• Korean Journal of Microbiology
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• v.44 no.3
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• pp.264-269
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• 2008

To develop an amylolytic industrial yeast strain producing $\beta$-amylase, the BAMY gene encoding Achlya bisexualis $\beta$-amylase was constitutively expressed under the control of the alcohol dehydrogenase gene promoter (ADC1p) in an industrial strain of Saccharomyces cerevisiae. Yeast transformation was carried out by an integration system containing $\delta$-sequences as the recombination site. The integrative cassette devoid of bacterial DNA sequences was constructed that contains the BAMY gene and $\delta$-sequences. Industrial S. cerevisiae transformed with this integrative cassette secreted 45 kDa $\beta$-amylase into the culture medium. The $\beta$-amylase activity of the transformant was approximately 18.5-times higher than that of A. bisexualis. The multi-integrated BAMY genes in the transform ant were stable after 100 generations of growth in nonselective medium. Hydrolysis of soluble starch and various starches with the enzyme released maltose but not glucose or oligosaccharides.

• Applied Biological Chemistry
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• v.35 no.1
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• pp.23-29
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• 1992

Bromelain was purified from Korean pineapple, Ananas comosus, L. The enzyme was purified about 21 fold by DEAF-cellulose ion-exchange chromatography and gel filtration on Sephadex G-150. Purified enzyme was confirmed as active single band by polyacrylamide electrophoresis and the molecular weight was estimated to be about 22,000 by SDS-PAGE. The optimum pH and temperature were 6.0 and $60^{\circ}C$, respectively. The range of its stability to the pH and temperature were respectively 5.0 to 7.0 and below $50^{\circ}C$. It was found that $Mn^{2+}$ increased the enzyme activity, whereas $Mg^{2+}\;and\;Fe^{2+}$ decreased it abruptly. The purified enzyme was inhibited by p-chloromercuribenzoic acid, indicating that reactive SH groups are required for the enzyme activity. The reaction of the enzyme followed typical Michaelis-Menten kinetics with Km value of $5.747{\times}10^{-4}\;M\;and\;Vmax\;of\;131.58\;{\mu}g/min$ for casein. When meat was treated with the enzyme, free soluble nitrogen and amino acid nitrogen increased as enzyme concentration increased.

• Applied Biological Chemistry
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• v.47 no.4
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• pp.379-383
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• 2004

For the elucidation of substrate specificity to the brown copra meal by Bacillus sp. ${\beta}-mannanase.$, the enzymatic hydrolysate after 24 hr of reaction was heated in a boiling water bath for 10 min, and then centrifuged to remove the insoluble materials from hydrolysates. The major hydrolysates composed of D.P 5 and 7 galactosyl mannooligosaccharides. For the separate of galactosyl mannooligosaccharides, the supernatant solution of 150 ml was put on a first activated carbon column. The column was then washed with 5 l of water to remove mannose and salts. The oligosaccharides in the column were eluted by a liner gradient of $0{\sim}30%$ ethanol, at the flow rate of 250 ml per hour. The sugar composition in each fraction tubes was examined by TLC and FACE analysis. The combined fraction from F3 was concentrated to 30 ml by vacuum evaporator. Then put on a second activated carbon column. The oligosaccharides in the column were eluted by a liner gradient of $0{\sim}30%$ ethanol (total volume: 5 l), at the flow rate of 250 ml per hour. The eluent was collected in 8 ml fraction tubes, and the total sugar concentration was measured by method of phenol-sulfuric acid. The major component of F2 separated by 2nd activated carbon column chromatography were identified $Gal^3Man_4(6^3-mono-{\alpha}-D-galactopyranosyl-{\beta}-mannotetraose)$. To investigate the effects of brown copra meal galactomannooligosaccharides on growth of Bifidobacterium longum, B. bifidum were cultivated individually on the modified-MRS medium containing carbon source such as $Gal^3Man_4$, compared to those of standard MRS medium.

