• Journal of Microbiology and Biotechnology
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• v.22 no.9
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• pp.1253-1257
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• 2012

A gene (acas) designated as ${\alpha}$-amylase was cloned from Arthrobacter chlorophenolicus A6. The multiple amino acid sequence analysis and functional expression of acas revealed that this gene really encoded an amylosucrase (ASase) instead of ${\alpha}$-amylase. In fact, the recombinant enzyme exhibited typical ASase activity by showing both sucrose hydrolysis and glucosyltransferase activities. The purified enzyme has a molecular mass of 72 kDa and exhibits optimal hydrolysis activity at $45^{\circ}C$ and a pH of 8.0. The analysis of the oligomeric state of ACAS with gel permeation chromatography revealed that the ACAS existed as a monomer.

• Asian-Australasian Journal of Animal Sciences
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• v.16 no.8
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• pp.1165-1170
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• 2003

A total of 250 laying hens were fed a diet containing 0, 1, 2.5 or 5% conjugated linoleic acid (CLA), and 5% Safflower seed oil (SSO) for 5 weeks, and eggs were collected by week to analyse lipid characteristics of egg yolk. Egg yolk from CLA-fed groups showed significant increase in CLA content with increased CLA in the diet. Dietary CLA also increased the ratio of saturated fatty acids and decreased unsaturated fatty acids in the egg yolk. The proportion of myristic, palmitic, stearic and CLA were increased, while those of oleic, linoleic, linolenic and arachidonic acid were decreased. The cholesterol content in egg yolk was significantly decreased by dietary CLA for 5 weeks feeding. After 7 days of feeding, 5% CLA-fed group showed the lowest cholesterol content in egg yolk. CLAfed groups showed significantly lower 2-thiobarbituric acid-reactive substances (TBARS) values compared to control and SSO-fed group after 14 days of storage. No significant differences in TBARS values among CLA-fed groups were observed at the 28 days of storage. Results suggested that lipid oxidation of egg yolk during cold storage could be inhibited by dietary CLA due not only to changes in fatty acid composition but also to the high concentration of CLA in egg yolk.

• Journal of Applied Biological Chemistry
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• v.58 no.3
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• pp.281-285
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• 2015

Human neutrophil elastase (HNE) represents a good therapeutic target for the treatment of inflammatory diseases as well as invasion of microorganism. The methanol extract of a aerial part of Chelidonium majus L. showed high activity against the neutrophil elastase with an $IC_{50}$ value of $100{\mu}g/mL$. Due to its potency, subsequent bioactivity-guided fractionation of methanol extract led to six alkaloids (1-6), which were identified as dihydrosanguinarine (1), (s)-stylopine (2), arnottianamide (3), (+)-chelidonine (4), spallidamine (5), and N-trans-feruloyltyramine (6). Among of them, three alkaloids (2, 5, and 6) inhibited HNE in a dose-dependent manner with $IC_{50}$ ranging between 11.6 and $51.0{\mu}M$. Lineweaver-Burk and Dixon plots, and their secondary replots showed that alkaloids (2, 5, and 6) were mixed inhibitors of HNE. The analysis of $K_I$ and $K_{IS}$ value proved that all inhibitors (2, 5, and 6) had reversible mixed type I mechanism.

• Microbiology and Biotechnology Letters
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• v.48 no.4
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• pp.439-446
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• 2020

Two Bacillus strains, K3 and K208, both demonstrating strong fibrinolytic activities were isolated from Kimchi, a traditional Korean preparation of fermented vegetables. Isolates were subjected to various molecular biology based identification methods including RAPD-PCR and identified as B. subtilis and B. velezensis, respectively. Tryptic soy broth (TSB) was found to best maintain both the growth and the fibrinolytic activity of these strains. Culture supernatants were analyzed by SDS-PAGE and fibrin zymography, and the results indicate that a 40 and 27 kDa band seem to be responsible for the fibrinolytic activities of these two isolates and the 27 kDa band was subsequently identified as the mature form of AprE, the major fibrinolytic enzyme. Thus the aprE genes were cloned and the translated amino acid sequences demonstrated 99.3% identity with each other, and 86.5% identity with BsfA, a fibrinolytic enzyme from B. subtilis ZA400 also isolated from Kimchi, and AprE2, a fibrinolytic enzyme from B. subtilis CH3-5 isolated from Cheonggukjang, a traditional Korean fermented soy. Given this B. subtilis K3 and B. velezensis K208 may be promising starter cultures in the production of fermented foods.

