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( Zhuang Yao ),( Xiaoming Liu ),( Jae Min Shim ),( Kang Wook Lee ),( Hyun-jin Kim ),( Jeong Hwan Kim ) 한국미생물 · 생명공학회 2017 Journal of microbiology and biotechnology Vol.27 No.1
Nine bacilli with fibrinolytic activities were isolated from doenjang, a traditional Korean fermented soy food. Among them, RSB34 showed the strongest activity and was identified as Bacillus amyloliquefaciens by 16S rRNA and recA gene sequencing. During growth on LB up to 96 h, RSB34 showed the highest fibrinolytic activity (83.23 mU/μl) at 48 h. Three bands of 23, 27, and 42 kDa in size were observed when the culture supernatant was analyzed by SDSPAGE and 27 and 42 kDa bands by fibrin zymography. The gene encoding the 27 kDa fibrinolytic enzyme AprE34 was cloned by PCR. BLAST analyses confirmed that the gene was a homolog to genes encoding AprE-type proteases. aprE34 was overexpressed in Escherichia coli BL21(DE3) using pET26b(+). Recombinant AprE34 was purified and examined for its properties. The K<sub>m</sub> and V<sub>max</sub> values of recombinant AprE34 were 0.131 ± 0.026 mM and 16.551 ± 0.316 μM/l/min, respectively, when measured using an artificial substrate, Nsuccinyl- ala-ala-pro-phe-p-nitroanilide. aprE34 was overexpressed in B. subtilis WB600 using pHY300PLK. B. subtilis transformants harboring pHYRSB34 (pHY300PLK with aprE34) showed higher fibrinolytic activity than B. amyloliquefaciens RSB34.
( Kang Wook Lee ),( Jae Min Shim ),( Zhuang Yao ),( Jeong A Kim ),( Hyun-jin Kim ),( Jeong Hwan Kim ) 한국미생물생명공학회(구 한국산업미생물학회) 2017 Journal of microbiology and biotechnology Vol.27 No.7
To develop starters for the production of functional foods or materials, lactic acid bacteria producing γ-aminobutyric acid (GABA) were screened from jeotgals, Korean fermented seafoods. One isolate producing a high amount of GABA from monosodium L-glutamate (MSG) was identified as Enterococcus avium by 16S rRNA gene sequencing. E. avium M5 produced 18.47 ± 1.26 mg/ml GABA when incubated for 48 h at 37℃ in MRS broth with MSG (3% (w/v)). A gadB gene encoding glutamate decarboxylase (GAD) was cloned and overexpressed in E. coli BL21 (DE3) using the pET26b (+) expression vector. Recombinant GAD was purified through a Ni-NTA column and the size was estimated to be 53 kDa by SDS-PAGE. Maximum GAD activity was observed at pH 4.5 and 55℃ and the activity was dependent on pyridoxal 5`-phosphate. The K<sub>m</sub> and V<sub>max</sub> values of GAD were 3.26 ± 0.21 mM and 0.0120 ± 0.0001 mM/min, respectively, when MSG was used as a substrate. Enterococcus avium M5 secretes a lot of GABA when grown on MRS with MSG, and the strain is useful for the production of fermented foods containing a high amount of GABA.
Properties of Kimchi Fermented with GABA-Producing Lactic Acid Bacteria as a Starter
( Kang Wook Lee ),( Jae Min Shim ),( Zhuang Yao ),( Jeong A Kim ),( Jeong Hwan Kim ) 한국미생물생명공학회(구 한국산업미생물학회) 2018 Journal of microbiology and biotechnology Vol.28 No.4
Kimchi (a traditional Korean fermented vegetable) was prepared with a starter, Lactobacillus zymae GU240 producing γ-aminobutyric acid (GABA), and one precursor of GABA (glutamic acid, glutamic acid monosodium salt (MSG), or kelp extract). L. zymae GU240, an isolate from kimchi, can grow at 7% NaCl and low temperature. Five different kimchi samples were fermented for 20 weeks at -1°C. Kimchi with starter alone could not produce GABA. The GABA content was highest in kimchi with co-inoculation of the starter and MSG (1% (w/w)). Kimchi co-inoculated with the starter and kelp extract powder (3% (w/w)) had the second highest GABA content. Addition of glutamic acid powder (1% (w/w)) caused a reduction in the pH level of kimchi and growth inhibition of lactic acid bacteria and yeasts. Kimchi samples with MSG or kelp extract showed improvement of sensory evaluation scores. The results demonstrate the possibility to produce kimchi with improved functionality and taste by using L. zymae GU240 as a starter along with a suitable precursor such as MSG or kelp extract.
