• Journal of Microbiology and Biotechnology
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• 제23권8호
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• pp.1060-1069
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• 2013

Genome organization near cyclomaltodextrinases (CDases) was analyzed and compared for four different hyperthermophilic archaea: Thermococcus, Pyrococcus, Staphylothermus, and Thermofilum. A gene (CL1_0884) encoding a putative CDase from Thermococcus sp. CL1 (tccd) was cloned and expressed in Escherichia coli. TcCD was confirmed to be highly thermostable, with optimal activity at $85^{\circ}C$. The melting temperature of TcCD was determined to be $93^{\circ}C$ by both differential scanning calorimetry and differential scanning fluorimetry. A size-exclusion chromatography experiment showed that TcCD exists as a monomer. TcCD preferentially hydrolyzed ${\alpha}$-cyclodextrin (${\alpha}$-CD), and at the initial stage catalyzed a ring-opening reaction by cleaving one ${\alpha}$-1,4-glycosidic linkage of the CD ring to produce the corresponding single maltooligosaccharide. Furthermore, TcCD could hydrolyze branched CDs (G1-${\alpha}$-CD, G1-${\beta}$-CD, and G2-${\beta}$-CD) to yield significant amounts (45%, 40%, and 46%) of isomaltooligosaccharides (panose and $6^2$-${\alpha}$-maltosylmaltose) in addition to glucose and maltose. This enzyme is one of the most thermostable maltogenic amylases reported, and might be of potential value in the production of isomaltooligosaccharides in the food industry.

• Journal of Microbiology and Biotechnology
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• 제30권8호
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• pp.1244-1251
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• 2020

Phospholipase A₂ (PLA₂) from Streptomyces violaceoruber is a lipolytic enzyme used in a wide range of industrial applications including production of lysolecithins and enzymatic degumming of edible oils. We have therefore investigated expression and secretion of PLA₂ in two workhorse microbes, Pichia pastoris and Escherichia coli. The PLA₂ was produced to an activity of 0.517 ± 0.012 U/ml in the culture broth of the recombinant P. pastoris. On the other hand, recombinant E. coli BL21 star (DE3), overexpressing the authentic PLA₂ (P-PLA₂), showed activity of 17.0 ± 1.3 U/ml in the intracellular fraction and 21.7 ± 0.7 U/ml in the culture broth. The extracellular PLA2 activity obtained with the recombinant E. coli system was 3.2-fold higher than the corresponding value reached in a previous study, which employed recombinant E. coli BL21 (DE3) overexpressing codon-optimized PLA₂. Finally, we observed that the extracellular PLA₂ from the recombinant E. coli P-PLA₂ culture was able to hydrolyze 31.1 g/l of crude soybean lecithin, an industrial substrate, to a conversion yield of approximately 95%. The newly developed E. coli-based PLA₂ expression system led to extracellular production of PLA₂ to a productivity of 678 U/l·h, corresponding to 157-fold higher than that obtained with the P. pastoris-based system. This study will contribute to the extracellular production of a catalytically active PLA₂.

• Journal of Microbiology and Biotechnology
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• 제25권6호
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• pp.856-862
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• 2015

The objective of this study was to optimize the slurry contents and salt concentrations for ethanol production from hydrolysates of the seaweed Eucheuma spinosum. A monosaccharide concentration of 44.2 g/l as 49.6% conversion of total carbohydrate of 89.1 g/l was obtained from 120 g dw/l seaweed slurry. Monosaccharides from E. spinosum slurry were obtained by thermal acid hydrolysis and enzymatic hydrolysis. Addition of activated carbon at 2.5% (w/v) and the adsorption time of 2 min were used in subsequent adsorption treatments to prevent the inhibitory effect of HMF. The adsorption surface area of the activated carbon powder was 1,400-1,600 m²/g and showed selectivity to 5-hydroxymethyl furfural (HMF) from monosaccharides. Candida tropicalis KCTC 7212 was cultured in yeast extract, peptone, glucose, and high-salt medium, and exposed to 80, 90, 100, and 110 practical salinity unit (psu) salt concentrations in the lysates. The 100 psu salt concentration showed maximum cell growth and ethanol production. The ethanol fermentations with activated carbon treatment and use of C. tropicalis acclimated to a high salt concentration of 100 psu produced 17.9 g/l of ethanol with a yield (Y_EtOH) of 0.40 from E. spinosum seaweed.

• 한국미생물·생명공학회지
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• 제17권2호
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• pp.136-141
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• 1989

Streptomyces sp. 4M-2의 생전분 분해효소를 황산 암모니움 염석과 이온교환 크로마토그라피 및 Gel 여과 등으로 비활성이 51.22 RSU/mg, 수율 4.5%로 정제할 수 있었다. 정제효소의 분자량은 102,000이었으며 생옥수수 전분에 대한 Km값은 44.44mg/m1 이었다 정제효소의 작용 최적온도는42$^{\circ}C$, pH는 5.5였고 $Ca^{2+}$와 $Ba^{2+}$의 첨가에 의해 효소활성이 증가되었다. 정제효소는 옥수수 amylose를 가장 잘 분해하고 감자전분도 비교적 잘 분해하였다. 이 효소에 의한 옥수수 생전분의 분해산물은 주로 maltose와 maltotriose였고 소량의 glucose와 oligosaccharide도 검출되었으므로 $\alpha$-amylase 계통의 효소로 추정된다.

