• 한국미생물·생명공학회지
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• 제24권5호
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• pp.567-573
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• 1996

Chitinase (EC 3.2.1.14) from culture fluid of Bacillus licheniformis KFB-C14 was purified 66-folds to homogenity in overall yield of 21% by ammonium sulfate fractionation, DEAE-Toyopearl, Butyl-Toyopearl and TSK-Gel HW-55F column chromatography. The enzyme protein had a molecular weight of about 86,000 and was composed of one subunit. The enzyme was significantly stable not only at high temperature but also on treatment with organic solvents and protein denaturants such as SDS, urea and guanidine-HC1. The optimum temperature and pH for reaction was 60$\circ $C and 6.0, respectively. The enzyme activity was inhibited by only Mn$^{2+}$ ion, but not inhibited by EDTA, N- ethylmaleimide and pCMB. The enzyme had high activity with colloidal chitin (V$_{max}$: 421) and commercial chitin (V$_{max}$: 480), but not with typical substrates of exo type chitinase. The thermostable chitinase had an useful reactivity for producing functional chitooligosaccharide, showing the production of (GlcNAc)$_{1}, (GlcNAc)$_{3}$, and (GlcNAc)$_{2}$ as major product.

• Asian-Australasian Journal of Animal Sciences
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• 제24권2호
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• pp.214-221
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• 2011

Effects of the cell wall-degrading enzymes derived from Acremonium cellulolyticus and Trichoderma viride on the silage fermentation and in sacco degradation of tropical grasses i.e. rhodesgrass (Chloris gayana Kunth. cv. Callide) and guineagrass (Panicum maximum Jacq. cv. Natsukaze) were investigated in laboratory-scale experiments. These two grasses were either treated with or without the enzymes before ensiling. Untreated rhodesgrass produced acetate fermentation silage (lactate, $13.0\;g\;kg^{-1}$ DM; acetate, $38.7\;g\;kg^{-1}$ DM) with high final pH value and $NH_3$-N content (5.84 and $215\;g\;kg^{-1}$ DM). Addition of enzymes significantly increased (p<0.01) the lactate production (lactate, 45.6; acetate, $34.0\;g\;kg-^{1}$ DM) and decreased (p<0.01) the pH and $NH_3$-N (4.80 and $154\;g\;kg^{-1}$ DM) in the ensiled forages when compared with the control silages. Untreated guineagrass was successfully preserved with a high lactate proportion (lactate, 45.5; acetate, $24.1\;g\;kg^{-1}$ DM), and the addition of enzymes further enhanced the desirable fermentation (lactate, $57.5\;g\;kg^{-1}$ DM; acetate, $19.4\;g\;kg^{-1}$ DM). The content of NDF was lowered (p<0.05) by enzymes in both silages, but the extent appeared greater in the enzyme-treated rhodesgrass (rhodesgrass, $48\;g\;kg^{-1}$ DM; guineagrass, $21\;g\;kg^{-1}$ DM). Changes in the kinetics of in sacco degradation showed that enzyme treatment increased (p<0.01) the rapidly degradable DM (rhodesgrass, 299 vs. $362\;g\;kg^{-1}$ DM; guineagrass, 324 vs. $343\;g\;kg^{-1}$ DM) but did not influence the potential degradation, lag time and degradation rate of DM and NDF in the two silages.

• Applied Biological Chemistry
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• 제30권2호
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• pp.169-178
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• 1987

