• Journal of Applied Biological Chemistry
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• 제55권1호
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• pp.13-17
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• 2012

In order to enzymatically produce chitooligosaccharide using the crude enzyme preparation from Bacillus cereus D-11, we first studied the optimal reaction conditions. It was found that the optimal temperature for hydrolysis of chitosan was $55^{\circ}C$. The ratio of enzyme/substrate should not be lower than 0.13 U/mg in the reaction mixture. The enzyme activity was stable below $50^{\circ}C$. The products of enzymatic reaction were analyzed by both thin layer chromatography and high performance liquid chromatography. Under the appropriate condition, chitosan was hydrolyzed using the enzyme preparation. The resulting chitooligosaccharides were purified and separated by Dowex ($H^+$) ion exchange chromatography. From 4 g soluble chitosan, 0.95 g $(GlcN)_2$, 1.43 g $(GlcN)_3$, and 1.18 g $(GlcN)_4$ were recovered.

• 농업과학연구
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• 제45권4호
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• pp.789-797
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• 2018

This study investigated the activity of crude enzymes obtained from Pyrus pyrifolia cv. Shingo on milk proteins. In the milk processing industry, there is an increasing interest in the addition of functional materials to dairy products or functional peptides isolated from milk proteins. First, Pyrus pyrifolia cv. Shingo was separated into core, flesh, and peel regions, and crude enzymes were obtained from the individual regions. The activity of the obtained crude enzymes was measured using casein and gelatin agar. The crude enzyme obtained from the flesh of Pyrus pyrifolia cv. Shingo decomposed gelatin, but the activity of the crude enzymes obtained from the peel and core regions was insignificant. On the other hand, the crude enzymes obtained from the flesh and core regions of Pyrus pyrifolia cv. Shingo had a remarkable enzymatic activity in casein agar. However, the activity of the crude enzyme obtained from the peel region was insignificant. In addition, the crude enzymes obtained from the individual regions were mixed with casein to induce reactions, and the degradation patterns were investigated through electrophoresis and high performance liquid chromatography (HPLC). According to the results, the crude enzymes from Pyrus pyrifolia cv. Shingo degraded milk proteins. Thus, the results of this study can be used in studies on functionality. Additionally, it is expected that the use of pear peels and cores in the milk processing industry would greatly contribute to the reduction of food waste.

• 미생물학회지
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• 제14권2호
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• pp.65-74
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• 1976

Atternaria sp. was isolated from soil and crude cellulases were prepared from wheat bran culture of the fungus. The activities of the crude enzyme were studied on five different subvstrates and some phsical properties were also examined, crude enzymes were purified by column chromatography on DEAE Sephadex and Sephadex, Isozymes were separated some of which were active specifically on DEAE cellulose and some were primarily active on cellulose and CM-cellulose. The optimal points of pH and temperature for the crude enzyme were varied depending on the substrates ; On cellulose they were at pH 6.0 and 40.deg.C, on CM-cellulose at pH's 4.0 and 6.0 and 60.deg.C, and on DEAW-cellulose at pH 5.0 and 50.deg.C. Two active fractions, F-1 and F-II on Na-CMC was used as substrate the Km values of crude enzyme, F-I and F-II were calculated to be $4{\times}10^{-5}$ , 1.1 * 10$^{-4}$ , and $1.25{\times}10^{-4}mN$ resepctively. The Ki value of $Cu^{++}$ for crude enzyme was$4{\times}^{-4}mN$ , while that of $Nm^{++}$ while in the same concentration of $Mn^{++}$ it reached to 91%. Some 57% activity of F-1 was inhibited in s mN $Cu^{++}$, whereas it was inhibited as much as 81% in the same concentration above the concentration of 0.3 mM with tis activity reaching up to 137% in 2 mM. On the other hand the F-11 was inhibited by the presence of M $n^{++}$ and some 67% activity was inhibited at 2mM.

• 한국식품과학회지
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• 제28권1호
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• pp.34-39
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• 1996

메밀(날 메밀, 볶은 메밀, 찐 메밀)에서 추출, 분리한 crude hemicellulose, alcohol-insoluble hemicellulose, residue 및 고분자 가용성 다당류(분자량 10 KDa 이상)와 저분자 가용성 다당류(분자량 10 KDa 이하)가 in vitro에서 췌장의 소화효소 활성에 미치는 영향을 조사하였다. 다당류-효소액을 $37^{\circ}C$에서 5분간 반응시킨 후 다당류를 제거하고 여액의 소화효소 활성을 측정함으로써 다당류가 소화효소의 활성에 미치는 영향을 조사하였다. 메밀의 crude hemicellulose, alcohol-in-soluble hemicellulose, residue는 ${\alpha}-amylase$ 활성을 저하시켰으며, 고분자 수용성 다당류와 저분자 수용성 다당류는 ${\alpha}-amylase$ 활성을 저해하지 않았다. 또한, 저분자 수용성다당류를 제외한 모든 다당류는 lipase의 활성을 감소시켰다. Crude hemicellulose, alcohol-insoluble hemicellulose, residue와 고분자 수용성 다당류는 trypsin과 chymotrypsin 활성을 현저히 저하시켰으나 저분자 수용성 다당류는 이들의 활성을 약간 감소시키는 것으로 나타났다.