• Preventive Nutrition and Food Science
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• 제22권1호
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• pp.37-44
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• 2017

Sulfur-containing amino acids play important roles in good flavor generation in Maillard reaction of non-enzymatic browning, so aqueous model systems of glucosamine and cysteine were studied to investigate the effects of reaction temperature, initial pH, reaction time, and concentration ratio of glucosamine and cysteine. Response surface methodology was applied to optimize the independent reaction parameters of cysteine and glucosamine in Maillard reaction. Box-Behnken factorial design was used with 30 runs of 16 factorial levels, 8 axial levels and 6 central levels. The degree of Maillard reaction was determined by reading absorption at 425 nm in a spectrophotometer and Hunter's L, a, and b values. ${\Delta}E$ was consequently set as the fifth response factor. In the statistical analyses, determination coefficients ($R^2$) for their absorbance, Hunter's L, a, b values, and ${\Delta}E$ were 0.94, 0.79, 0.73, 0.96, and 0.79, respectively, showing that the absorbance and Hunter's b value were good dependent variables for this model system. The optimum processing parameters were determined to yield glucosamine-cysteine Maillard reaction product with higher absorbance and higher colour change. The optimum estimated absorbance was achieved at the condition of initial pH 8.0, $111^{\circ}C$ reaction temperature, 2.47 h reaction time, and 1.30 concentration ratio. The optimum condition for colour change measured by Hunter's b value was 2.41 h reaction time, $114^{\circ}C$ reaction temperature, initial pH 8.3, and 1.26 concentration ratio. These results can provide the basic information for Maillard reaction of aqueous model system between glucosamine and cysteine.

• 대한한의학회지
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• 제27권1호
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• pp.91-103
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• 2006

Objectives : Etiological studies of diabetes and its complications showed that oxidative stress might play a major role. Therefore, many effects have been fried to regulate oxygen free radicals for treating diabetes and its complications. Because Holotrichia has been known to be effective for the treatment of diabetes, the methanol extract of Holotrichia was tested for its effectiveness in reducing the oxidative stress induced by streptozotocin. Methods : Holotrichia was washed, dried in the shade and crushed. The crushed Holotrichia was extracted 3 times, each time with 3 volumes of methyl alcohol at $60^{\circ}C$ for 24h. The extract was filtered and evaporated under a reduced pressure using a rotary evaporator to yield 17 g. Holotrichia extract was oral-administed to the diabetic rats induced by streptozotocin 50 mg per 1 kg of body weight for 20 days. The efficacy of the Holotrichia extract was examined with regard to the enzymatic pathways involved in the oxygen free radical production and the glutathione balance. Results : The Effects of the methanol extract of Holotrichia in streptozotocin-induced diabolic rats with regards to body weight, blood glucose level, hepatic lipid peroxide level, hepatic superoxide anion radical content. hepatic xanthine oxidase activity and type conversion rate, hepatic glutathione level, hepatic aldose reductase activity, and hepatic sorbitol dehydrogenase activity were shown to be good enough to cure and prevent the diabetes and its complications. Conclusions : These results indicated that Holotrichia might reduce the oxidative stress in the tissues and organs by regulating the production of oxygen free radicals. Especially, Holotrichia might prevent and cure the diabetes and its complications by reducing the oxidative stress in the ${\beta}$-cells of pancreas. Some suggestions on biophoton experiments were made.

• 생명과학회지
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• 제8권5호
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• pp.497-503
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• 1998

GTase와 CDase를 함께 분비$\cdot$생산하는 B. stearother-mophilus KJl6 균주의 CDase를 ammonium sulfate 침전, DBAE-cellulose, Sephadex G-100 column chromatogra-phy, 및 FPLC로 수율 7%, 비활성 12.4 units/mg, 정제도 87.6배로 정제된 CDase를 얻었으며 SDS-PAGE 상 단일 band를 확인하였다. 정제된 CDase의 분자량은 약 68,000 dalton 이었고 활성 최적 pH와 온도는 6.0와 55$^{\circ}C$였다. pH 안정성은 5.5~8.5의 범위에서 비교적 안정하였으며, 온도 안정성은 5$0^{\circ}C$에서 2시간까지는 안정하였고, 7$0^{\circ}C$에서 1시간 전처리하여도 80% 이상의 잔존활성을 나타내었다. 효소 활성은 $Cu^{+2}$와 $Hg^{+2}$와 같은 금속이온과 p-chlorome-rcuribenzoate, N-bromosuccinimide, mercaptoethanol, dithiothreitol에 의해서 효소활성이 강하게 저해되었다. 기질에 대한 반응 특이성은 $\gamma$ -CD를 가장 잘 분해하였으며, 그 외에 soluble starch나 amylose, amylopectin 등의 기질도 잘 분해하나 이들의 분해속도는 $\gamma$-CD에 비해서는 늦었다. 이들 기질의 최종 분해산물은 maltose였으며, maltose는 거의 분해되지 않았다.

