• 한국산림과학회지
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• 제39권1호
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• pp.57-63
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• 1978

Trichoderma viride SANK호(號) cellulase에 의(依)한 목재(木材) 가수분해(加水分解)에 관(關)한 연구(硏究)로서 재료(材料)는 산오리나무재(材)를 사용(使用)하였다. 그리고 당생성(糖生成)을 위한 cellulase의 최적(最適) 반응조건(反應條件)을 조사(調査)하였다. Trichoderma viride 조효소액(粗酵素液)은 진탕배양법(振盪培養法)에 의(依)하여 생산(生產)하였고, 이것을 염석효소액(鹽析酵素液)을 조제(調製)하여 사용(使用)하였다. 기질(基質)은 산오리나무재(材) 톱밥을 과초산법(過醋酸法)에 의(依)하여 탈(脫)리그닌 한것을 사용(使用)하였다. 환원당(還元糖) 정량(定量)은 DNS법(法)에 의(依)하였다. 결과(結果)는 다음과 같다. 1. 최적(最適) pH는 5.0이며, pH 안정성(安定性)의 범위(範圍)는 4.0~6.0으로 나타났다. 2. 반응온도(反應溫度)는 $40^{\circ}C$가 최적(最適)이였으며, 온도(溫度)의 안정성(安定性)은 $40^{\circ}C{\sim}50^{\circ}C$ 범위(範圍)로 나타났다. 3. 효소농도(酵素濃度)는 높을수록 환원당(還元糖) 생성율(生成率)은 증가(增加)되었다. 4. 기질농도(基質濃度)는 높을수록 단위(單位) 기질(基質)에 대(對)한 환원당(還元糖) 생성율(生成率)은 감소(減少)되었다. 5. 당류중(糖類中) pructose가 가장 강(强)한 조해제(阻害劑)로 나타났으며, glucose의 조해작용(阻害作用)은 약(弱)하게 나타났다.

• 한국균학회지
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• 제25권4호통권83호
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• pp.330-339
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• 1997

식물 병원진균 Botrytis cinerea가 분비하는 polygalacturonase의 동위효소 중에서 endo-type의 polygalacturonase를 Phenyl-Toyopearl column chromatography와 DEAE-, Mono Q-, heparin-high performance liquid chromatography법으로 약 68배 정제하였고 sodium dodecylsulfate(SDS)를 포함하는 polyacrylamide gel electrophoresis로 분석하여 단일 띠를 확인하였다. 정제한 효소의 분자량은 SDS-polyacrylamide gel에서 37 kDa로 측정되었으며 약산성인 조건과 $30^{\circ}C$ 이하에서 안정하였으며 최고 활성은 pH 4.5와 $55^{\circ}C$의 온도에서 관찰되었다. 정제한 효소는 polygalacturonic acid를 endo-type으로 가수분해하였고, polygalacturonic acid에 대한 $K_m$값과 $V_{max}$값은 각각 1.1 mg/ml과 250 nmol/min이었다. N-말단의 아미노산 서열을 결정하여 본 결과, 식물이나 다른 진균류에서 보고된 아미노산 서열과 유사성이 없었다.

• 한국산림과학회지
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• 제59권1호
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• pp.67-91
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• 1983