• Journal of Microbiology and Biotechnology
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• 제20권5호
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• pp.893-903
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• 2010

A cellulolytic and xylanolytic enzyme complex-producing alkalothermoanaerobacterium strain, Tepidimicrobium xylanilyticum BT14, is described. The cell was Grampositive, rod-shaped, and endospore-forming. Based on 16S rRNA gene analysis and various lines of biochemical and physiological properties, the strain BT14 is a new member of the genus Tepidimicrobium. The strain BT14 cells had the ability to bind to Avicel, xylan, and corn hull. The pH and temperature optima for growth were 9.0 and $60^{\circ}C$, respectively. The strain BT14 was able to use a variety of carbon sources. When the bacterium was grown on corn hulls under an anaerobic condition, a cellulolytic and xylanolytic enzyme complex was produced. Crude enzyme containing cellulase and xylanase of the strain BT14 was active in broad ranges of pH and temperature. The optimum conditions for cellulase and xylanase activities were pH 8.0 and 9.0 at $60^{\circ}C$, respectively. The crude enzyme had the ability to bind to Avicel and xylan. The analysis of native-PAGE and native-zymograms indicated the cellulosebinding protein showing both cellulase and xylanase activities, whereas SDS-PAGE zymograms showed 4 bands of cellulases and 5 bands of xylanases. Evidence of a cohesinlike amino acid sequence seemed to indicate that the protein complex shared a direct relationship with the cellulosome of Clostridium thermocellum. The crude enzyme from the strain BT14 showed effective degradation of plant biomass. When grown on corn hulls at pH 9.0 and $60^{\circ}C$ under anaerobic conditions, the strain BT14 produced ethanol and acetate as the main fermentation products.

• 한국미생물·생명공학회지
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• 제25권4호
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• pp.346-353
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• 1997

During the screening of cellulase producing microorganisms, a fungal strain C-4 was selected from etiolated leaves. Based on taxonomic studies, the fungus could be classified as a strain of Trichoderma sp. When the strain C-4 was cultured in Mandels' media at 28$circ$C for 6 days, the enzyme activities detected in broth were as follows: 8.2 U/ml (28.1 U/mg) of CMCase activity, 0.75 U/ml (2.58 U/mg) of Avicelase activity, 1.67 U/ ml (5.68 U/mg) of $eta$-glucosidase activity. The optimum pH for enzyme induction was 6.2. The crude enzyme retained 100% of its original CMCase activity at 50$circ$C for 1 hr (pH 5.0), and at 4$circ$C for 24 hrs (pH 5.0). There was no effect on the CMCase activity in the presence of 1 mM of CsCl, LiCl, MgCl$_{2}$, and FeCl$_{2}$, respectively. When the crude enzyme was treated with trypsin and chymotrypsin (2% W/w) for 10 minutes, the remaining CMCase activity was 70%, but there was no further loss of activity for 60 minutes treatment at 30$circ$C. The crude enzyme showed the synergism with rumen fluid for the hydrolysis of Avicel and CMC by 118% and 130%, respectively.

• 한국식품조리과학회지
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• 제13권5호
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• pp.617-622
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• 1997

닥나무 열매가 쇠고기의 연화와 맛에 미치는 영향을 알아보기 위하여 닥나무 열매의 처리형태(농축액, 조효소)와 첨가량을 달리하여 쇠고기에 첨가하였을 때의 효과를 실험한 결과는 다음과 같다. 1. 전단력은 닥나무 열매의 농축액와 조효소의 첨가량이 증가함에 따라 감소하여 쇠고기의 연화도가 증가하였는데 처리형태에 따라 살펴보면, 0∼8%의 농축액을 첨가하였을 때 4.8∼27%, 0∼1%의 조효소를 첨가하였을 때 7.8∼34.2%까지의 연화율을 나타내었다. 2. 중량감소율은 농축액의 형태로 첨가시는 닥나무열매를 첨가하는 양이 증가할수록 다소 감소하였으나 조효소형태로 첨가하였을 때에는 큰 차이를 나타내지 않았다. 3. 색도는 닥나무 열매를 첨가하여 조리함에 따라 장조림육 표면의 “a”값이 대체로 증가하는 경향으로 나타났다. 4. 유리아미노산을 보면 닥나무 열매의 농축액과 조효소 첨가시 단백질이 연화되어 국물속으로 용출되어 나오는 유리아미노산함량이 점차로 증가하는 것을 알 수 있었는데 농축액은 8%까지 첨가시 37%, 조효소는 1%까지 첨가시 31.2%의 증가율을 보였다. 5. 장조림육의 무기질 함량과 조성을 분석한 결과 농축액의 형태로 첨가하였을 때에는 첨가량이 증가할 수록 대부분의 무기질이 감소하였다. 반면에 조효소 형태로 첨가하였을 때는 Na, K, P, Ca, Mg의 순으로 높은 함량분포를 보였으며 Mn과 Cu가 비교적 낮았다. 첨가량이 증가함에 따라 대부분의 무기질들이 증가하는 경향으로 나타났다. 6. 장조림육의 관능검사 결과에서는 닥나무 열매 농축액과 조효소를 첨가함에 따라 전반적으로 높은 기호도를 나타내었다. 농축액 첨가시에는 4% 처리구, 그리고 조효소 첨가시에는 0.1% 처리구가 종합적인 기호도에서 가장 높은 점수를 얻었다. 이상의 결과로 닥나무 열매를 농축액이나 조효소의 형태로 첨가하는 양을 달리하여 장조림을 하였을 때 연화도와 기호도가 증가하였는데 처리형태에 따라 연화율이 조금씩 다르게 나타나는 것을 알 수 있었다.

