prevention of organ damage. Oral administration of RCP reduced the level of toxicity markers, alanine transaminase (ALT), aspartate transaminase (AST), and lactate dehydrogenase (LDH) without affecting body weight and diet intake. The oxidative stress marker glutathione (GSH) increased about 45% and malonaldehyde (MDA) decreased about 27% compared to the IPN group with RCP-H (3%) administration. By histological analysis, IPN induced significant myocardial damage in the heart and vascular injury in the liver, and RCP administration ameliorated the damages in a dose-dependent manner. Taken together, RCP activated the antioxidant system leading to prevention of damage to organs by IPN in rats, making it possible to expect beneficial efficacies by consuming the whole fructus of Rubus coreanus Miquel.

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8Bioconversion of Untreated Corn Hull into L-Malic Acid by Trifunctional Xylanolytic Enzyme from Paenibacillus curdlanolyticus B-6 and Acetobacter tropicalis H-1

저자 : Thi Bich Huong Duong , Prattana Ketbot , Paripok Phitsuwan , Rattiya Waeonukul , Chakrit Tachaapaikoon , Akihiko Kosugi , Khanok Ratanakhanokchai , Patthra Pason

발행기관 : 한국미생물생명공학회 간행물 : Journal of Microbiology and Biotechnology 31권 9호 발행 연도 : 2021 페이지 : pp. 1262-1271 (10 pages)

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L-Malic acid (L-MA) is widely used in food and non-food products. However, few microorganisms have been able to efficiently produce L-MA from xylose derived from lignocellulosic biomass (LB). The objective of this work is to convert LB into L-MA with the concept of a bioeconomy and environmentally friendly process. The unique trifunctional xylanolytic enzyme, PcAxy43A from Paenibacillus curdlanolyticus B-6, effectively hydrolyzed xylan in untreated LB, especially corn hull to xylose, in one step. Furthermore, the newly isolated, Acetobacter tropicalis strain H1 was able to convert high concentrations of xylose derived from corn hull into L-MA as the main product, which can be easily purified. The strain H1 successfully produced a high L-MA titer of 77.09 g/l, with a yield of 0.77 g/g and a productivity of 0.64 g/l/h from the xylose derived from corn hull. The process presented in this research is an efficient, low-cost and environmentally friendly biological process for the green production of L-MA from LB.

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9Aqueous Extract of Lysimachia christinae Hance Prevents Cholesterol Gallstone in Mice by Affecting the Intestinal Microflora

저자 : Shijia Liu , Quji Luorong , Kaizhi Hu , Weiguo Cao , Wei Tao , Handeng Liu , Dan Zhang

발행기관 : 한국미생물생명공학회 간행물 : Journal of Microbiology and Biotechnology 31권 9호 발행 연도 : 2021 페이지 : pp. 1272-1280 (9 pages)

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With changes in human dietary patterns, the proportion of high-fat and high-cholesterol foods in the daily diet has increased. As a result, the incidence rate of cholelithiasis is increasing rapidly. Many studies have reported on the crucial role that the intestinal microflora plays in the progression of gallstones. Although the whole herb of Lysimachia christinae, a traditional Chinese medicine, has long been extensively used as a remedy for cholelithiasis in China, its effects on the intestinal microflora remain unknown. Hence, in this study, we investigated the ability of the aqueous extract of L. christinae (LAE) to prevent cholesterol gallstones (CGSs) in model animals by affecting the intestinal microflora. The effects of LAE on body weight, serum lipid profile, visceral organ indexes, and histomorphology were studied in male C57BL/6J mice, which were induced by a lithogenic diet. After the 8-week study, CGSs formation was greatly reduced after LAE treatment. LAE also reduced body weight gain and hyperlipidemia and restored the histomorphological changes. Moreover, the intestinal microflora exhibited significant variation. In the model group fed the lithogenic diet, the abundances of the genera unclassified Porphyromonadaceae, Lactobacillus and Alloprevotella decreased, but in contrast, Akkermansia dramatically increased compared with the control check group, which was fed a normal diet; the administration of LAE reversed these changes. These results imply that L. christinae can be considered an efficient therapy for eliminating CGSs induced by a high-fat and high-cholesterol diet, which may be achieved by influencing the intestinal microflora.