침엽수(針葉樹)인 소나무, 리기다소나무, 일본잎갈나무 3수종(樹種)과 활엽수(闊葉樹)인 오리나무, 밤나무, 이태리포푸라 3수종(樹種)의 목재(木材)로부터 alcohol을 생산(生産)하기 위하여 목재(木材)의 화학성분(化學成分) 분석(分析)과 산(酸) 가수분해(加水分解) 조건(條件)을 확립(確立)하고 또한 섬유소(纖維素) 분해력(分解力)이 강(强)한 균주(菌株)를 분리(分離) 동정(同定)하여 균주(菌株)의 효소(酵素) 생산조건(生産條件), 효소(酵素)의 특성(特性) 및 alcohol 발효조건(醱酵條件) 등(等)을 연구조사(硏究調査)한 바 다음과 같은 결과(結果)를 얻었다. 1) 수종별(樹種別) 산(酸) 가수분해(加水分解)에서 HCl은 1.0%와 2.0%, $H_2SO_4$는 2.0%에서 높은 당화율(糖化率)을 보였고 수종(樹種)은 오리나무가 23.4%로 제일 좋았다. 2) HCl과 $H_2SO_4$으로 산(酸) 가수분해(加水分解)할 때 압력(壓力)은 $1.5kg/cm^2$, 산(酸)의 첨가량(添加量)은 30배량(倍量), 분해시간(分解時間)은 HCl 45분과 $H_2SO_4$ 30분에서 당화율(糖化率)이 양호(良好)하였다. 3) 전처리(前處理)에 있어서 농(濃) HCl 보다는 농(濃) $H_2SO_4$이 더 효과적(効果的)이었고 그 농도(濃度)는 50~60%이었으며 열처리(熱處理)는 $190^{\circ}C$에서 30분간(分間) 처리(處理)함으로써 무처리(無處理)보다 당화율(糖化率)이 약(約) 1.5% 증가(增加)하였다. 4) 목분(木粉) 1g에 있어서 산(酸) 가수분해액(加水分解液)의 당조성(糖組成)은 소나무는 glucose 137.78mg, arabinose 68.24mg이었고 오리나무는 glucose 162.22mg, arabinose 65.89mg을 함유(含有)하고 있었으며, xylose와 galactose도 검출(檢出)되었다. 5) 10개(個)의 균주(菌株) 중에 alcohol 효효력(醗酵力)이 왕성한 균주(菌株)는 Sacch. cerevisiae JAFM 101과 Sacch. cerevisiae var. ellipsoideus JAFM 125이었다. 6) 산(酸) 가수분해액(加水分解液)은 $CaCO_3$와 NaOH로 중화(中和)하는 것이 alcohol 생산(生産)과 효모생육(酵母生育)이 양호(良好)하였고 alcohol 생산(生産)은 pH 4.5~5.5, $30^{\circ}C$, 균주생육(菌株生育)에는 pH 5.0~6.0, $24^{\circ}C$에서 발효(醱酵)시키는 것이 좋았다. 7) Alcohol 생산(生産)에는 0.02%의 $(NH_2)_2CO$와 $(NH_4)_2SO_4$, 0.1%의 $KH_2PO_4$, 0.05%의 $MgSO_4$ 그 밖에 0.025%의 $CaCl_2$, 0.002%의 $MnCl_2$ 첨가(添加)가 효과적(効果的)이었고 효모증식(酵母增殖)에는 0.04~0.06%의 $(NH_2)_2CO$와 $(NH_4)_2SO_4$, 0.2%의 $K_2HPO_4$와 $K_3PO_4$, 0.05%의 $MgSO_4$ 그 밖에 0.025%의 $CaCl_2$, 0.002%의 NaCl 첨가(添加)가 효과적(効果的)이다. 8) 여러 가지 vitamin 중에서 alcohol 생산(生産)은 pyridoxine과 riboflavin, 효모증식(酵母增殖)에는 thiamin과 Ca-pantothenate, biotin과 inositol의 첨가(添加)가 좋았고 tannin과 furfural은 0.01% 농도(濃度)에서 alcohol 생산(生産)이 오히려 증가(增加)하였으며 그 이상(以上)의 농도(濃度)에서는 저해(沮害)를 받았다. 9) 발효액(醱酵液) 100ml에서 소나무는 2.201~2.275ml의 alcohol과 168~228mg의 효모(酵母)가 생산(生産)되었고 잔류당(殘溜糖)은 0.55~0.60g이었다. 오리나무는 2.075~2.125ml의 alcohol과 208~256mg의 효모(酵母)를 생산(生産)했으며 잔류당(殘溜糖)은 0.60~0.65g이었고 pH는 3.3~3.6으로 변화(變化)했다. 10) Trichoderma viride JJK107을 섬유소(纖維素) 분해력(分解力)이 강한 균주(菌株)로서 선정(選定)하여 동정(同定)하였다. 11) 효소(酵素) 생산조건(生産條件)으로 CMCase는 pH 6.0, $30^{\circ}C$, xylanase는 pH 5.0, $35^{\circ}C$에서 5일간 배양(培養)할 때 최고(最高)의 효소생산(酵素生産)을 나타냈고 효소작용(酵素作用)의 최적조건(最適條件)은 CMCase는 pH 4.5, $50^{\circ}C$, xylanase는 pH 4.5, $40^{\circ}C$에서 최고(最高)의 활성도(活性度)를 보였다. 12) 목분(木粉)을 peracetic acid로 탈(脫) lignin하고 효소(酵素) 가수분해(加水分解)하여 발효(醱酵)시킬 때가 alcohol 생산(生産)이 좋았으며 peracetic acid 첨가비(添加比)는 10배량(倍量) 이도(移度) 첨가(添加)하는 것이 좋았다. 13) 발효(醱酵)에 있어서 본분(本粉)과 밀기울 Koji의 혼합비율(混合比率)은 10:8일 때에 alcohol 생산(生産)이 좋았고 본분(本粉) 10g에서 소나무 2.01~2.14ml, 오리나무는 2.11~2.20ml의 alcohol을 생산(生産)하였다.

