• Title/Summary/Keyword: isolation of enzymes

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Isolation and Characterization of Putrescine-producing Bacteria in Commercially Available Sauces Made from Salted and Fermented Sand Lance Ammodytes personatus (시판 까나리(Ammodytes personatus) 액젓에서 Putrescine 생성균의 분리 및 특성)

  • Um, In-Seon;Kim, Tae-Ok;Park, Kwon-Sam
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.49 no.5
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    • pp.573-581
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    • 2016
  • Bacterial decarboxylation of amino acids in food leads to the production of biogenic amines, which can cause reactions in human that include headaches, nausea, palpitations, chills, and severe respiratory distress. The amine putrescine is an especially effective inhibitor of metabolizing enzymes and amplifies histamine intoxication and tyramine poisoning. Using an L-ornithine decarboxylating medium, we isolated 14 putrescine-producing bacteria from sand lance, Ammodytes personatus, sauces. The isolates were identified, using an API kit and 16S rRNA analysis, as Lysinibacillus fusiformis (1 strain), Lysinibacillus xylanilyticus (6 strains), Lysinibacillus macroides (1 strain), Lysinibacillus sphaericus (3 strains), Bacillus fusiformis (1 strain), Paenibacillus favisporus (1 strain), and Staphylococcus caprae (1 strain). These strains produced between 1.66 to 236.97 μg/mL of putrescine after 48 h incubation. Lysinibacillus spp. were the dominant putrescine-producing bacteria in sand lance sauces, which produced 236.97 μg/mL of putrescine from a culture broth containing 0.5% L-ornithine. This is the first report on the isolation and identification of putrescine-producing bacteria from sand lance sauces.

Rapid Detection and Isolation of Known and Putative $\alpha-L-Arabinofuranosidase$ Genes Using Degenerate PCR Primers

  • Park, Jung-Mi;Han, Nam-Soo;Kim, Tae-Jip
    • Journal of Microbiology and Biotechnology
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    • v.17 no.3
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    • pp.481-489
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    • 2007
  • [ $\alpha$ ]-L-Arabinofuranosidases (AFases; EC 3.2.1.55) are exo-type enzymes, which hydrolyze terminal nonreducing arabinose residues from various polysaccharides such as arabinan and arabinoxylan. Genome-wide BLAST search showed that various bacterial strains possess the putative AFase genes with well-conserved motif sequences at the nucleotide and amino acid sequence levels. In this study, two sets of degenerate PCR primers were designed and tested to detect putative AFase genes, based on their three highly conserved amino acid blocks (PGGNFV, GNEMDG; and DEWNVW). Among 20 Bacillus-associated species, 13 species were revealed to have putative AFase genes in their genome and they share over 67% of amino acid identities with each other. Based on the partial sequence obtained from an isolate, an AFase from Geobacillus sp. was cloned and expressed in E. coli. Enzymatic characterization has verified that the resulting enzyme corresponds to a typical AFase. Accordingly, degenerate PCR primers developed in this work can be used for fast, easy, and specific detection and isolation of putative AFase genes from bacterial cells.

Roles of Carbohydrate-Binding Module (CBM) of an Endo-β-1,4-Glucanase (Cel5L) from Bacillus sp. KD1014 in Thermostability and Small-Substrate Hydrolyzing Activity

  • Lee, Jae Pil;Shin, Eun-Sun;Cho, Min Yeol;Lee, Kyung-Dong;Kim, Hoon
    • Journal of Microbiology and Biotechnology
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    • v.28 no.12
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    • pp.2036-2045
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    • 2018
  • An endo-${\beta}$-1,4-glucanase gene, cel5L, was cloned using the shot-gun method from Bacillus sp.. The gene, which contained a predicted signal peptide, encoded a protein of 496 amino acid residues, and the molecular mass of the mature Cel5L was estimated to be 51.8 kDa. Cel5L contained a catalytic domain of glycoside hydrolase (GH) family 5 and a carbohydrate-binding module family 3 (CBM_3). Chromatography using HiTrap Q and CHT-II resulted in the isolation of two truncated forms corresponding to 50 (Cel5L-p50) and 35 kDa (Cel5L-p35, CBM_3-deleted form). Both enzymes were optimally active at pH 4.5 and $55^{\circ}C$, but had different half-lives of 4.0 and 22.8 min, respectively, at $70^{\circ}C$. The relative activities of Cel5L-p50 and Cel5L-p35 for barley ${\beta}$-glucan were 377.0 and 246.7%, respectively, compared to those for carboxymethyl-cellulose. The affinity and hydrolysis rate of pNPC by Cel5L-p35 were 1.7 and 3.3 times higher, respectively, than those by Cel5L-p50. Additions of each to a commercial enzyme set increased saccharification of pretreated rice straw powder by 17.5 and 21.0%, respectively. These results suggest CBM_3 is significantly contributing to thermostability, and to affinity and substrate specificity for small substrates, and that these two enzymes could be used as additives to enhance enzymatic saccharification.