• 한국수산과학회지
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• 제50권6호
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• pp.650-655
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• 2017

Extraction methods for cultured sea mussels Mytilus edulis and the quality characteristics of resulting extracts were investigated. The crude protein, carbohydrate and volatile basic nitrogen content of raw sea mussels was 15.2%, 1.9%, and 11.2 mg/100 g, respectively. Extracts were prepared using three different methods: hot-water extract (WE), scrap enzymatic hydrolysate extraction (SE), and complex extraction (CE). The respective extracts contained 5.5%, 8.6%, and 6.6% crude protein; 281.7, 366.0, and 343.0 mg/100 g amino nitrogen,: and 2.0%, 1.1% and 1.8% salinity. Their extraction yields were 689, 323, and 1,012 mL/kg. The CE method was superior to the traditional WE method in terms of extraction yield, amino-nitrogen content, and organoleptic qualities, but not odor. Active taste components were evaluated and the total free amino acid content of the WE and CE methods was 5,667.0 and 7,006.3 mg/100 g, respectively. The concentrations of major components (for WE and CE methods, respectively) were as follows: glutamic acid (1,244.0 and 955.4 mg/100 g), taurine (987.9 and 746.8 mg/100 g), glycine (721.2 and 847.0 mg/100 g), alanine (341.9 and 423.8 mg/100 g), arginine (265.5 and 376.5 mg/100 g), lysine (199.8 and 270.4 mg/100 g), and proline (253.9 and 220.3 mg/100 g). In conclusion, these results demonstrate that there is potential for using the CE method to expand the commercial utilization of sea mussels as a flavoring substance resource.

• 한국미생물·생명공학회지
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• 제24권1호
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• pp.50-58
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• 1996

Interspecific hybrids between Aspergillus oryzae var oryzae and A. nidulans 514 were obtained by nuclear transfer technique. Several autotrophic mutants isolated from conidiospores of the two strains were mutagenized with ultraviolet and N-methyl-N-nitrosoguanidine. Optimal conditions for formation of intergeneric hybrids were investigated. Frequencies of hybrid formation by nuclear transfer were $3{\times}10^{-5}{\sim}1{\times}10^{-5}$. From observation of genetic stability, conidial size, DNA content, and nuclear stain, it was suggested that their karyptypes are aneuploid. The hybrids showed 1.1~1.4 fold higher xylanase activities than parental strains did. The xylanase of Aspergiilus sp. TAVD514-3 was purified and some of it's enzymatc characteristics were investigated. The enzyme was purified about 85 fold with an overall yield of 17% from the culture medium by ammonium sulfate fractionation, Sephadex G-75 gel permeation chromatography, and CM-sephadex A-50 ion exchange chromatography. The purified enzyme functions optimally at pH 9.0 and 80$^{\circ}C$. The enzymatic activity was increased by the presence of $Mg^{2+}$ and $Mn^2$ ions.

• 대한한의학방제학회지
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• 제17권2호
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• pp.163-174
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• 2009

Reactive oxygen species(ROS) can induce hepatotoxicity and trigger apoptosis in the liver. In this study, we investigated the sulfated polysaccharide A-1 from Opuntia ficus-indica against alcoholic oxidative stress in human liver Hep G2 cell. An antioxidant substance A-1 obtained from the enzymatic extract of Opuntia ficus-indica fruit was purified by DEAE-cellulose ion exchange and sephadex G-100 gel permeation chromatography. The purification yield and molecular weight were 14.3% and 1.8 KDa, respectively. The A-1 predominately contained arabinose, galactose, rhamnose and also sulfate group. The structure of A-1 was investigated by periodate oxidation, FT-IR spectroscopy, $^1H$-NMR spectroscopy. The A-1 mainly composed of alternating unit of ${\rightarrow}4$)-$\alpha$-L- Rapp-2-$SO_3^-$-$\alpha$-L-Galp-($1{\rightarrow}$ and branched linkage of $\beta$-D-Arbp- ($5{\rightarrow}$. The antioxidative activity was measured using the SOD, CAT activity and GSH assay, respectively. The expression of Nrf2 protein was analyzed by western blotting. The viable cell count analyzed by autofluorescence. Oxidative stress induced by ethanol(1 M) were dramatically reduced by A-1 treatment. A-1 also prevented cell death induced by oxidative stress. It also increased expression Nrf2 protein level. We concluded that sulfated polysaccharide A-1 from Opuntia ficus-indica effectively protect Hep G2 liver cell from alcoholic oxidative stress.