• Applied Biological Chemistry
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• 제43권2호
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• pp.86-94
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• 2000

대두 단백질의 효소적 가수분해에 의한 분해물의 맛과 향을 개선시킬 수 있는 효소적 가수분해 system을 확립하기 위하여, 전통메주로부터 대두단백질 분해능이 우수한 균주를 분리하여, 분해 양상이 상이한 효소를 생산하는 곰팡이와 세균 3균주를 선별하여 동정을 실시하였다. 단백질 분해능이 우수한 균주 중 곰팡이 M4는 leucine 활성이, 곰팡이 M5는 azodye 활성이 높았으며, 세균은 leucine활성이 가장 낮은 B16을 선별하여 효소의 작용에 의한 대두 단백의 분해 양상이 상이한 균주로 분리하였다. 분리한 곰팡이 M4는 배양배지에서 균사는 백색이었으며 균핵은 흰색에서 검은 색으로 변화하였다. Slide배양 결과, 분생자두는 방사형이었고, 정낭은 곤봉형이었다. 분생자는 반구형으로 기경자와 단경자가 존재하였다. 분생자병은 무색으로 매끈한 표면이었다. 분류학상 Aspergillus oryzae(ahlburg)종의 특징과 유사하여 이 종으로 분류 동정하고 Aspergillus oryzae M4로 명명하였다. 곰팡이 M5는 MEA배지에서 흰색과 검은색의 균사가 혼재되어 있었다. Slide배양 결과, 회녹색의 균총과 긴(7 mm) 포자낭병이 존재하였다. 포자낭은 회갈색이었고, 큰 포자낭과 기저막에서 작은 포자낭들이 떨어져 나오는 형태를 보였다. 중축은 구형으로 절단된 유리질의 형태이었고, 포자낭 포자는 직경이 작은$(80\;{\mu}m)$ 타원형이었다. 따라서 분류학상 접합균의 Mucor circinelloides의 종과 유사하여 이 종으로 분류 동정하고 Mucor circinelloides M5라 명명하였다. 세균 B16은 백색의 불투명한 colony로서 circular, lobate한 형태였고, 간상형으로 내생포자를 함유하였다. 또한, gram염색, catalase, oxidase, ${\beta}-glucosidase$, V-P test 등에서 양성이었고, D-fructose, ${\alpha}-D-glucose$, maltose, D-mannose, D-raffinose, stachyose, sucrose등의 이용성이 있었다. B16의 형태학적, 생리학적인 특징의 결과, Bacillus megaterium의 특징과 일치하였다. 그러나, 균체 지방산은 Paenibacillus marcerans의 균종과 유사하여 B16은 더 자세한 동정방법과 분류체계를 통해 명확한 동정이 필요하리라 사료되었다. 따라서 B16은 잠정적으로 Bacillus megaterium B16이라 명명하였다.