• Korean Journal of Food Science and Technology
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• v.49 no.5
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• pp.538-543
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• 2017

Astringent persimmon (Diospyros kaki Thunb. cv. Cheongdo-Bansi) peel with the astringency removed, which is a by-product of dried persimmon (gotgam), was investigated for its antioxidant and neuroprotective properties. A mixture of peel and 40% (v/v) aqueous ethanol was subjected to ultrasonication and then thermal and nonthermal treatments, to produce thermally-treated and nonthermally-treated persimmon peel extracts (TPE and NTPE, respectively). The total phenolic and flavonoid contents and the antioxidant capacity of TPE was approximately 1.3-1.8 times higher than those of NTPE. TPE resulted in the increased viability of neuronal PC-12 cells compared with NTPE. Furthermore, intracellular oxidative stress in PC-12 cells was more decreased by treatment with TPE than NTPE. Cholinesterases, such as acetylcholinesterase and butyrylcholinesterase, were more inhibited by treatment with TPE than NTPE. These results suggest that TPE is useful as a functional material to decrease oxidative stress in neuronal cells and to inhibit cholinesterases.

• Proceedings of the Korean Society of Postharvest Science and Technology of Agricultural Products Conference
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• 2000.04a
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• pp.6-7
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• 2000

개발도상국의 급격한 인구 증가에 의해 세계 60억 이라는 인구도 2050년을 맞아 약 100억에 도달할 것이라고 일컬어진다. 이로 인한 장래의 식량 위기에 대비하여 벼, 밀, 옥수수 등의 증산, 품종 개발도 물론 필요하지만 선진국을 중심으로 시장성 높은 작물의 소비가 우선되어지는 상황에 맞추어 세계적으로 주식이 될 수 있는 새로운 곡류의 확보와 생산체제도 중요한 문제이다. 한편으로 생활의 향상에 따른 식물의 다양화와 건강지향의 관점으로 본 다 품목 소량형의 식생활을 하는 것이 식물성 allergy의 방지 측면으로서의 곡류 특히 잡곡류의 유효 이용이 부각되어진다. 이들 중 amarans, quinoa는 벼과 식물에 비교해서 광합성능이 좋은 C4식물로서 생장이 빠르고 동시에 비타민, 무기질, 지질이 풍부하고 구성 단백질 중에 필수 아미노산을 많이 함유하여 아미노산 등급도 높고 특히 영양 발란스도 우수하다. 또 cholesterol 저하작용, 식물섬유에 의한 대장암의 억제 작용 등이 잘 알려져 있다. 그리l고 quinoa에 대해서는 아메리카 항공우주국(NASA)에서 CELSS(Controlled Ecological Life Support System; 장기간 우주특무비행의 승선원을 위한 공기중의 이산화탄소를 제거하고 식량·산소·물을 만들어 내기 위해 식물을 이용하는 방법)에 적합한 작물 후보로써 선택되어 신규 식품소재로써 주목받고 있다. 이상과 같은 견지로부터 amarans, quinoa를 일상식화되고 있는 빵에 이용하기 위해 제빵성 및 혼합중의 반죽의 모든 성질에 대해서 검토했다. amarans는 초과의 Amaranthus에 속하고 주요 생산국은 아메리카, 멕시코, 페루등이지만 일본에서는 주로 A.hypochondriacus가 수입되어 이용 되어지고 있다.amarans의 가루는 단독으로는 점탄성 있는 반죽을 형성하지 않기 때문에 밀가루에 일부를 대용한 wheat flour dough를 사용하고 가정용 제빵기로 구워 최종 단계에까지의 제빵성 결과를 산출했다. amarans folur 5%의 대체에는 빵의 비용적이 비교적 증대했지만 그 이상 amarans flour을 대처하면 확연히 비용적은 감소했다. amarans flour 10% 대체에 hemicellulase 1250U 이상을 첨가하면 비용적은 눈에 띄게 증대했다. farinograph에 있어서 반죽의 안정성은 amarans flour 10% 대용에 현저히 감소했다. 반죽의 점탄성(아축응력, 탄성률, 점성계수)는 amarans flour 10%를 대용한 것이 무첨가한 것보다 많이 단단해졌음을 알 수 있었다. 혼합중의 반죽의 조사형 전자현미경 관찰로 amarans flour로 대체한 gluten이 단단해졌음을 알수 있었다. 유화제 stearly 칼슘, 혹은 hemicellulase를 amarans 10% 대체한 밀가루에 첨가하면 확연히 비용적을 증대시킬 수 있다는 사실을 알 수 있었다. quinoa는 명아주과 Chenopodium에 속하고 페루, 볼리비아 등의 고산지에서 재배 되어지는 것을 시료로 사용하였다. quinoa 분말은 중량의 5-20%을 quinoa를 대체하고 더욱이 분말중량에 대하여 0-200ppm의 lipase를 lipid(밀가루의 2-3배)에 대하여 품질개량제로서 이용했다. 그 결과 quinoa 대량 7.5%에서 비용적, gas cell이 가장 긍정적 결과를 산출했고 반죽의 조직구조가 강화되었다. 또 quinoa 대체에 의해 전분-지질 복합제의 흡열량이 증대된 것으로부터 전분-지질복합제의 형성 촉진이 시사되었다.이것으로 인하여 호화억제에 의한 노화 방지효과가 기대되었지만 실제로 빵의 노화는 현저히 진행되었다. 이것은 quinua 대체량 증가에 따른 반죽의 안정성이 저하되어 버린 것으로 생각되어진다. 더욱이 lipase를 첨가하면 반죽이 분화하는 경향이 보여졌지만 첨가량 75ppm에 있어서 상당히 비용적의 증대가 보였다. 이것은 lipase의 가수분해에 의해 생긴 monogliceride에 의한 유화각 일어나서 보존성이 개선되어진 것으로 quinoa를 보다 많이 빨에 이용하기 위해서는 lipaserk 품질개량제로서 유효하다는 것을 알 수 있었다. 또 lipase는 quinoa의 대체량이 비교적 많은 10-20%의 섭취가 곧 allergy 질환 문제의 개선책이 되는 것은 물론 amarans, quinoa에는 lysine, 함황아미노산이 많고 지질중의 지방산조성도 좋고 무기질도 많이 함유되어 있다. 이와같이 우리들 개인의 건강에 대한 배려도 있고 amarans, quinoa등의 식품재료를 적극적으로 사용할 수 있도록 유념해 두었으면 하는 바램이다.