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10The Effect of Lactobacillus gasseri BNR17 on Postmenopausal Symptoms in Ovariectomized Rats

저자 : Sol Lee , Dong Hoon Jung , Miri Park , Seung-woo Yeon , Sang-hyuk Jung , Sung-il Yun , Han-oh Park , Wonbeak Yoo

발행기관 : 한국미생물생명공학회 간행물 : Journal of Microbiology and Biotechnology 31권 9호 발행 연도 : 2021 페이지 : pp. 1281-1287 (7 pages)

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Clinical and preclinical studies have reported that Lactobacillus gasseri BNR17, a probiotic bacterial strain isolated from human breast milk, reduces body weight and white adipose tissue volume. In order to further explore the actions of L. gasseri BNR17, we investigated the anti-menopausal effects of L. gasseri BNR17 in an ovariectomized (OVX) rat model. The serum alanine aminotransferase levels of the rats in the OVX-BNR17 group were lower than those of the rats in the OVX-vehicle only (OVX-Veh) group. Upon administration of L. gasseri BNR17 after ovariectomy, calcitonin and Serotonin 2A levels increased significantly, whereas serum osteocalcin levels showed a decreasing tendency. Compared to the rats in the OVX-Veh group, those in the OVX-BNR17 group showed lower urine deoxypyridinoline levels, lower pain sensitivity, and improved vaginal cornification. Furthermore, L. gasseri BNR17 administration increased bone mineral density in the rats with OVX-induced femoral bone loss. These results suggest that L. gasseri BNR17 administration could alleviate menopausal symptoms, indicating that this bacterium could be a good functional probiotic for managing the health of older women.

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1Bacillus subtilis Spore Surface Display Technology: A Review of Its Development and Applications

저자 : Guoyan Zhang , Yingfeng An , Hossain M. Zabed , Qi Guo , Miaomiao Yang , Jiao Yuan , Wen Li , Wenjin Sun , Xianghui Qi

발행기관 : 한국미생물생명공학회 간행물 : Journal of Microbiology and Biotechnology 29권 2호 발행 연도 : 2019 페이지 : pp. 179-190 (12 pages)

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Bacillus subtilis spore surface display (BSSD) technology is considered to be one of the most promising approaches for expressing heterologous proteins with high activity and stability. Currently, this technology is used for various purposes, such as the production of enzymes, oral vaccines, drugs and multimeric proteins, and the control of environmental pollution. This paper presents an overview of the latest developments in BSSD technology and its application in protein engineering. Finally, the major limitations of this technology and future directions for its research are discussed.

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2Effects of Starter Candidates and NaCl on the Production of Volatile Compounds during Soybean Fermentation

저자 : Do-won Jeong , Hyundong Lee , Keuncheol Jeong , Cheong-tae Kim , Sun-taek Shim , Jong-hoon Lee

발행기관 : 한국미생물생명공학회 간행물 : Journal of Microbiology and Biotechnology 29권 2호 발행 연도 : 2019 페이지 : pp. 191-199 (9 pages)

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We inoculated different combinations of three starter candidates, Bacillus licheniformis, Staphylococcus succinus, and Tetragenococcus halophilus, into sterilized soybeans to predict their contributions to volatile compound production through soybean fermentation. Simultaneously, we added NaCl to soybean cultures to evaluate its effect on the volatile compounds profile. Cells in soybean cultures (1.5% NaCl) nearly reached their maximum growth in a day of incubation, while cell growth was delayed by increasing NaCl concentrations in soybean cultures. The dominance of B. licheniformis and S. succinus in the mixed cultures of three starter candidates switched to T. halophilus as the NaCl concentration increased from 1.5% to 14% (w/w). Seventeen volatile compounds were detected from the control and starter candidateinoculated soybean cultures with and without the addition of NaCl. Principal component analysis of these volatile compounds concluded that B. licheniformis and S. succinus made major contributions to producing a specific volatile compound profile from soybean cultures where both species exhibited good growth. 3-Hydroxybutan-2-one, butane-2,3-diol, and 2,3,5,6-tetramethylpyrazine are specific odor notes for B. licheniformis, and 3-methylbutyl acetate and 2-phenylethanol are specific for S. succinus. Octan-3-one and 3-methylbutan-1-ol were shown to be decisive volatile compounds for determining the involvement of S. succinus in the soybean culture containing 7% NaCl. 3-Methylbutyl acetate and 3-methylbutan-1-ol were also produced by T. halophilus during soybean fermentation at an appropriate level of NaCl. Although S. succinus and T. halophilus exhibited growth on the soybean cultures containing 14% NaCl, species-specific volatile compounds determining the directionality of the volatile compounds profile were not produced.