Kluyveromyces marxianus로 부터 inulase를 생산하고 정제하며 intra 및 extracellular inulase의 성질을 조사하였다. 본 균주는 stationary phase인 24시간째 intra 및 extracelullar enzyme의 생산이 최고에 달했으며 유기 질소원으로 YNB를 사용하고 배양 중 pH를 조절해 줌으로써 효소 생산을 향상시킬 수 있었다. 조효소는 DEAE-cellulose에 의해 intra 및 extracellular inulase 모두 2개의 fraction으로 분리되었고 각 fraction의 전기영동 양상은 비슷하여 주 band를 비롯 모두 3개의 glycoprotein band가 관찰되었으며 이중 주 band만 inulase 및 invertase activity를 보유하고 있었다. 정제 효소의 inulase 및 invertase의 최적 pH는 각각 5.0과 4.5였고 intra가 extracellular enzyme 에 비해 다소 넓은 범위의 pH에서 높은 활성을 나타내었다. 모든 fraction의 최적 온도는 inulase가 $40^{\circ}C$, invertase가 $50^{\circ}C$였으며 intracellular enzyme이 더 넓은 범위의 온도에서 안정하였고 열에 대한 안정성도 intracellular inulase가 extracellular inulase보다 높게 나타났다. Km value는 intra가 $16{\sim}19mM$, extracellular inulase가 $9{\sim}11mM$로써 extracellular inulase가 inulin에 대한 친화력이 더 높았으나 모두 exo-type의 inulase로 판명되었다.

• Journal of Microbiology and Biotechnology
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• 제31권2호
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• pp.327-337
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• 2021

Fibrinolytic enzymes with a direct mechanism of action and safer properties are currently requested for thrombolytic therapy. This paper reports on a new enzyme capable of degrading blood clots directly without impairing blood coagulation. This enzyme is also non-cytotoxic and constitutes an alternative to other thrombolytic enzymes known to cause undesired side effects. Twenty-four Bacillus isolates were screened for production of fibrinolytic enzymes using a fibrin agar plate. Based on produced activity, isolate S127e was selected and identified as B. subtilis using the 16S rDNA gene sequence. This strain is of biotechnological interest for producing high fibrinolytic yield and consequently has potential in the industrial field. The purified fibrinolytic enzyme has a molecular mass of 27.3 kDa, a predicted pI of 6.6, and a maximal affinity for Ala-Ala-Pro-Phe. This enzyme was almost completely inhibited by chymostatin with optimal activity at 48℃ and pH 7. Specific subtilisin features were found in the gene sequence, indicating that this enzyme belongs to the BPN group of the S8 subtilisin family and was assigned as AprE127. This subtilisin increased thromboplastin time by 3.7% (37.6 to 39 s) and prothrombin time by 3.2% (12.6 to 13 s), both within normal ranges. In a whole blood euglobulin assay, this enzyme did not impair coagulation but reduced lysis time significantly. Moreover, in an in vitro assay, AprE127 completely dissolved a thrombus of about 1 cc within 50 min and, in vivo, reduced a thrombus prompted in a rat tail by 11.4% in 24 h compared to non-treated animals.

• Journal of Microbiology and Biotechnology
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• 제24권2호
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• pp.177-187
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• 2014

Microbial phytases are enzymes with biotechnological interest for the feed industry. In this article, the effect of spray-drying conditions on the stability and activity of extracellular phytase produced by R. microsporus var. microsporus biofilm is described. The phytase was spray-dried in the presence of starch, corn meal (> $150{\mu}m$), soy bean meal (SB), corn meal (< $150{\mu}m$) (CM), and maltodextrin as drying adjuvants. The residual enzyme activity after drying ranged from 10.7% to 60.4%, with SB and CM standing out as stabilizing agents. Water concentration and residual enzyme activity were determined in obtained powders as a function of the drying condition. When exposed to different pH values, the SB and CM products were stable, with residual activity above 50% in the pH range from 4.5 to 8.5 for 60 min. The use of CM as drying adjuvant promoted the best retention of enzymatic activity compared with SB. Spray drying of the R. microsporus var. microsporus phytase using different drying adjuvants showed interesting results, being quite feasible with regards their biotechnological applications, especially for poultry diets.