Characterization of Two Metagenome-Derived Esterases That Reactivate Chloramphenicol by Counteracting Chloramphenicol Acetyltransferase

  • Tao, Weixin;Lee, Myung-Hwan;Yoon, Mi-Young;Kim, Jin-Cheol;Malhotra, Shweta;Wu, Jing;Hwang, Eul-Chul;Lee, Seon-Woo
    • Journal of Microbiology and Biotechnology
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    • v.21 no.12
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    • pp.1203-1210
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    • 2011
  • Function-driven metagenomic analysis is a powerful approach to screening for novel biocatalysts. In this study, we investigated lipolytic enzymes selected from an alluvial soil metagenomic library, and identified two novel esterases, EstDL26 and EstDL136. EstDL26 and EstDL136 reactivated chloramphenicol from its acetyl derivates by counteracting the chloramphenicol acetyltransferase (CAT) activity in Escherichia coli. These two enzymes showed only 27% identity in amino acid sequence to each other; however both preferentially hydrolyzed short-chain p-nitrophenyl esters (${\leq}C_5$) and showed mesophilic properties. In vitro, EstDL136 catalyzed the deacetylation of 1- and 3-acetyl and 1,3-diacetyl derivates; in contrast, EstDL26 was not capable of the deacetylation at $C_1$, indicating a potential regioselectivity. EstDL26 and EstDL136 were similar to microbial hormone-sensitive lipase (HSL), and since chloramphenicol acetate esterase (CAE) activity was detected from two other soil esterases in the HSL family, this suggests a distribution of CAE among the soil microorganisms. The isolation and characterization of EstDL26 and EstDL136 in this study may be helpful in understanding the diversity of CAE enzymes and their potential role in releasing active chloramphenicol in the producing bacteria.

Isolation and Enzymatic Characterization of Bacteria from Livestock Manure (축산분뇨에서 분리한 세균의 동정 및 효소학적 특성)

  • 김진선;정소선;이준석;최미영;서승염
    • Korean Journal of Microbiology
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    • v.37 no.3
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    • pp.214-220
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    • 2001
  • To develop the effective composting system, we isolated bacteria that have the abilities to degrade organic matters such as cellulose, carbohydrate, protein and lipid during the compositing of livestock manureAmong 24 strains, 6 bacteria have all the enzymatic activities of protease, amylase, cellulase and lipase.These microorganisms were identified as Corynebacterium varibilis, Bacillus spp., Pseudomonas spinosa,Acetobacter calcoaceticus and Athrobacter cumminsii All the enzymes produced by the bacteria showedactivities at the broad pH range and the maximal activities were obtained at $60^{\circ}C$. It seemed that after theincrease of temperature caused by fermentation of livestock manure, the enzymes started to degrade the rawmaterials, which are added for the control of humidity. However cellulase activity was maximum at $37^{\circ}C$,suggesting that the cellulase-producing bacteria work at an early stage of livestock manure fermentation toprovide the organic material for the growth of other bacteria. The production of the enzymes were growth-associated and maximal activities appeared at the early stationary phase of growth.

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Selection of High Efficient Enzyme for Protoplasts Isolation from Mushrooms (버섯류의 원형질체 나출을 위한 고효율 효소 선발)

  • Kim, Jong-Kun;Kim, Jin-Hee;Kong, Won-Sik;Kang, Hee-Wan
    • The Korean Journal of Mycology
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    • v.38 no.1
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    • pp.21-24
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    • 2010
  • This study was carried out to select cell wall degrading enzymes for maximizing protoplast yield from Basidiomycetes. The protoplasts were released from spore suspension, mycelia cultured on cellophane membrane, and homogenized mycelia of Flammulina velutipes using commercial cell wall degrading enzymes. The highest yield of protoplasts was obtained from the homogenized mycelia treated with the enzyme combination of $Glucanex^R$ 200G and cellulase onozuka R-10. The protocol was also available for Pleurotus ostreatus, P. eryngii, and Hypsizygus marmoreus.

