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Letícia Maria Zanphorlin,Fernanda Dell Antonio Facchini,Filipe Vasconcelos,Rafaella Costa Bonugli-Santos,André Rodrigues,Lara Durães Sette,Eleni Gomes,Gustavo Orlando Bonilla-Rodriguez 한국미생물학회 2010 The journal of microbiology Vol.48 No.3
Thermophilic fungi produce thermostable enzymes which have a number of applications, mainly in biotechnological processes. In this work, we describe the characterization of a protease produced in solidstate (SSF) and submerged (SmF) fermentations by a newly isolated thermophilic fungus identified as a putative new species in the genus Myceliophthora. Enzyme-production rate was evaluated for both fermentation processes, and in SSF, using a medium composed of a mixture of wheat bran and casein, the proteolytic output was 4.5-fold larger than that obtained in SmF. Additionally, the peak of proteolytic activity was obtained after 3 days for SSF whereas for SmF it was after 4 days. The crude enzyme obtained by both SSF and SmF displayed similar optimum temperature at 50°C, but the optimum pH shifted from 7 (SmF) to 9 (SSF). The alkaline protease produced through solid-state fermentation (SSF), was immobilized on beads of calcium alginate, allowing comparative analyses of free and immobilized proteases to be carried out. It was observed that both optimum temperature and thermal stability of the immobilized enzyme were higher than for the free enzyme. Moreover, the immobilized enzyme showed considerable stability for up to 7 reuses.
Sondor Ganbat,Dariimaa Ganbat,Ga Eul Jeong,Kee-Sun Shin,이용직,박미화,Jin Hyun Koh,이상재 한국산업식품공학회 2022 산업 식품공학 Vol.26 No.2
Natural environmental resources are considered a prospective source of microorganisms capable of producing biocatalysts with great potential in industrial areas. Arable soil fertilized with peat moss is a habitat for various microorganisms. The present research focused on the isolation and identification of hydrolase-producing bacteria that thrive at a broad temperature range. In this study, a total of 33 strains were isolated from arable soil fertilized with peat moss (Silla Garden in Busan, South Korea). The isolated bacteria were mesophiles and thermophiles with a wide temperature range. Taxonomic identification showed that the isolated strains belonged to 2 phyla, 5 families, 10 genera, and 24 species. Subsequently, the isolated strains were screened for hydrolase (amylase, lipase, and protease) activity. All isolates possessed activity of at least one enzyme and six bacterial isolates produced combined extracellular enzymes. Diversity of soil bacteria species in the present study suggest the potential of soil bacteria in the various industrial applications.
숯가마 주변 토양에서 분리한 호염성을 가지는 고온균의 생리학적 특성과 효소 생산능 분석
이용직 ( Yong-jik Lee ) 강원대학교 농업생명과학연구원 2021 강원 농업생명환경연구 Vol.33 No.3
본 연구는 호염성 고온균의 분리 가능성을 확인하기 위해 숯가마 주변의 토양을 사용하였다. 호염성 세균의 순수 분리를 위하여 marine agar 배지를 사용하였으며 37℃에서 호기적으로 배양하였다. 순수분리를 통하여 5균주를 분리하였으며 16S-based ID 분석 프로그램으로 동정을 시행한 결과, 모든 분리 균주는 Bacillus 속으로 확인되었다. 그리고 분리한 균주들이 식품, 화장품과 기타 산업에 활용이 가능한지를 확인하기 위해 고온에서의 생육 가능성과 amylase, lipase, protease 같은 효소 생산능을 확인한 결과, 5균주 모두 55도에서 생육이 가능하였으며 최소 1종류 이상의 효소 활성을 타내는 것을 확인하였다. 이 결과는 분리한 균주들이 호염성 고온균임을 나타냄과 함께 식품 및 화장품 산업에서의 고온 생물 공정에서의 활용 가능성을 나타내었다. 또한 이번 연구는 국내 유전자원 확보 및 가수분해 효소 생산능 개선을 위한 균주 개량에 본 연구를 통하여 순수분리된 균주의 활용이 도움이 될 것으로 생각된다. Soil samples around charcoal kilns were analyzed in this study to confirm the possibility of isolating halophilic thermophiles from them. A marine agar medium was used for the isolation of halobacteria with cultivation at 37℃ for several days. Five pure colonies were isolated using single colony isolation techniques. A 16S-based ID service was then performed to confirm the species of each strain identified. Results indicated that all the strains isolated were Bacillus species. Further study of these isolated strains was then undertaken to confirm whether they can be used industrially in the food, cosmetics, and other industries. This included examining whether these strains could be grown at high temperatures, as well as testing for enzyme productivity including amylase, lipase, and protease. Results demonstrated that five of these strains could grow at 55℃ and possessed at least one enzyme activity. Findings also indicated that the isolated strains were halophilic thermophiles. These results have shown the possibility of the use of these strains in biological processes at high temperatures in the food and cosmetics industries. This study also contributes to securing domestic biological resources and the improvement of hydrolytic enzyme activity by using isolated strains of halophilic thermophiles.
