• 한국미생물·생명공학회지
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• 제51권1호
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• pp.26-31
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• 2023

A map gene encoding methionyl-specific aminopeptidase (MAP; EC 3.4.11.18) was cloned from Tetragenococcus halophilus CY54. Translated amino acid sequence of CY54 MAP showed high similarities with those from Enterococcus faecalis (83.8%) and Streptococcus salivarius (62.2%) but low similarities with MAPs from Lactobacillus and Lactococcus genera. The map gene was overexpressed in E. coli BL21(DE3) using pET26b(+),pET26b(+), and the recombinant MAP was purified by using an Ni-NTA column. The size of recombinant MAP was 29 kDa as determined by SDS-PAGE. The optimum pH and temperature of CY54 MAP were pH 5.0 and 60℃, respectively. The activity of CY54 MAP was most significantly increased by Co²⁺ ion (159%), and showed the highest activity at 12% NaCl. K_m and V_max were 0.64 ± 0.006 mM and 10.12 ± 0.014 U/mg protein, respectively when met-pNA was used as the substrate. This is the first report on a MAP from Tetragenococcus species.

• Journal of Microbiology and Biotechnology
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• 제5권5호
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• pp.280-284
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• 1995

Aspergi11us niger F-580, a potent producer of aminopeptidase M inhibitor, was isolated from the brown spots of plant leaves with a pathological trait. The inhibitory activity was found only in the fungal mat formed by surface culture of Aspergi11us niger F-580, but not in the culture supernatant or cell pellet. The inhibitor was purified from the hot water extract of this fungal mat by using chromatographies on Diaion HP-20, DEAE-cellulose, Sephadex G-l0 and YMC-ODS-AQ columns. The purified inhibitor was analyzed by UV, mass, and NMR spectroscopies, and identified as OF494911, which had been isolated as an aminopeptidase B inhibitor from Penicillium rugulosum OF4949

• 한국수산과학회지
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• 제54권5호
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• pp.724-732
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• 2021

This study used response surface methodology to investigate the optimal conditions to reduce the bitterness of alcalase-treated anchovy hydrolysate (AAH) by the aminopeptidase active fraction (AAF) derived from the common squid Todarodes pacificus hepatopancreas. The central composite design selected AAF/AAH ratio (X₁, %) and hydrolysis time (X₂, h) as independent variables, and the degree of hydrolysis (Y₁) and bitterness (Y₂) as dependent variables. The uncoded values of the multiple response optimization for independent variables were 3.4% for the AAF/AAH ratio and 9.2 h for the hydrolysis time. The predicted values of the yield and bitterness score of alcalase-AAF continuously treated anchovy hydrolysate (AAAH) under the optimized conditions were 68.9% and 4.6 points, respectively. Their measured values of 69.5% for yield and 4.6±0.5 points for bitterness were similar to the predicted values. The food components of AAAH were 91.4% (moisture), 7.5% (protein), 0.1% (lipid) and 0.6% (ash). The findings indicate the potential value for use as an anchovy seasoning base. The results also confirm that the bitterness of AAH was remarkably improved by AAF and implicates AAF derived from squid hepatopancreas as a good enzyme to catalyze reduced bitterness.

• 한국미생물·생명공학회지
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• 제24권3호
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• pp.336-343
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• 1996

Maximum amount of the aminopeptidase M inhibitors produced by Streptomyces griseoplanus SL20209 in 500 ml-Erlenmeyer flask was accumulated after cultivation for 3 days at 28$\circ$C, thereafter the amount of inhibitors decreased slowly with a pH change to alkaline. Arabinose, xylose, mannose and soluble starch were good carbon sources for the production of the inhibitors. On the other hand, glucose was only good for the cell growth but potently inhibited the production of inhibitors. Natural organic nitrogen sources such as soybean meal, fish meal, gluten meal and peanut powder were good for the production of inhibitors, however, soytone, peptone and inorganic nitrogens such as NH$_{4}$C1 and NH$_{4}$NO$_{3}$ were poor. Inclusion of yeast extract (0.5%, w/v) or K$_{2}$HPO$_{4}$ (0.05%) into the production medium increased the production of inhibitors by accelerating cell growth. The production of inhibitors was slightly increased on the medium containing CaCO$_{3}$ (0.3%) and zeolite (0.5%), respectively. Optimal temperature and initial pH range for the production ot inhibitors were determined to be 28$\circ$C and 6.0-7.0, respectively. Employing two improved production media consisting of 3% arabinose or soluble starch, 2.5% soybean meal, 0.5% yeast extract, 0.05% K$_{2}$HP0$_{4}$, 0.1% CaCO$_{3}$ and 0.3% zeolite (pH 6.8), 1.8-fold increase in the amount of inhibitors was achieved, comparing with the basal medium used.