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3Oral Delivery of Probiotics Using pH-Sensitive Phthalyl Inulin Tablets

저자 : Whee-soo Kim , Chong-su Cho , Liang Hong , Geon Goo Han , Bum Ju Kil , Sang-kee Kang , Dae-duk Kim , Yun-jaie Choi , Chul Sung Huh

발행기관 : 한국미생물생명공학회 간행물 : Journal of Microbiology and Biotechnology 29권 2호 발행 연도 : 2019 페이지 : pp. 200-208 (9 pages)

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Probiotics show low cell viability after oral administration because they have difficulty surviving in the stomach due to low pH and enzymes. For the oral delivery of probiotics, developing a formula that protects the probiotic bacteria from gastric acidity while providing living cells is mandatory. In this study, we developed tablets using a new pH-sensitive phthalyl inulin (PI) to protect probiotics from gastric conditions and investigated the effects of different compression forces on cell survival. We made three different tablets under different compression forces and measured survivability, disintegration time, and kinetics in simulated gastric-intestinal fluid. During tableting, there were no significant differences in probiotic viability among the different compression forces although disintegration time was affected by the compression force. A higher compression force resulted in higher viability in simulated gastric fluid. The swelling degree of the PI tablets in simulated intestinal fluid was higher than that of the tablets in simulated gastric fluid due to the pH sensitivity of the PI. The probiotic viability formulated in the tablets was also higher in acidic gastric conditions than that for probiotics in solution. Rapid release of the probiotics from the tablet occurred in the simulated intestinal fluid due to the pH sensitivity. After 6 months of refrigeration, the viability of the PI probiotics was kept. Overall, this is the first study to show the pH-sensitive properties of PI and one that may be useful for oral delivery of the probiotics.

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4Characteristics of Potential Gamma-Aminobutyric Acid-Producing Bacteria Isolated from Korean and Vietnamese Fermented Fish Products

저자 : Thi Thu-thao Vo , Jong-hyun Park

발행기관 : 한국미생물생명공학회 간행물 : Journal of Microbiology and Biotechnology 29권 2호 발행 연도 : 2019 페이지 : pp. 209-221 (13 pages)

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Gamma-aminobutyric acid (GABA) is a neurotransmitter that exerts several physiological functions and positive effects on human health. The aim of this study was to isolate and characterize the strains that had GABA-producing abilities from various fermented fish products. A total of 91 acid-producing strains were isolated from 41 samples of fermented fish products, and 27 strains showing GABA-producing abilities were identified by the 16S rDNA sequences. Among the strains, 31% strains tolerated at high-salt environment of 10-20% throughout the fermentation of fish sauces. The 27 isolates that produced GABA at various concentrations did so in the range of 5 to 454 mM. These GABA-producing isolates were identified as lactic acid bacteria of 14 strains, which included twelve Lactococcus lactis, one Enterococcus faecium, and one Lactococcus pentosus; eight Bacillus cereus group, which included seven B. thuringiensis and one B. cereus; and five Staphylococcus spp. Interestingly, with Vietnamese fish sauces, we mostly identified species of B. thuringiensis and Staphylococcus spp., while with Korean fermented fish products, the majority of the strains identified belonged to L. lactis. Among the strains, B. thuringiensis LH2134 produced the highest levels of GABA at 366 mM among the strains identified from Vietnamese fish sauces, whereas L. lactis LA43, a new strain isolated from Korean jeotgal (salted shrimp paste), produced the highest amount of GABA at 454 mM and the glutamate concentration in the medium was essential for GABA accumulation. Therefore, such the isolates might serve as good starters for development of more GABA-reinforced foods among fermented fish products.