• Journal of Applied Biological Chemistry
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• v.59 no.4
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• pp.285-288
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• 2016

Proteolytic enzymes perform hydrolysis of the peptide bonds in the protein and most commonly use in the industry. Pseudoxanthomonas sp. WD12 and WD32 were previously isolated as protease producers from a rotten wood sample. Here, we report the secreted proteolytic enzymes. The optimum enzyme reaction temperature for the secreted crude enzyme from the strain WD12 and WD32 were $50^{\circ}C$ at pH 9.0 and $45^{\circ}C$ at pH 8.0, respectively. The enzyme activities of both strains were increased by addition of KCl, NaCl, $CaCl_2$ or $MnSO_4$, and decreased by addition of $AgNO_3$, $CuSO_4$, $FeCl_3$ or $AlCl_3$. Secreted enzymes of both strains were most strongly inhibited by addition of $FeCl_3$ or $CuSO_4$. Taken together these results, WD12 could be a candidate strain of industrial alkaline protease production.

• Microbiology and Biotechnology Letters
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• v.26 no.4
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• pp.316-322
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• 1998

An ${\alpha}$-D-galactosidase (${\alpha}$-D-galactoside galactohydrolase, EC 3. 2. 1. 22) from sunflower seed was purified by affinity chromatography using N-$\varepsilon$-aminocaproyl-${\alpha}$-D-galactopyranosylamine coupled to sepharose and its properties were examined. The specific activity of the purified enzyme, tested with p-nitrophenyl-${\alpha}$-D-galactopyranoside as substrate, was 291.66 units/mg protein, representing an 115-folds purification of the original crude extract. The final preparation obtained from by Sephadex G-25 chromatography showed a single band on SDS-polyacrylamide gel electrophoresis. The molecular weight was determined to be 42,000 by SDS-polyacrylamide gel electrophoresis. The purified galactosidase showed maximum activity at pH 4.5 and 55$^{\circ}C$, and was stable in the pH and temperature ranges of 4.0 to 5.0 and 30 to 55$^{\circ}C$, respectively. The enzyme activity was inhibited by Ag$\^$2+/, Hg$\^$2+/ and Co$\^$2+/. The enzyme activity was not affected considerably by treatment with other metal compounds. The enzyme liberated galactose from melibiose, raffinose, copra galactomannan, guar gum and locust bean gum by TLC, and also the hydrolysis rate of substrate was compared by HPLC.