• Journal of Microbiology and Biotechnology
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• 제28권12호
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• pp.2036-2045
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• 2018

An endo-${\beta}$-1,4-glucanase gene, cel5L, was cloned using the shot-gun method from Bacillus sp.. The gene, which contained a predicted signal peptide, encoded a protein of 496 amino acid residues, and the molecular mass of the mature Cel5L was estimated to be 51.8 kDa. Cel5L contained a catalytic domain of glycoside hydrolase (GH) family 5 and a carbohydrate-binding module family 3 (CBM_3). Chromatography using HiTrap Q and CHT-II resulted in the isolation of two truncated forms corresponding to 50 (Cel5L-p50) and 35 kDa (Cel5L-p35, CBM_3-deleted form). Both enzymes were optimally active at pH 4.5 and $55^{\circ}C$, but had different half-lives of 4.0 and 22.8 min, respectively, at $70^{\circ}C$. The relative activities of Cel5L-p50 and Cel5L-p35 for barley ${\beta}$-glucan were 377.0 and 246.7%, respectively, compared to those for carboxymethyl-cellulose. The affinity and hydrolysis rate of pNPC by Cel5L-p35 were 1.7 and 3.3 times higher, respectively, than those by Cel5L-p50. Additions of each to a commercial enzyme set increased saccharification of pretreated rice straw powder by 17.5 and 21.0%, respectively. These results suggest CBM_3 is significantly contributing to thermostability, and to affinity and substrate specificity for small substrates, and that these two enzymes could be used as additives to enhance enzymatic saccharification.

• 한국수산과학회지
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• 제37권5호
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• pp.359-365
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• 2004

To study the lipid adsorption characteristic of chitosans with different molecular weights and the degrees of deacetylation, in vitro test and near-infrared (NIR) spectroscopic analysis have been performed for the measurement of lipid adsorption characteristics of chitosan. The degrees of deacetylation in chitosans were $70{\%},\;85{\%}\;and\;92{\%}$ at different deacetylation times (1 hr, 2 hrs, 3 hrs), respectively. The molecular weight of each chitosan was controlled by enzymatic hydrolysis, and then the molecular weight of the chitosan was 4 kDa. The bulk density, water holding capacity and fat binding capacity of each chitosan powder were $96.2-504.0{\%},\;374.4-1217.9{\%},\;and\;307.0-659.3{\%}$, respectively. The higher molecular weight of chitosan was exhibited the lower bulk density and the higher water and fat binding capacities. Bindinf capacities of chitosan powders to bile salts, cholesterol and linoleic acid were $41.2-63.3{\%},\;40.8-67.4{\%},\;42.6-72.6{\%}$, respectively. In NIR spectrum of lipid adsorbed chitosan the occurrence static eletronical binding between chitosan and lipid was identified by NIR spectrum peak induced from combination of carboxylic group in lipid and amino group in chitosan. In conclusion, the higher degree of deacetylation and molecular weight of chitosan showed the higher lipid binding capacity and the lipid adsorption of chitosan were occurred by combination of carboxylic group in lipids and amino group in chitosan.

• Journal of Microbiology and Biotechnology
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• 제17권11호
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• pp.1789-1796
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• 2007

A ${\beta}$-glucosidase with the molecular mass of 160,000 Da was purified to homogeneity from cell extract of a cellulolytic bacterium, Cellulomonas uda CS1-1. The kinetic parameters ($K_m$ and $V_{max}$) of the enzyme were determined with pNP-cellooligosccharides (DP 1-5) and cellobiose. The molecular orbital theoretical studies on the cellulolytic reactivity between the pNP-cellooligosaccharides as substrate (S) molecules and the purified ${\beta}$-glucosidase (E) were conducted by applying the frontier molecular orbital (FMO) interaction theory. The results of the FMO interaction between E and S molecules verified that the first stage of the reaction was induced by exocyclic cleavage, which occurred in an electrophilic reaction based on a strong charge-controlled reaction between the highest occupied molecular orbital (HOMO) energy of the S molecule and the lowest occupied molecular orbital (LUMO) energy of the hydronium ion ($H_3O^+$), more than endocyclic cleavage, whereas a nucleophilic substitution reaction was induced by an orbital-controlled reaction between the LUMO energy of the oxonium ion ($SH^+$) protonated to the S molecule and the HOMO energy of the $H_2O_2$ molecule. A hypothetic reaction route was proposed with the experimental results in which the enzymatic acid-catalyst hydrolysis reaction of E and S molecules would be progressed via $SN_1$ and $SN_2$ reactions. In addition, the quantitative structure-activity relationships (QSARs) between these kinetic parameters showed that $K_m$ has a significant correlation with hydrophobicity (logP), and specific activity has with dipole moment, respectively.