• Asian-Australasian Journal of Animal Sciences
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• 제33권1호
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• pp.24-34
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• 2020

Objective: The effects of Pleurotus ostreatus on the feed utilization of broad bean stalks (BBS), rape straw (RS), paddy straw (PS), and corn stalk (CS) was examined. Methods: The four roughages were co-cultured with Pleurotus ostreatus. The chemical composition; enzyme activities of laccase, carboxymethylcellulase (CMCase) and xylanase; carbohydrate and protein fractions (based on The Cornell Net Carbohydrate and Protein System [CNCPS]) were assessed at different days after inoculation (7, 14, 21, 28 d) and un-inoculated roughages (control, 0 d). The digestibility of nutrient components and the gas production of roughage with various incubation times were monitored at 0, 2, 4, 6, 9, 12, 24, 36, 48, 60, and 72 h using an in vitro ruminal fermentation method. Results: A higher CMCase activity (0.1039 U/mL) and earlier time to peak (14 d) were detected in Pleurotus ostreatus cultured with CS (p<0.05). Significantly, the incubation length-dependent responses of cumulative gas production were observed from 24 to 72 hours post fermentation (p<0.05), and these incubation length-dependent effects on cumulative gas production of PS and CS appeared earlier (24 h) for PS and CS than those (48 h) for BBS and RS (p<0.05). The fast-degradable carbohydrate (CA) content for all four roughages significantly increased over time (p<0.05). Nonetheless, increased degradation efficiency for CA treated with Pleurotus ostreatus was detected at both 21 and 28 days of incubation (p<0.05). With the exception of PS (p<0.05), there were no significant difference among the roughages (p>0.05) in slowly-degradable carbohydrate (CB2) at different incubation times (p<0.05). Conclusion: Assessment of the alterations in chemical composition, CNCPS system fractions, and the fermentation kinetics after biological pretreatment may yield a valuable database for evaluating the biological pretreatment of Pleurotus ostreatus in ruminant feed.

• Asian-Australasian Journal of Animal Sciences
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• 제16권11호
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• pp.1610-1613
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• 2003

The aim of the experiment was to study the changes in the activities of various rumen fibre degrading enzymes due to the feeding of chemically treated mustard (Brassica campestris) straw in sheep. Mustard straw (MS) (<5 cm particle size) was treated either with urea (4% (w/w), or with 2% sodium hydroxide (NaOH), or with alkaline hydrogen peroxide (2% NaOH and 1.5% hydrogen peroxide ($H_2O_2$)) and/or supplemented with 2% (w/w) urea. Seven maintenance type rations were prepared using MS (70 parts) with molasses (5 parts) and concentrate (25 parts). They were untreated MS (CMS), urea treated MS (UMS), urea supplemented MS (MSUS), alkali treated MS (AMS), alkali treated and urea supplemented MS (AMS-US), alkali $H_2O_2$ treated MS (AHMS) and alkali $H_2O_2$ treated and urea supplemented MS (AHMS-US). They were then compressed into a complete feed block with the help of block making machine. Forty two male hoggets of Malpura breed sheep were equally distributed into each treatment group and (were) offered feed and water ad libitum. At the end of 21 days of feeding trial, rumen liquor was collected through stomach tube from three animals in each group at 0 h, 4 h, 8 h, 12 h of post feeding. Results showed that the level of enzyme varied from 8.52 to 11.12, 40.85 to 50.37, 3.22 to 3.78, 2.09 to 2.77 and 31.44 to 44.24 units/100 ml SRL respectively for carboxymethyl cellulase (CMCase), $\alpha$-amylase, microcrystalline cellulase (MCCase), filter paper (FP) degrading enzyme and $\alpha$-glucosidase. Processing of MS affected the enzyme activities, in a way, that NaOH and AHP treatment significantly reduced CMCase and FP degrading enzyme. The effect of urea treatment showed an increase in the activity of MCCase and $\alpha$-glucosidase. But the supplementation of urea increased the activity of CMCase, FP degrading enzyme and $\alpha$-glucosidase. The CMCase, $\alpha$-amylase, $\alpha$-glucosidase activities were highest at 4hr whereas MCCase and FP degrading enzyme had maximum activities at 12 h post feeding Results suggested that MS might need longer time in the rumen for its effective degradation.