• KSBB Journal
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• v.10 no.4
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• pp.374-385
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• 1995

Nutritional requirements and cultural conditions for the production of extracellular gelatin-degrading proteolytic enzyme by Bacillus subtilis B0021 were investigated. Optimum carbon source for proteolytic enzyme production was salicin, but it was substituted by glucose for economical reason. The fermentation medium giving a maximum proteolytic enzyme activity was consisted of 1.5%(w/v) glucose, 2.5%(w/v) yeast extract, and 0.001%(w/v) manganese sulfate and 0.002%(w/v) ferrous sulfate. Proteolytic enzyme activity of B. subtilis B0021 was completely inhibited by 0.5%(w/v) tannic acid. Initial pH was optimal at 7.0 and the enzyme activity in the flask culture usually reached a maximal level after 36 hours of fermentation at $30^{\circ}C$. In the $5\ell$ fermentor fermentation at $30^{\circ}C$, enzyme activity was maximum at 36 hour of cultivation but after this enzyme activity was decreased rapidly. Initial viscosity of 45%(w/v) gelatin(2,800mPas) was decreased rapidly to 96%(mPas) after hydrolysis for 4hr at $40^{\circ}C$ by crude enzyme of B. subtilis B0021.

• Journal of the Korean Chemical Society
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• v.54 no.2
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• pp.208-214
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• 2010

In the presence of alcohol, phospholipase D (PLD) is known to perform transphosphatidylation activity, during which the overall reaction rate of PLD increased. To elucidate the reaction mechanism of transphosphatidylation further, we investigated rate constants of transphosphatidylation reaction of the purified ${\alpha}$-type PLD from cabbage in the presence of various alcohols. The second-oder rate constants of PLD transphosphatidylation showed a large increase with the primary alcohols examined as expected. In the case of butanol we observed the second-oder rate constant of $33.33{\pm}1.33M^{-1}sec^{-1}$. This second-order rate constant of transphosphatidylation was as 400 times greater as the second-order hydrolysis rate constant of $0.078M^{-1}sec^{-1}$ which was adjusted for the water concentration. A linear free energy relationship between the $pK_a$ of alcohol and transphosphatidylation rate gives a Br${\o}$nsted slope of ${\beta}_{nu}$ = 0.12 ${\pm}$ 0.03. This small ${\beta}_{nu}$ value implicates that the transition state of break down of phosphatidyl-enzyme intermediate (E-P) is likely dissociative. Finally, a reaction mechanism of cabbage PLD is suggested on the basis of our results presented here and the histidine residue known to be located in the active site of cabbage PLD.

• Korean Journal of Food Science and Technology
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• v.32 no.1
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• pp.161-167
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• 2000

The protease produced by a newly isolated Aspergillus wentti from Korean traditional Meju was purified and characterized. The optimal medium composition and culture conditions for maximum protease production were ; bran :1% glucose solution =1 : 1, pH 9.0, $30^{\circ}C$, and 4 days of fermentation. Protease was purified by QAE-Sephadex, SP-Sephadex ion exchange chromatography and Sephadex G-100 chromatography. The specific activity and the purification fold of the purified enzyme were 213 unit/mg protein and 27.3, respectively. The molecular weight of purified protease was found to be 32 kDa by SDS-PAGE. Km and Vmax value's for hammastein milk casein were $3.049{\times}10^{-4}\;M\;and\;151.1\;{\mu}g/min$, respectively. Kinetic parameters showed that the enzyme has higher affinity to casein than isolated soybean protein, hemoglobin and bovine serum albumin. Optimal pH and temperature for reaction of the purified enzyme were 9.0 and $50^{\circ}C$, respectively. The enzyme was stable at pH 4.0-11.0, below $40^{\circ}C$, and the activity was not stimulated by metal ions. 1mM phenylmethylsulfonyl fluoride inhibited the enzyme activity by 98.5%. It means that the enzyme is one of serine protease.