DNA Shuffling of aprE Genes to Increase Fibrinolytic Activity and Thermostability
Yao, Zhuang,Jeon, Hye Sung,Yoo, Ji Yeon,Kang, Yun Ji,Kim, Min Jae,Kim, Tae Jin,Kim, Jeong Hwan The Korean Society for Microbiology and Biotechnol 2022 Journal of microbiology and biotechnology Vol.32 No.6
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.
조민정 ( Min Jeong Cho ),심재민 ( Jae Min Shim ),이재용 ( Jae Yong Lee ),이강욱 ( Kang Wook Lee ),야오좡 ( Zhuang Yao ),류샤오밍 ( Xiaoming Liu ),김정환 ( Jeong Hwan Kim ) 한국미생물생명공학회(구 한국산업미생물학회) 2016 한국미생물·생명공학회지 Vol.44 No.2
대두에 B. amyloliquefaciens 2 균주들과 P. farinose SY80, 그리고 R. oryzae 총 4 균주를 접종한 메주를 만들었다(메주 1). 대조구로 B. subtilis KACC16450과 A. oryzae를 접종한 메주(메주 2)와 볏짚을 균원시료로 접종한 메주(메주 3)들을 제조하여 총 56일 발효시켰다. 발효 기간중 메주 1과 2의 pH와 적정산도는 메주 3보다 각각 높고 그리고 낮았다. 메주의 혈전용해능은 메주 1이 가장 높았다. 발효 기간중 모든 메주에서 B. cereus는 검출되지 않았으나 메주 2와 3에서 여러 모양과 색을 띈 잡균들이 검출되었다. 메주 2에서 histamine이 그리고 메주 3에서 tyramine이 저 농도로 검출되었으나 메주 1에서는 둘 다 검출되지 않았다. B. amyloliquefaciens EMD17이나 MJ1-4와 같은 항균력 균주들을 접종하여 메주를 제조하면 미생물학적으로 보다 안전한 장류제품들 생산이 가능할 것이다. A test meju (meju 1) was prepared by inoculating two strains of Bacillus amyloliquefaciens (EMD17, MJ1-4), Pichia farinosa SY80, and Rhizopus oryzae into cooked soybeans. A control (meju 2) was prepared by inoculating Bacillus subtilis KACC16450 and Aspergillus oryzae. Another control (meju 3) was prepared using rice straw as the source of microorganisms. Three different mejus were fermented for 56 days outdoors. Meju 1 and meju 2 showed higher pH values than meju 3, whereas meju 3 showed higher titratable acidity than meju 1 and meju 2. Meju 1 showed the highest fibrinolytic activity. Bacillus cereus was not detected in any mejus, but various microorganisms were detected in meju 2 and 3. Histamine was detected in meju 2 and tyramine in meju 3 at lower concentration, which were not detected in meju 1. It was concluded that microbially safe, fermented soybean products could be produced from meju fermented with starters such as B. amyloliquefaciens EMD17 and B. amyloliquefaciens MJ1-4.
Lee, Kang Wook,Shim, Jae Min,Kim, Dong Wook,Yao, Zhuang,Kim, Jeong A.,Kim, Hyun-Jin,Kim, Jeong Hwan Korean Society of Food Science and Technology 2018 Food Science and Biotechnology Vol.27 No.2
Kimchi was prepared with different types of salts: purified salt (PS), solar salt aged for 1 year (SS1), aged for 3 years (SS3), and bamboo salt (BS). Kimchi inoculated with Leuconostoc mesenteroides P30 (starter kimchi), and control kimchi (non-starter kimchi) were prepared, and stored at - $1^{\circ}C$ for 20 weeks. Titratable acidity values increased slowly and reached 0.96-1.01% (pH 3.73-3.83) at 20 weeks. Proportions of coccus-type lactic acid bacteria (LAB) among total LAB were higher in SS kimchi than PS kimchi. Among non-starter kimchi, the proportions were 44.7, 41.6, 29.7, and 32.1% for SS3, SS1, BS, and PS kimchi, respectively, at 2 weeks, and 11.5, 12.8, 6.7, and 5.8%, respectively, at 20 weeks. SS kimchi had much less yeast counts than PS kimchi. Among starter kimchi, yeasts were detected from PS kimchi at 10 weeks but not detected until 18 weeks from SS1 and BS kimchi and 20 weeks from SS3 kimchi.