• 한방안이비인후피부과학회지
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• 제24권1호
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• pp.86-95
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• 2011

Objectives : Ulmus davidiana (UD) has been widely used in Korean herbal medicines used for treatment of acute and chronic inflammatory diseases, such as rhinitis, asthma, and abscess. In this study, To investigated the protective effect of UD on type 1 allergic response, we determined whether UD inhibits early and late allergic response. Methods : The effect of UD was analyzed by ELISA and RT-PCR in RBL-2H3 cells. Levels of ${\beta}$ -hexosaminidase, interleukin (IL)-4 and TNF-${\alpha}$ were measured using enzyme-linked immunosorbent assays (ELISAs). mRNA levels of COX-2 and T-helper type 2(Th2) cytokines were analyzed with RT-PCR. Results : We found that UD suppressed ${\beta}$-hexosaminidase release in RBL-2H3 not only by the PMA plus A23187 stimulation, but also by the IgE-DNP-HSA stimulation at the antigen-antibody binding stage and antibody-receptor binding stage. UD also significantly inhibited COX2 level, along with reduced Th2 cytokine levels, such as IL-3, IL-4, IL-5, IL-13, GM-CSF, and TNF-${\alpha}$ in RBL-2H3. Conclusions : Our results indicate that UD protects against type 1 allergic response and exerts an anti-inflammatory effect through the inhibition of degranulation and expression of COX2 and Th2 cytokines.

• Asian-Australasian Journal of Animal Sciences
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• 제20권6호
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• pp.914-919
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• 2007

The effect of the addition of exogenous fibrolytic enzymes (mainly xylanase and cellulase activities, 15 ml/15 kg of fresh forage), on intake, total tract digestibility and nylon bag degradability of a chopped fresh Digitaria decumbens grass was studied at 2 stages of regrowth ( 21 and 56-day old grasses). Moreover, comparisons between ground and chopped grass were done using the nylon bag degradability method. DM intake (g/kg $BW^{0.75}$) and organic matter total tract digestibility for control and enzyme treatments respectively were 69.1 vs. 65.9 (p>0.05) and 0.723 vs. 0.727 (p>0.05) with the 21-day old regrowth. Based on the same parameters, values for the 56-day old grass were 58.1 vs. 52.7 (p>0.05) and 0.621 vs. 0.591 (p>0.05). Nylon bag degradation at 24 h of the dry matter for control versus enzyme treatments were 0.653 vs. 0.70 (p<0.05) and 0.644 vs. 0.733 (p<0.0001) for the 21-day old chopped and ground forage respectively, whereas with the 56-day old grass, corresponding values were 0.321 vs. 0.392 (p<0.0001) and 0.463 vs. 0.481 (p>0.05). The positive impact of exogenous fibrolytic enzymes (EFE) on degradability of the young and ground pangola grass may suggest that in some cases, enzyme accessibility to potentially digestible cell wall is a limiting factor in their digestion.

• 한국미생물·생명공학회지
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• 제40권3호
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• pp.243-249
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• 2012

부산 송정 연안에서 해조류 및 해수로부터 알긴산 분해 미생물을 분리동정하고 미생물의 생육 조건 및 조효소액의 알긴산 분해 특성을 확인하였다. Sargassum thunbergii로부터 분리한 알긴산 분해균을 동정한 결과, Vibrio crassostreae strain로 확인 되었으며, V. crassostreae PKA 1002로 명명 하였다. V. crassostreae PKA 1002 최적 생육 조건을 확인한 결과, pH 9, 2% NaCl, $30^{\circ}C$ 및 배양 24 hr인 것으로 나타났으며, 최적 생육 조건에서 7% 알긴산과 1:1 배양시 환원당이 가장 많이 생성되는 것으로 나타났다. V. crassostreae PKA 1002를 최적 생육 조건으로 대량배양 후, 원심분리하여 얻은 상층액을 조효소액으로 하였으며, V. crassostreae PKA 1002 유래 알긴산 분해 조효소액은 pH 9, $30^{\circ}C$에서 분해 활성이 최대이며, 4% 알긴산 용액에서 1 hr 반응시 3.035 g/L의 환원당을 생성하는 것으로 나타나 추후 효소를 정제하고 알긴산 분해 특성을 구명하여 알긴산 올리고당 제조에 이용할 수 있을 것으로 사료되어 진다.