• Microbiology and Biotechnology Letters
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• v.51 no.1
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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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• v.5 no.5
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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

• Korean Journal of Fisheries and Aquatic Sciences
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• v.54 no.5
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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.

• Microbiology and Biotechnology Letters
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• v.24 no.3
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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.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.42 no.2
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• pp.129-133
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• 1997

A total 943 accession of soybeans (G. max) and 50 wild soybeans (G. soja) were examined for leucine aminopeptidase (LAP) isozyme variation by 5％ polyacrylamide gel electrophoresis(PAGE) and isoelectric focusing(IEF) of pH 4~6.5. The Lap1*b by PAGE was the most common phenotype in both G. max and G. soja. The frequency of Lap1*b allele was observed to be higher in G. max(1.00) than in G. soja(0.96) of Korea. This result shows that G. max is fixed for Lapl*b allele at the Lap1 locus. LAP isozyme band type I and II were found using IEF of pH 4~6.5 in G. max and G. soja of Korea. Type I was observed from 92.8％ in G. max and 92.0％ in G. soja, and type II was discovered in 7.2％ G. max and 8.0％ G. soja. This result suggested the possibility to be found more various band types.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.54 no.6
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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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• v.19 no.2
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• pp.51-64
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• 2002

We have compared(using the same series of experimental tissue samples) the levels of proteolytic enzyme activities and free radical-induced protein damage in synovial fluid from RA and CPH cases. Many protease types showed significantly increased (typically by a factor of approximately 2-3-fold) activity in RA, compared to normal rats. However, CPH significantly reduced the cytoplasmic enzyme activities of arginyl aminopeptidase, leucyl aminopeptidase, pyroglutamyl aminopeptidase, tripeptidyl aminopeptidase, and proline endopeptidase to almost about 1/10 each. For the Iysosomal proteases, synovial fluid samples from RA rats, CPH significantly reduced the enzyme activities of cathepsin B, dipeptidyl aminopeptidase I and dipeptidyl aminopeptidase II. In extracellular matrix degrading(collagenase, tissue elastase) and leukocyte as sociated proteases (leukocyte elastase, cathepsin G), CPH decreased these enzyme activities of collagenase, tissue elastase and leukocyte associated elastase in RA. In cytoplasmic and lysosomal protease activities in plasma from RA. CPH and normal plasma samples were not significantly different, suggesting that altered activity of plasma proteases (particularly those enzymes putatively involved in the immune response) is not a contributory factor in the pathogenesis of RA. In addition, the level of free radical induced damage to synovial fluid proteins was approximately twice that in RA, compared with CPH. CPH significantly decreased the level of ROS induced oxidative damage to synovial fluid proteins (quantified as protein carbonyl derivative). Therefore we conclude that both proteolytic enzymes and free radicals are likely to be of equal potential importance as damaging agents in the pathogenesis of inflammatory joint disease, and that the design of novel therapeutic strategies for patients with the latter disorder should include both protease inhibitory and free radical scavenging elements. In addition, the protease inhibitory element should be designed to inhibit the action of a broad range of protease mechanistic types (i.e. cysteine-, metallo- and serine- proteinases and peptidases). However, increased protein damage induced by ROS could not be rationalised in terms of compromised antioxidant total capacity, since the latter was not significantly altered in RA synovial fluid or plasma compared with CPH.

• The Korean Journal of Soil Zoology
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• v.3 no.2
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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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• v.27 no.2
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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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• v.28 no.12
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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.