• 한국작물학회지
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• 제42권2호
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• pp.129-133
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• 1997

한국 재래종 대두(Glycine max) 943계통과 강원도 지성 수집종 야생종(G.soja) 50계통을 polyacrylamide gel electrophoresis와 isoelectric focusing 전기영동으로 LAP 동립효소를 분석한 결과를 요약하면 다음과 같다. PAGE에 의한 Lap1*b는 재래종과 야생종에서 가장 흔한 대립인자였다. Lap1*b의 대립인자 빈도는 한국 야생종(0.94)에서보다 재래종(1.00)에서 더 높았다. 이러한 결과는 한국 재래종 대두는 Lap1 유부자좌에서 Lap1*b 대립인자로 고정되어 있음을 나타내는 것이다. IEF 전기영동법을 이용하여 pH 4∼6.5의 gel에서 LAP 동립효소의 band type을 확인한 결과 재래종과 야생종에서 I type과 II type의 band type을 확인하였다. 재래종과 야생종에서 II type이 6계통(7.2％)과 4계통(8.0％)으로 각각 나타났고 I type은 79계통(92.8％)과 46계통(92.0％)으로 각각 나타났다. 이러한 결과는 다양한 band type이 나타날 수 있는 가능성을 제시하고 있다.

• 한국수산과학회지
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• 제54권6호
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• pp.849-860
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• 2021

This study investigated the preparation of seasoned anchovy sauce (SAS) and its functional characteristics by using aminopeptidase active fractions (AAFs) derived from squid Todarodes pacificus hepatopancreas as a bitter taste improver. As the base of the SAS, a hydrolysate (AAAH) prepared by continuously treating raw anchovies with Alcalase-AAF was used. The high-performance liquid chromatography profile of the AAAH suggested that the action of AAFs decreased the hydrophobicity of the N-terminal peptide related to bitterness in the protein hydrolysates. SAS was prepared by blending with the AAAH and other ingredients. The crude protein (2.5%), carbohydrates (18.4%), amino acid-nitrogen (1,325.1 mg/100 mL), and total free and released amino acids (FRAAs, 700.2 mg/100 mL) of SAS were higher than those of commercial anchovy sauce (CAS). Sensory evaluation revealed that SAS was superior to CAS in flavor, color, and taste. The main FRAAs of SAS were glycine (16.8%), alanine (13.2%), glutamic acid (7.8%), and leucine (7.3%). The amino acids that had a major influence on the taste according to the SAS taste values were glutamic acid, aspartic acid, alanine, and histidine. The angiotensin-converting enzyme inhibitory (2.21 mg/mL) and antioxidant activities (3.58 mg/mL) of SAS were superior to those of CAS.

• Journal of Acupuncture Research
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• 제19권2호
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• pp.51-64
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• 2002

류마티스 관절염의 쥐의 활액에서 단백분해효소, 산화제와 유리기에 대한 녹용약침의 비특이적 면역억제효과를 연구하였다. 일련의 실험표본으로서 여러가지 세포질, 리소좀, 기질 백분해효소의 제 활성을 RA대조군과 녹용약침군의 활액에서 카르보닐기 유도로 생성되는 유리기-유발 단백질손상과 항산화를 비교하였다. 전반적으로 단백분해효소활성이 정상군과 비교하여 RA대조군에서 유의성 있게 증가하였다. 세포질 단백분해효소들은 정상군과 RA군의 차이에서는 유의성이 없었다. 녹용약침처리($100{\mu}g/kg$)결과 세포질, 리소좀, 기질 단백분해효소생성을 억제하였으며, RA군과 녹용약침군 또는 정산군 사이에 활액 또는 세포질 항산화에서 유의성 있는 차이가 없음에도 불구하고, RA군 활액의 단백질손상을 유발하는 유리기는 녹용약침군과 정산군에 비교하여 약 2배 정도 높았다. 이상의 결과에서 단백분해효소와 유리기는 RA유발시 단백질손상을 유도하는 물질로 밝혀졌으며, 따라서 단백분해효소 저해와 유리기소거능을 갖는 치료법개발이 새로운 RA예방치료법으로 제시되었다. 나아가서 여러가지 기질특이성을 갖는 활액내 단백분해효소류(cysteine, serine, metallo proteinases와 peptidases)에 대한 효과적인 저해제개발이 필요한 것으로 보인다. 따라서 본 녹용약침은 이와 같은 새로운 개념의 2가지(유리기제거, 단백분해활성) 관절염치료 요소를 충족하는 약리활성을 포함하는 훌륭한 제제로 평가된다.