Isolation of Bacillus subtilis CK-2 Hydrolysing Various Organic Materials (다양한 유기물을 분해하는 Bacillus subtilis CK-2의 분리)

  • Kim, Chul-Ho;Lee, Sang-Hyup
    • Journal of Life Science
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    • v.21 no.12
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    • pp.1716-1720
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    • 2011
  • A bacterium hydrolysing various organic materials including cellulose, protein, starch and lipid was isolated. The isolate was identified as Bacillus subtilis, and named Bacillus subtilis CK-2 in this paper. This bacterium showed optimal growth at $40\sim45^{\circ}C$, pH 6~9, and 0~3% of NaCl. B. subtilis CK-2 seemed to synthesis highly active autolysin. The hydrolytic enzymes produced by B. subtilis CK-2 were primary enzymes because extracellular enzyme activities varied similarly to the growth curve. The hydrolytic enzymes seemed to be stable at basic pH conditions. From these results, B. subtilis CK-2 was found to bea useful bacterial agent for composting, or for use in feed-production waste in agriculture, fishery, forest materials, livestock farming, and food.

Isolation, Screening and Identification of Swine Gut Microbiota with Ochratoxin A Biodegradation Ability

  • Upadhaya, Santi Devi;Song, Jae-Yong;Park, Min-Ah;Seo, Ja-Kyeom;Yang, Liu;Lee, Chan-Ho;Cho, Kyung-J.;Ha, Jong-K.
    • Asian-Australasian Journal of Animal Sciences
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    • v.25 no.1
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    • pp.114-121
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    • 2012
  • The potential for ochratoxin A (OTA) degradation by swine intestinal microbiota was assessed in the current study. Intestinal content that was collected aseptically from swine was spiked with 100 ppb OTA and incubated for 6 and 12 h at $39^{\circ}C$. An OTA assay was conducted using the incubated samples, and it was found that 20% of the OTA toxin was detoxified, indicating the presence of microbes capable of OTA degradation. Twenty-eight bacterial species were isolated anaerobically in M 98-5 media and 45 bacterial species were isolated using nutrient broth aerobically. Screening results showed that one anaerobic bacterial isolate, named MM11, detoxified more than 75% of OTA in liquid media. Furthermore, 1.0 ppm OTA was degraded completely after 24 h incubation on a solid 'corn' substrate. The bacterium was identified by 16S rDNA sequencing as having 97% sequence similarity with Eubacterium biforme. The isolation of an OTA-degrading bacterium from the swine natural flora is of great importance for OTA biodegradation and may be a valuable potential source for OTA-degradation enzymes in industrial applications.

Culture of Rat Salivary Acinar Cells (백서 타액선 선포 세포의 배양)

  • Lee, Sung-Woo;Han, Song;Kho, Hong-Seop
    • Journal of Oral Medicine and Pain
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    • v.24 no.2
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    • pp.163-169
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    • 1999
  • We investigated the culture condition and effects of various growth factors on the culture of salivary gland acinar cells. Male, Sprague-Dawley rats (about 6 weeks old) were sacrificed and their submandibular, sublingual, and parotid glands were used as specimens. High oxygen level more than 90% and coating of Matrigel on culture dish were important factors to help increase the survival time of acinar cells, Proper concentration of enzymes such as collagenase and hyaluronidase during isolation steps was also important. Addition of various growth factors such as dexamethasone, insulin, transferrin, selenous acid, reduced glutathione, epidermal growth factor, isoproterenol, and putrescine in culture medium helped to increase lifetime of cultured salivary acinar cells.

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Isolation and Characterization of cDNA Encoding Pyridoxal Kinase from Ovine Liver

  • Lee, Hyun-Shik;Choi, Soo-Young;Kwon, Oh-Shin
    • BMB Reports
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    • v.32 no.5
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    • pp.502-505
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    • 1999
  • cDNA fragments of ovine liver pyridoxal kinase were amplified by PCR using degenerate oligonucleotide primers derived from partial amino acids sequences of the enzyme. Using PCR products as probes, several overlapping cDNA clones were isolated independently from an ovine liver and a human brain cDNA library. The largest cDNA clone for each was selected for sequence analysis. The ovine liver cDNA encodes a polypeptide of 297 amino acid residues with Mr of 32,925, whereas the human clone is comprised of an open reading frame encoding 312 amino acid residues with Mr of 35,102. The deduced sequence of the human brain enzyme is completely identical to that of human testes cDNA recently reported (Hanna et al., 1997). The ovine enzymes have approximately 77% sequence identity with the human enzyme although the two sequences are completely different in the N-terminus comprising 32 residues. This result suggests that pyridoxal kinase is highly homologous in mammalian species.

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