• Journal of Microbiology and Biotechnology
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• v.31 no.6
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• pp.833-839
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• 2021

Bacillus subtilis CH3-5 isolated from cheonggukjang secretes a 28 kDa protease with a strong fibrinolytic activity. Its gene, aprE3-5, was cloned and expressed in a heterologous host (Jeong et al., 2007). In this study, the promoter of aprE3-5 was replaced with other stronger promoters (P_cry3A, P₁₀, P_SG1, P_srfA) of Bacillus spp. using PCR. The constructed chimeric genes were cloned into pHY300PLK vector, and then introduced into B. subtilis WB600. The P10 promoter conferred the highest fibrinolytic activity, i.e., 1.7-fold higher than that conferred by the original promoter. Overproduction of the 28 kDa protease was confirmed using SDS-PAGE and fibrin zymography. RT-qPCR analysis showed that aprE3-5 expression was 2.0-fold higher with the P10 promoter than with the original promoter. Change of the initiation codon from GTG to ATG further increased the fibrinolytic activity. The highest aprE3-5 expression was observed when two copies of the P₁₀ promoter were placed in tandem upstream of the ATG initiation codon. The construct with P10 promoter and ATG and the construct with two copies of P10 promoter in tandem and ATG exhibited 117% and 148% higher fibrinolytic activity, respectively, than that exhibited by the construct containing P10 promoter and GTG. These results confirmed that significant overproduction of a fibrinolytic enzyme can be achieved by suitable promoter modification, and this approach may have applications in the industrial production of AprE3-5 and related fibrinolytic enzymes.

• Microbiology and Biotechnology Letters
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• v.50 no.1
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• pp.63-70
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• 2022

Two small cryptic plasmids, pHG1 and pHG2, were isolated from Levilactobacillus zymae (formerly Lactobacillus zymae) GU240 and characterized. pHG1 is 1,814 bp in size with a GC content of 37.4% and contains two open reading frames. orf1 can potentially encode a protein of 101 amino acids (aa) with 99% identity with the copy number control protein of Lacticaseibacillus paracasei. orf2 can potentially encode a protein of 230 aa with 99% identity with a replication protein from multiple species. Six inverted repeats (IR I-VI) and six direct repeats (DR I-VI) were found in pHG1. pHG2 is 2,864 bp in size, with a GC content of 39.6%. pHG2 has two orfs. orf1 might encode a protein with 99% identity with the TrsL transmembrane protein. orf2 might encode a protein with 99% identity with plasmid recombination proteins from lactic acid bacteria. Both pHG1 and pHG2 may be useful as frames for constructing lactic acid bacteria-Escherichia coli shuttle vectors.

• Journal of Microbiology and Biotechnology
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• v.32 no.6
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• pp.800-807
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• 2022

Four aprE genes encoding alkaline serine proteases from B. subtilis strains were used as template genes for family gene shuffling. Shuffled genes obtained by DNase I digestion followed by consecutive primerless and regular PCR reactions were ligated with pHY300PLK, an E. coli-Bacillus shuttle vector. The ligation mixture was introduced into B. subtilis WB600 and one transformant (FSM4) showed higher fibrinolytic activity. DNA sequencing confirmed that the shuffled gene (aprEFSM4) consisted of DNA mostly originated from either aprEJS2 or aprE176 in addition to some DNA from either aprE3-5 or aprESJ4. Mature AprEFSM4 (275 amino acids) was different from mature AprEJS2 in 4 amino acids and mature AprE176 in 2 amino acids. aprEFSM4 was overexpressed in E. coli BL21 (DE3) by using pET26b(+) and recombinant AprEFSM4 was purified. The optimal temperature and pH of AprEFSM4 were similar to those of parental enzymes. However, AprEFM4 showed better thermostability and fibrinogen hydrolytic activity than the parental enzymes. The results indicated that DNA shuffling could be used to improve fibrinolytic enzymes from Bacillus sp. for industrial applications.

• Proceedings of the Korean Society of Plant Biotechnology Conference
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• 2003.04a
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• pp.105-105
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• 2003

• 한국생물공학회:학술대회논문집
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• 2005.10a
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• pp.642-643
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• 2005

• 한국생물공학회:학술대회논문집
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• 2005.10a
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• pp.715-717
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• 2005