• 한국토양동물학회지
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• 제3권2호
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• pp.51-57
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• 1998

A new rod-shaped endospore-forming bacterium isolated from the hindgut flora of the termite, Reticulitermes speratus kyushuenesis Morimoto is described. The isolate stained Gram positive, but the KOH test and the test for L-alanine aminopeptidase were negative. The length of a single cell varies from 2.5-9.0 $\mu $m, and the cell is about 0.5-0.7$\mu $m thick. The isolate had a high cellulolytic ability and was identified as Bacillus amyloliquefaciens.

• Journal of Microbiology and Biotechnology
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• 제27권2호
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• pp.251-261
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• 2017

Initially, we screened 18 Aspergillus sojae-like strains from Aspergillus spp. isolated from meju (Korean traditional fermented soybean brick) according to their morphological characteristics. Because members of Aspergillus section Flavi are often incorrectly identified because of their phylogenetic similarity, we re-identified these strains at the morphological and molecular genetic levels. Fourteen strains were finally identified as A. sojae. The isolates produced protease and ${\alpha}-amylase$ with ranges of 2.66-10.64 and 21.53-106.73 unit/g-initial dry substrate (U/g-IDS), respectively, which were equivalent to those of the koji (starter mold) strains employed to produce Japanese soy sauce. Among the isolates and Japanese koji strains, strains SMF 127 and SMF 131 had the highest leucine aminopeptidase (LAP) activities at 6.00 and 6.06 U/g-IDS, respectively. LAP plays an important role in flavor development because of the production of low-molecular-weight peptides that affect the taste and decrease bitterness. SMF 127 and SMF 131 appeared to be non-aflatoxigenic because of a termination point mutation in aflR and the lack of the polyketide synthase gene found in other A. sojae strains. In addition, SMF 127 and SMF 131 were not cyclopiazonic acid (CPA) producers because of the deletion of maoA, dmaT, and pks/nrps, which are involved in CPA biosynthesis. Therefore, A. sojae strains such as SMF 127 and SMF 131, which have high protease and LAP activities and are free of safety issues, can be considered good starters for soybean fermentations, such as in the production of the Korean fermented soybean products meju, doenjang, and ganjang.

• Bulletin of the Korean Chemical Society
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• 제28권12호
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• pp.2303-2309
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• 2007

Expressed protein ligation (EPL) technique, joining recombinantly expressed proteins to polypeptides, has been widely adopted for addressing various biological questions and for drug discovery. However, joining two recombinant proteins together is sometimes difficult when proteins are expressed insoluble and unrefoldable, because ligation-active proteins via intein-fusion are obtainable when they are folded correctly. We overcame this limitation coexpressing target protein with additional methionine aminopeptidase (MAP) which enhances removal of the initiation methionine of recombinantly expressed protein. Our approach demonstrated that two domains of 46 kDa 5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase, a target of herbicide glyphosate, were successfully joined by native chemical ligation, although its C-terminal domain was expressed as an inclusion body. The intein-fused N-terminal fragment of EPSP synthase (EPSPSN, residues 1-237) was expressed and the ligation-active thioester tagged N-terminal fragment (EPSPSN-thioester) was purified using a chitin affinity chromatography and mercapto-ethanesulphonate (MESNA) as intein thiolysis reagent. Its Cterminal fragment (EPSPSC, residues Met237-238CYS-427), expressed as an inclusion body, was prepared from an additional MAP-expressing strain. Protein ligation was initiated by mixing ~1 mM of EPSPSN-thioester with ~2 mM of EPSPSCCYS (residues 238CYS-427). Also we found that addition of 2% thiophenol increased the ligation efficiency via thiol exchange. The ligation efficiency was ~85%. The ligated full-length EPSP synthase was dissolved in 6 M GdHCl and refolded. Circular dichroism (CD) and enzyme activity assay of the purified protein showed that the ligated enzyme has distinct secondary structure and ~115% specific activity compared to those of wild-type EPSP synthase. This work demonstrates rare example of EPL between two recombinantly expressed proteins and also provides hands-on protein engineering protocol for large proteins.