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5Change of Ginsenoside Profiles in Processed Ginseng by Drying, Steaming, and Puffing

저자 : Ji-hye Shin , Young Joon Park , Wooki Kim , Dae-ok Kim , Byung-yong Kim , Hyungjae Lee , Moo-yeol Baik

발행기관 : 한국미생물생명공학회 간행물 : Journal of Microbiology and Biotechnology 29권 2호 발행 연도 : 2019 페이지 : pp. 222-229 (8 pages)

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Korean ginseng (Panax ginseng Meyer) was processed by drying, steaming, or puffing, and the effects of these processes on the ginsenoside profile were investigated. The main root of 4-year-old raw Korean ginseng was dried to produce white ginseng. Steaming, followed by drying, was employed to produce red or black ginseng. In addition, these three varieties of processed ginseng were puffed using a rotational puffing gun. Puffed ginseng showed significantly higher extraction yields of ginsenosides (49.87-58.60 g solid extract/100 g of sample) and crude saponin content (59.40-63.87 mg saponin/g of dried ginseng) than nonpuffed ginseng, respectively. Moreover, puffing effectively transformed the major ginsenosides (Rb1, Rb2, Rc, Rd, Re, and Rg1) of ginseng into minor ones (F2, Rg3, Rk1, and Rg5), comparable to the steaming process effect on the levels of the transformed ginsenosides. However, steaming takes much longer (4 to 36 days) than puffing (less than 30 min) for ginsenoside transformation. Consequently, puffing may be an effective and economical technique for enhancing the extraction yield and levels of minor ginsenosides responsible for the major biological activities of ginseng.

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6A Dual Selection Marker Transformation System Using Agrobacterium tumefaciens for the Industrial Aspergillus oryzae 3.042

저자 : Yunlong Sun , Yali Niu , Bin He , Long Ma , Ganghua Li , Van-tuan Tran , Bin Zeng , Zhihong Hu

발행기관 : 한국미생물생명공학회 간행물 : Journal of Microbiology and Biotechnology 29권 2호 발행 연도 : 2019 페이지 : pp. 230-234 (5 pages)

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Currently, the genetic modification of Aspergillus oryzae is mainly dependent on protoplast-mediated transformation (PMT). In this study, we established a dual selection marker system in an industrial A. oryzae 3.042 strain by using Agrobacterium tumefaciens-mediated transformation (ATMT). We first constructed a uridine/uracil auxotrophic A. oryzae 3.042 strain and a pyrithiamine (PT)-resistance binary vector. Then, we established the ATMT system by using uridine/uracil auxotrophy and PT-resistance genes as selection markers. Finally, a dual selection marker ATMT system was developed. This study demonstrates a useful dual selection marker transformation system for genetic manipulations of A. oryzae 3.042.

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7Isolation and Characterization of an Eosinophilic GH 16 β-Agarase (AgaDL6) from an Agar-Degrading Marine Bacterium Flammeovirga sp. HQM9

저자 : Yan Liu , Xiaoxu Tian , Chao Peng , Zongjun Du

발행기관 : 한국미생물생명공학회 간행물 : Journal of Microbiology and Biotechnology 29권 2호 발행 연도 : 2019 페이지 : pp. 235-243 (9 pages)

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A special eosinophilic agarase exo-type β-agarase gene, AgaDL6, was cloned from a marine agar-degrading bacterium, Flammeovirga sp. HQM9. The gene comprised 1,383-bp nucleotides encoding a putative agarase AgaDL6 of 461 amino acids with a calculated molecular mass of 52.8 kDa. Sequence analysis revealed a β-agarase domain that belongs to the glycoside hydrolase family (GH) 16 and a carbohydrate-binding module (CBM_4_9) unique to agarases. AgaDL6 was heterologously expressed in Escherichia coli BL21 (DE3). Enzyme activity analysis of the purified protein showed that the optimal temperature and pH of AgaDL6 were 50°C and 3.0, respectively. AgaDL6 showed thermal stability by retaining more than 98% of activity after incubation for 2 h at 50°C, a feature quite different from other agarases. AgaDL6 also exhibited outstanding acid stability, retaining 100% of activity after incubation for 24 h at pH 2.0 to 5.0, a property distinct from other agarases. This is the first agarase characterized to have such high acid stability. In addition, we observed no obvious stimulation or inhibition of AgaDL6 in the presence of various metal ions and denaturants. AgaDL6 is an exo-type β-1,4 agarase that cleaved agarose into neoagarotetraose and neoagarohexaose as the final products. These characteristics make AgaDL6 a potentially valuable enzyme in the cosmetic, food, and pharmaceutical industries.

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8Crystal Structure and Functional Characterization of a Xylose Isomerase (PbXI) from the Psychrophilic Soil Microorganism, Paenibacillus sp.