• Journal of the Korean Wood Science and Technology
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• 제19권2호
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• pp.22-29
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• 1991

The endo-1,4-${\beta}$-xylanase was extracted and purified from the extracellular xylanolytic systems of Trichoderma viride. The crude enzyme was chromatographed with ion-exchange reins of DEAE Sepharose CL-6B, Sepharose, S-Sepharose CL-6B and the resulting xylanase was turned out to be a single protein as 20KD hy SDS-polyacrylamide gel electrophoresis. The xylooligomers were obtained from xylan by incubation with the purified xylanase up to 50%. The ${\beta}$-xylosidase lost its activity completely by incubation of crude enzyme for 24hr with buffer solution of pH 2.8 at $27^{\circ}C$. And also, the xylooligomers were obtained from xylan as a main product by incubation with the crude enzyme treated with acidic buffer.

• Journal of Ginseng Research
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• 제39권3호
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• pp.221-229
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• 2015

Background: Minor ginsenosides, those having low content in ginseng, have higher pharmacological activities. To obtain minor ginsenosides, the biotransformation of American ginseng protopanaxadiol (PPD)-ginsenoside was studied using special ginsenosidase type-I from Aspergillus niger g.848. Methods: DEAE (diethylaminoethyl)-cellulose and polyacrylamide gel electrophoresis were used in enzyme purification, thin-layer chromatography and high performance liquid chromatography (HPLC) were used in enzyme hydrolysis and kinetics; crude enzyme was used in minor ginsenoside preparation from PPD-ginsenoside; the products were separated with silica-gel-column, and recognized by HPLC and NMR (Nuclear Magnetic Resonance). Results: The enzyme molecular weight was 75 kDa; the enzyme firstly hydrolyzed the C-20 position 20-O-${\beta}$-D-Glc of ginsenoside Rb1, then the C-3 position 3-O-${\beta}$-D-Glc with the pathway $Rb1{\rightarrow}Rd{\rightarrow}F2{\rightarrow}C-K$. However, the enzyme firstly hydrolyzed C-3 position 3-O-${\beta}$-D-Glc of ginsenoside Rb2 and Rc, finally hydrolyzed 20-O-L-Ara with the pathway $Rb2{\rightarrow}C-O{\rightarrow}C-Y{\rightarrow}C-K$, and $Rc{\rightarrow}C-Mc1{\rightarrow}C-Mc{\rightarrow}C-K$. According to enzyme kinetics, $K_m$ and $V_{max}$ of Michaelis-Menten equation, the enzyme reaction velocities on ginsenosides were Rb1 > Rb2 > Rc > Rd. However, the pure enzyme yield was only 3.1%, so crude enzyme was used for minor ginsenoside preparation. When the crude enzyme was reacted in 3% American ginseng PPD-ginsenoside (containing Rb1, Rb2, Rc, and Rd) at $45^{\circ}C$ and pH 5.0 for 18 h, the main products were minor ginsenosides C-Mc, C-Y, F2, and C-K; average molar yields were 43.7% for C-Mc from Rc, 42.4% for C-Y from Rb2, and 69.5% for F2 and C-K from Rb1 and Rd. Conclusion: Four monomer minor ginsenosides were successfully produced (at low-cost) from the PPD-ginsenosides using crude enzyme.

• BMB Reports
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• 제28권5호
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• pp.443-450
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• 1995

Cloned staphylococcal peptidoglycan hydrolase was used in determining the physiological characteristics of peptidoglycan hydrolase. This enzyme hydrolyzed the bacterial cell walls and released the N-terminal alanine, but not the reducing groups. This cloned gene product was localized in the cytoplasm of transformed Escherichia coli. Activity gels indicated the enzyme had an Mr of about 54,000, which was consistent with the deduced Mr from sequencing of the cloned gene. The activity bound to CM-cellulose but not DEAE-cellulose resin, indicating it as a basic protein. Enhanced enzyme activity in a low concentration of cations, and inhibited enzyme activity in a solution with dissolved phospholipids, suggested that the activity and the availability of this basic protein may be regulated between negatively charged and positively charged cellular molecules. The activity against boiled crude cell wall was much greater than against purifed cell wall, suggesting protein associated with crude cell wall may aid in the binding of the peptidoglycan hydrolase The cloned peptidoglycan hydrolase showed positive activity on whole cells of some lysostaphin-resistant coagulase-negative staphylococci. The cloned enzyme may be an alternative for lysostaphin for lysis of staphylococci.