( Eunhye Jo ),( Jihye Kim ),( Areum Lee ),( Keumok Moon ),( Jaeho Cha ) 한국미생물생명공학회(구 한국산업미생물학회) 2021 Journal of microbiology and biotechnology Vol.31 No.3
Two putative genes, lip29 and est29, encoding lipolytic enzymes from the thermophilic bacterium Geobacillus thermocatenulatus KCTC 3921 were cloned and overexpressed in Escherichia coli. The recombinant Lip29 and Est29 were purified 67.3-fold to homogeneity with specific activity of 2.27 U/mg and recovery of 5.8% and 14.4-fold with specific activity of 0.92 U/mg and recovery of 1.3%, respectively. The molecular mass of each purified enzyme was estimated to be 29 kDa by SDS-PAGE. The alignment analysis of amino acid sequences revealed that both enzymes belonged to GDSL lipase/esterase family including conserved blocks with SGNH catalytic residues which was mainly identified in plants before. While Est29 showed high specificity toward short-chain fatty acids (C4-C8), Lip29 showed strong lipolytic activity to long-chain fatty acids (C12-C16). The optimal activity of Lip29 toward p-nitrophenyl palmitate as a substrate was observed at 50oC and pH 9.5, respectively, and its activity was maintained more than 24 h at optimal temperatures, indicating that Lip29 was thermostable. Lip29 exhibited high tolerance against detergents and metal ions. The homology modeling and substrate docking revealed that the long-chain substrates showed the greatest binding affinity toward enzyme. Based on the biochemical and in silico analyses, we present for the first time a GDSL-type lipase in the thermophilic bacteria group.
Wang, Yi,Wang, Chen,Chen, Yonglun,Cui, MingYu,Wang, Qiong,Guo, Peng The Korean Society for Microbiology and Biotechnol 2022 Journal of microbiology and biotechnology Vol.32 No.6
α-Galactosidase is a debranching enzyme widely used in the food, feed, paper, and pharmaceuticals industries and plays an important role in hemicellulose degradation. Here, T26, an aerobic bacterial strain with thermostable α-galactosidase activity, was isolated from laboratory-preserved lignocellulolytic microbial consortium TMC7, and identified as Parageobacillus thermoglucosidasius. The α-galactosidase, called T26GAL and derived from the T26 culture supernatant, exhibited a maximum enzyme activity of 0.4976 IU/ml when cultured at 60℃ and 180 rpm for 2 days. Bioinformatics analysis revealed that the α-galactosidase T26GAL belongs to the GH36 family. Subsequently, the pET-26 vector was used for the heterologous expression of the T26 α-galactosidase gene in Escherichia coli BL21 (DE3). The optimum pH for α-galactosidase T26GAL was determined to be 8.0, while the optimum temperature was 60℃. In addition, T26GAL demonstrated a remarkable thermostability with more than 93% enzyme activity, even at a high temperature of 90℃. Furthermore, Ca<sup>2+</sup> and Mg<sup>2+</sup> promoted the activity of T26GAL while Zn<sup>2+</sup> and Cu<sup>2+</sup> inhibited it. The substrate specificity studies revealed that T26GAL efficiently degraded raffinose, stachyose, and guar gum, but not locust bean gum. This study thus facilitated the discovery of an effective heat-resistant α-galactosidase with potent industrial application. Meanwhile, as part of our research on lignocellulose degradation by a microbial consortium, the present work provides an important basis for encouraging further investigation into this enzyme complex.
Jian Yang,Jie Li,Yunfeng Hu,Lizhen Li,Lijuan Long,Fazuo Wang,Si Zhang 한국생물공학회 2015 Biotechnology and Bioprocess Engineering Vol.20 No.1
High value utilization of byproduct of slaughterhousesis regarded as beneficial to both economy andenvironment. A hemoglobin degrading protease PA720was purified from fermentation broth of a Porites luteaassociated strain Streptomyces rutgersensis SCSIO 11720(SCSIO 11720) and exhibited maximal activity at 70°C,pH 10.5. The isolated strain was identified by genotypicmethod and 16 s RNA gene sequence was deposited inGenBank under accession No. KC414842. The enzymeexhibited excellent thermostability since it was stable at 70°Cwith t1/2 value of 1,386.3 min. Kinetic and thermodynamicstudy showed the activation energy (Ea), ΔG* (free energy),ΔH* (enthalpy) and ΔS* (entropy) for protease deactivationwere 62.71 kJ/mol, 74.26 kJ/mol, 59.86 kJ/mol, and-41.97 J/mol/K, respectively. Highest specific activity ofprotease PA720 was observed when using casein (9,953 U/mg)and hemoglobin (9,854 U/mg) as substrates. Hemoglobinhydrolysate prepared by protease PA720 showed significantantibacterial activity towards Escherichia coli, Staphylococcusaureus and Bacillus subtilis, indicating this protease couldbe used as an instrumental enzyme for production ofhemoglobin-based antibacterial peptides. Based on partialamino acid sequences of the enzyme PA720, the full geneencoding this protease was obtained by degenerate primerPCR and has been deposited in GenBank under accessionNo. KC414842. The deduced amino acid sequence exhibitshomology with other microbial thermophilic proteases insubtilisin family.
( Woo Soo Jeong ),( Dong Ho Seo ),( Jong Hyun Jung ),( Dong Hyun Jung ),( Dong-woo Lee ),( Young-seo Park ),( Cheon-seok Park ) 한국미생물 · 생명공학회 2017 Journal of microbiology and biotechnology Vol.27 No.2
A highly thermostable β-(1-4)-glucanase (NA23_08975) gene (fig) from Fervidobacterium islandicum AW-1, a native-feather degrading thermophilic eubacterium, was cloned and expressed in Escherichia coli. The recombinant FiG (rFiG) protein showed strong activity toward β-D-glucan from barley (367.0 IU/mg), galactomannan (174.0 IU/mg), and 4- nitrophenyl-cellobioside (66.1 IU/mg), but relatively weak activity was observed with hydroxyethyl cellulose (5.3 IU/mg), carboxymethyl cellulose (2.4 IU/mg), and xylan from oat spelt (1.4 IU/mg). rFiG exhibited optimal activity at 90oC and pH 5.0. In addition, this enzyme was extremely thermostable, showing a half-life of 113 h at 85oC. These results indicate that rFiG could be used for hydrolysis of cellulosic and hemicellulosic biomass substrates for biofuel production.