• Journal of Microbiology and Biotechnology
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• v.33 no.12
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• pp.1681-1691
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• 2023

Flavin mononucleotide-binding proteins or domains emit cyan-green fluorescence under aerobic and anaerobic conditions, but relatively low fluorescence and less thermostability limit their application as reporters. In this work, we incorporated the codon-optimized fluorescent protein from Chlamydomonas reinhardtii with two different linkers independently into the redox-responsive split intein construct, overexpressed the precursors in hyperoxic Escherichia coli SHuffle T7 strain, and cyclized the target proteins in vitro in the presence of the reducing agent. Compared with the purified linear protein, the cyclic protein with the short linker displayed enhanced fluorescence. In contrast, cyclized protein with incorporation of the long linker including the myc-tag and human rhinovirus 3C protease cleavable sequence emitted slightly increased fluorescence compared with the protein linearized with the protease cleavage. The cyclic protein with the short linker also exhibited increased thermal stability and exopeptidase resistance. Moreover, induction of the target proteins in an oxygen-deficient culture rendered fluorescent E. coli BL21 (DE3) cells brighter than those overexpressing the linear construct. Thus, the cyclic reporter can hopefully be used in certain thermophilic anaerobes.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.10 no.1
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• pp.194-199
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• 2009

An efficient production method of food-grade heamatococcus extract was developed based on stepwise enzymatic hydrolysis. In the first step, Haematococcus pluvialis cells hydrolysis carried out with commercially available exopeptidase(Flavourzyme) and endopeptidase (Alcalase), resulted in increased astaxanthin content. In the second step, proteolytic hydrolyzed H. pluvialis cells treated with hetero-polysaccharides hydrolytic enzyme (Viscozyme). By two-stage treatments using Alcalase and Flavourzyme and Viscozyme, the highest astaxanthin content was obtained. The astaxanthin content was remarkably enhanced by 320% $(529{\mu}g/g\rightarrow2,256{\mu}g/g)$ than that of the non-treated extract. And then, antioxidative activities determined by DPPH method were increased with increasing the astaxanthin content in haematococcus extract prepared by enzymatic hydrolysis.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.36 no.3
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• pp.210-219
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• 2003

To develop natural flavoring substances, optimal two stage enzyme hydrolysis conditions and flavor compounds of short-neck clam (Tapes philippinarum) enzyme hydrolysates were examined. The optimal enzyme hydrolysis conditions for two stage enzyme hydrolysate (TSEH) of short-neck clam were revealed in temperature at $55^{\circ}C$ for 4 hours digestion with alcalase at the 1st stage and 4 hours digestion at $45^{\circ}C$ with exopeptidase type neutrase at the 2nd stage. In quality tests of hot-water extracts, steam extracts and 4 kinds of enzyme hydrolysates, TSEH processing method was superior to other methods in yield, nitrogen contents, organoleptic taste such as umami intensity and inhibition of off-flavor formation, and transparency of extract. Total free amino acid contents in hot-water extract, steam extract and TSEH were 1,352.1 mg/100 g, 1,174.1 mg/100 g and 2,122.4 mg/100 g, respectively, Major free amino acids in TSEH were glutamic acid, glycine, alanine, tyrosine, phenylalanine and arginine. As for nucleotides and other bases, betaine, TMAO and creatinine were principal components in TSEH. The major inorganic ions in TSEH were Na, K, P and Cl. TSEH also revealed very higher angiotensin-I converting enzyme inhibition effect $(70.7\%)$ than those of hot-water and steam extract. We conclude that TSEH from short-neck clam was more flavorable compared with the seasoning materials on the market, it could be utilized as the instant soup base and the seasoning substances for fisheries processing.

• Journal of Life Science
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• v.21 no.5
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• pp.761-765
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• 2011

Salt stability of enzymes is a crucial practical factor in the food industry. Previously, leucine aminopeptidase (LAP) was purified from Bacillus sp. N2. Here, we present the salt effect of LAP using synthetic substrates. LAP had a hydrolytic activity for L-leucine-${\rho}$-nitroanilide in high concentrations of NaCl (up to 4 M), but not for other neutral salts (LiBr, LiCl, NaBr, KBr, and KCl). It hydrolyzed various synthetic di-peptide substrates with hydrophobic and hydrophilic amino acids at the C-terminal Xaa region, in the presence of 0-4 M NaCl. The result indicated that the hydrolytic action of LAP is not dependent on the hydrophobicity of the amino acid side chain at the scissile bond of the substrate. Remarkably, the hydrolytic activity of LAP was 1-3 folds higher than those of other LAPs and aminopeptidases in 4.5 M NaCl, suggesting that NaCl-tolerant LAP might be used in the food industry as cheese and anchovy sauce.

• The Korean Journal of Food And Nutrition
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• v.15 no.4
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• pp.364-369
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• 2002

Streptomyces shows a eukaryotic characteristic that vegetative cell can grow into mycelial form and has morphological and physiological differentiation at a certain period during its life cycle. Streptomyces has been used for the production of many biologically active compounds, such as antibiotics and pronase. Production of second metabolites and differentiation of the vegetative cell share the certain period of its lift cycle. Therefore, second metabolites may affect the differentiation of the vegetative cell. One of the microbial hormone, called A-factor, regulates the production of second metabolites, sporulation and differentiation of the cells. Streptomyces griseus produces streptomycin as well as many different kinds of proteinase. As mentioned, period of proteinases production overlaps with the period of differentiation of the vegetative cells. Protease may play a important role for the differentiation of the cells. In this paper, function of the SGPD gene cloned from S. griseus IFO 13350 tested whether it affects for the differentiation of A-factor mutated S. griseus HH1 and S. griseus IFO13350. pWHM3 and pWHM3-sprD plasmid was transformed into S. griseus HH1 and S. griseus IFO13350. Chymotrypsin activity of the cultured medium of the transformants with pWHM3-sprD plasmid didn't show any change with that of the transformants with plasmid only. The transformants with pWHM3-sprD plasmid didn't show the increase of the production of actinorhodin as well as morphological change in S. griseus IFO 13350 and HH1, as well. The promoter sequences of the SGPA and SGPB gene which encode chymotrypsin-like protease, were compared with that of SGPD gene. Regulatory mechanism of gene expression of proteinase genes will be studied for the development of high production system for protease as well as the function of the proteases.

• Microbiology and Biotechnology Letters
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• v.37 no.3
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• pp.183-188
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• 2009

Seven isolates showing strong proteolytic activity, KYC 1028, 1100, 1134, 1139, 1151, 1159, and 1182, were collected. Out of them, the broth of KYC 1134 and KYC 1139 showed the high proteolytic activity measured by azocazein. To determine 16S rDNA sequences for identification, 16S rDNA of seven isolates were amplified and compared with the 16S rDNA sequences of other myxobacteria at NCBI. It is evident from the phylo-genetic tree that the isolates belong to the genus Myxococcus. Sharing high percentage similarity values with myxobacteria, the 16S rDNA sequences were involved in two species, Myxococcus macrospores and M. Fulvus. Biochemical characteristics of KYC 1134 broth, which showed the highest proteolytic activity, showed increased activity 8 times to seven days after culture, and protein production were increased gradually and stopped at five days. The broth had optimal temperature at $60^{\circ}C$ for proteolytic activity, and stability of pH was ranged from pH 5 to 10, at $50^{\circ}C$ and 60, respectively. To classify proteases being in the broth, ten inhibitors were determined and only bestatin showed 27% inhibition effect. The inhibition result demonstrates that the broth contains kinds of amino peptidases and other exopeptidases.