Properties of Doenjang (Soybean Paste) Prepared with Different Types of Salts
( Jae Min Shim ),( Kang Wook Lee ),( Zhuang Yao ),( Hyun Jin Kim ),( Jeong Hwan Kim ) 한국미생물 · 생명공학회 2016 Journal of microbiology and biotechnology Vol.26 No.9
Samples of doenjang (a fermented soybean paste) were prepared with different types of salts; purified salt (PS), 3-year-aged solar salt (SS3), 1-year-aged solar salt (SS1), and bamboo salt (BS, 3rd processing product). For starter doenjang samples, selected starters comprising two bacilli, one yeast, and one fungus were inoculated, whereas for non-starter doenjang samples, microorganisms present in rice straw were inoculated after enrichment. The doenjang samples were fermented for 13 weeks at 25°C. During the fermentation period, SS and BS doenjang samples showed higher bacilli counts as well as much lower yeast counts than PS doenjang. At 13 weeks, yeast counts of starter doenjang samples were 7.75, 5.69, 6.08, and 4.74 log CFU/g for PS, SS3, SS1, and BS doenjang, respectively. For non-starter doenjang samples, counts were 7.17, 5.05, 5.92, and 4.54 log CFU/g for PS, SS3, SS1, and BS doenjang, respectively. SS and BS promoted growth of bacilli but inhibited growth of yeasts compared with PS. Debaryomyces hansenii was the dominant yeast in PS doenjang, whereas Candida guilliermondii and Pichia sorbitophila were dominant in SS and BS doenjang. In the sensory evaluation, SS and BS doenjang scored better than PS doenjang. In conclusion, SS and BS seem better than PS for production of high-quality doenjang.
( Jae Min Shim ),( Kang Wook Lee ),( Zhuang Yao ),( Jeong A Kim ),( Hyun-jin Kim ),( Jeong Hwan Kim ) 한국미생물생명공학회(구 한국산업미생물학회) 2017 Journal of microbiology and biotechnology Vol.27 No.10
Myeolchi jeotgals (MJs) were prepared with purified salt (PS), solar salt aged for 1 year (SS), and bamboo salt (BS) melted 3 times at 10% and 20% (w/w) concentrations, and fermented for 28 weeks at 15℃. BS MJ showed higher pH and lower titratable acidities than the other samples because of the alkalinity of bamboo salt. Lactic acid bacteria counts increased until 4-6 weeks and then decreased gradually, and were not detected after 20 weeks from MJs with 10% salt. Yeast counts of PS MJs were higher than those of BS and SS MJs. Bacilli were detected in relatively higher numbers throughout the 28 weeks, like marine bacteria, but archae were detected in lower numbers during the first 10 weeks. When 16S rRNA genes were amplified from total DNA from PS MJ (10% salt) at 12 weeks, Tetragenococcus halophilus was the major species. However, Staphylococcus epidermidis was the dominant species for BS MJ at the same time point. In SS MJ, T. halophilus was the dominant species and S. epidermidis was the next dominant species. BS and SS MJs showed higher amino-type nitrogen, ammonia-type nitrogen, and volatile basic nitrogen contents than PS MJs. SS and BS were better than PS for the production of high-quality MJs.
Junbo Wu,Kang Sun Park,Lili Zhuang,Dong Eon Kim,Eun Do Lee,Chi Sun Yun,Eun Ji Lee,Beom Sik Kim,Min Kyu Kim 한국수정란이식학회 2017 한국수정란이식학회 학술대회 Vol.2017 No.05
Somatic cell nuclear transfer (SCNT) is the technique which generates embryos by transferring diploid nucleus into an enucleated oocyte, it has produced specific animals successfully in a variety of species. However, the developmental capacity of SCNT embryos is still relatively lower than that of embryos produced in vivo. Oocyte is a kind of lipid rich cells, its quality limits the efficiency of embryo production. L-carnitine is a co-enzyme facilitating the transportation of long chain fatty acids across the inner mitochondria membrane where fatty acids are used for generating adenosine triphosphate (ATP) via beta-oxidation. It also has antioxidant actions which may protect mitochondrial membranes and DNA against damage induced by reactive oxygen species (ROS). Whether L-carnitine is functional in bovine SCNT embryos are unknown. Therefore, the objective of this study was to examine the effects of L-carnitine on oocyte maturation and developmental competence of subsequent SCNT embryos. L-carnitine was supplemented during IVM, then intracellular ROS and GSH levels, mitochondrial activity, gene expression of COCs were analyzed at the end of IVM. SCNT embryos were produced subsequently, apoptosis detection and gene expression evaluation were performed in blastocysts. In the results, treatments with 1.5 mM and 3 mM L-carnitine significantly improved maturation rates (P<0.05). Treatments with 3 mM L-carnitine effectively induced improvement in nuclear maturation, intracellular GSH levels and mitochondrial activity, as well as a reduction in intracellular ROS levels (P<0.05). mRNA levels of CPT1A, ACAA1, ACAA2, AREG, EREG, SOD1, GPX4, GLUT1 and CDC2 transcripts were effectively up-regulated by 3 mM L-carnitine treatments (P < 0.05). Similarly, 3mM L-carnitine induced an increase in blastocyst developmental rates and an improvement in blastocyst quality (P<0.05). Our study indicates that L-carnitine treatment during IVM improves oocyte nuclear maturation and subsequent SCNT embryo development.