저자 : Sun-ha Park , Sunghark Kwon , Chang Woo Lee , Chang Min Kim , Chang Sook Jeong , Kyung-jin Kim , Jong Wook Hong , Hak Jun Kim , Hyun Ho Park , Jun Hyuck Lee

발행기관 : 한국미생물생명공학회 간행물 : Journal of Microbiology and Biotechnology 29권 2호 발행 연도 : 2019 페이지 : pp. 244-255 (12 pages)

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Xylose isomerase (XI; E.C. 5.3.1.5) catalyzes the isomerization of xylose to xylulose, which can be used to produce bioethanol through fermentation. Therefore, XI has recently gained attention as a key catalyst in the bioenergy industry. Here, we identified, purified, and characterized a XI (PbXI) from the psychrophilic soil microorganism, Paenibacillus sp. R4. Surprisingly, activity assay results showed that PbXI is not a cold-active enzyme, but displays optimal activity at 60°C. We solved the crystal structure of PbXI at 1.94-A resolution to investigate the origin of its thermostability. The PbXI structure shows a (β/α)8-barrel fold with tight tetrameric interactions and it has three divalent metal ions (CaI, CaII, and CaIII). Two metal ions (CaI and CaII) located in the active site are known to be involved in the enzymatic reaction. The third metal ion (CaIII), located near the β4-α6 loop region, was newly identified and is thought to be important for the stability of PbXI. Compared with previously determined thermostable and mesophilic XI structures, the β1-α2 loop structures near the substrate binding pocket of PbXI were remarkably different. Site-directed mutagenesis studies suggested that the flexible β1-α2 loop region is essential for PbXI activity. Our findings provide valuable insights that can be applied in protein engineering to generate lowtemperature purpose-specific XI enzymes.

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9Single Cell Oil Production from Undetoxified Arundo donax L. hydrolysate by Cutaneotrichosporon curvatus

저자 : Nicola Di Fidio , Federico Liuzzi , Silvio Mastrolitti , Roberto Albergo , Isabella De Bari

발행기관 : 한국미생물생명공학회 간행물 : Journal of Microbiology and Biotechnology 29권 2호 발행 연도 : 2019 페이지 : pp. 256-267 (12 pages)

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The use of low-cost substrates represents one key issue to make single cell oil production sustainable. Among low-input crops, Arundo donax L. is a perennial herbaceous rhizomatous grass containing both C5 and C6 carbohydrates. The scope of the present work was to investigate and optimize the production of lipids by the oleaginous yeast Cutaneotrichosporon curvatus from undetoxified lignocellulosic hydrolysates of steam-pretreated A. donax. The growth of C. curvatus was first optimized in synthetic media, similar in terms of sugar concentration to hydrolysates, by applying the response surface methodology (RSM) analysis. Then the bioconversion of undetoxified hydrolysates was investigated. A fed-batch process for the fermentation of A. donax hydrolysates was finally implemented in a 2-L bioreactor. Under optimized conditions, the total lipid content was 64% of the dry cell weight and the lipid yield was 63% of the theoretical. The fatty acid profile of C. curvatus triglycerides contained 27% palmitic acid, 33% oleic acid and 32% linoleic acid. These results proved the potential of lipid production from A. donax, which is particularly important for their consideration as substitutes for vegetable oils in many applications such as biodiesel or bioplastics.

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10Altering UDP-Glucose Donor Substrate Specificity of Bacillus licheniformis Glycosyltransferase towards TDP-Glucose

저자 : Kye Woon Cho , Tae-su Kim , Tuoi Thi Le , Hue Thi Nguyen , So Yeong Oh , Ramesh Prasad Pandey , Jae Kyung Sohng

발행기관 : 한국미생물생명공학회 간행물 : Journal of Microbiology and Biotechnology 29권 2호 발행 연도 : 2019 페이지 : pp. 268-273 (6 pages)

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The specificity of a Bacillus licheniformis uridine diphosphate (UDP) glycosyltransferase, YjiC, was increased towards thymidine diphosphate (TDP)-sugar by site-directed mutagenesis. The Arg-282 of YjiC was identified and investigated by substituting with Trp. Conversion rate and kinetic parameters were compared between YjiC and its variants with several acceptor substrates such as 7-hydroxyflavone (7-HF), 4',7-dihydroxyisoflavone, 7,8-dihydroxyflavone and curcumin. Molecular docking of TDP-glucose and 7-HF with YjiC model showed pi-alkyl interaction with Arg-282 and His-14, and pi-pi interaction with His14 and thymine ring. YjiC (H14A) variant lost its glucosylation activity with TDP-glucose validating significance of His- 14 in binding of TDP-sugars.

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