• Journal of Microbiology and Biotechnology
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• 제28권2호
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• pp.284-292
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• 2018

A novel ${\beta}$-agarase, AgaJ5, was identified from an agar-degrading marine bacterium, Gayadomonas joobiniege G7. It belongs to the glycoside hydrolase family 86 and is composed of 805 amino acids with a 30-amino-acid signal peptide. Zymogram analysis showed that purified AgaJ5 has agarase activity. The optimum temperature and pH for AgaJ5 activity were determined to be $30^{\circ}C$ and 4.5, respectively. AgaJ5 was an acidic ${\beta}$-agarase that had strong activity at a narrow pH range of 4.5-5.5, and was a cold-adapted enzyme, retaining 40% of enzymatic activity at $10^{\circ}C$. AgaJ5 required monovalent ions such as $Na^+$ and $K^+$ for its maximum activity, but its activity was severely inhibited by several metal ions. The $K_m$ and $V_{max}$ of AgaJ5 for agarose were 8.9 mg/ml and 188.6 U/mg, respectively. Notably, thin-layer chromatography, mass spectrometry, and agarose-liquefication analyses revealed that AgaJ5 was an endo-type ${\beta}$-agarase producing neoagarohexaose as the final main product of agarose hydrolysis. Therefore, these results suggest that AgaJ5 from G. joobiniege G7 is a novel endo-type neoagarohexaose-producing ${\beta}$-agarase having specific biochemical features that may be useful for industrial applications.

• BMB Reports
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• 제30권1호
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• pp.46-54
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• 1997

Alkaliphilic Bacillus sp. S-1 secretes a large amount (approximately 80% of total pullulanase activity) of an extracellular pullulanase (PUL-E). The pullulanase exists in two forms: a precursor form (PUL-I: $M_r$ 180,000), and a processed form (PUL-E: $M_r$ 140,000). Two forms were purified to homogeneity and their properties were compared. PUL-I was different in molecular weight, isoelectric point, $NH_2$-terminal amino acid sequence, and stabilities over pH and temperature ranges. The catalytic activities of PUL-I were also distinguishable in the $K_m$ and $V_{max}$ values for various substrates, and in the specific activity for pullulan hydrolysis. PUL-E showed 10-fold higher specific activities than PUL-I. However. PUL-I is immunologically identical to PUL-E, suggesting that PUL-I is initially synthesized and proteolytically processed to the mature form of PUL-E. Processing was inhibited by PMSF, but not by pepstatin, suggesting that some intracellular serine proteases could be responsible for processing of the PUL-I. PUL-I has a different conformational structure for antibody recognition from that of PUL-E. It is also postulated that the translocation of alkaline pullulanase(AP) in the bacterium possibly requires processing of the $NH_2$-terminal region of the AP protein. Processing of the precursor involves a conformational shift. resulting in a mature form. Therefore. precursor processing not only cleaves the signal peptide, but also induces conformational shift. allowing development of active form of the enzyme.

• 한국축산식품학회지
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• 제34권6호
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• pp.844-851
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• 2014

This study focused on the anti-oxidative and collagenase- and elastase inhibition effects of low molecular weight peptides (LMP) from commercial Jeju horse leg bone hydrolysates (JHLB) on pancreatin, via enzymatic hydrolysis. Cell viability of dermal fibroblasts exposed to UVB radiation upon treatment with LMP from JHLB was evaluated. Determination of the antioxidant activity of various concentrations of LMP from JHLB were carried out by assessing 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2-azino-bis-3-ethybenzothiazoline-6-sulphonic acid (ABTS) radical scavenging activity, ferric reducing antioxidant power (FRAP), and oxygen radical absorbance capacity (ORAC). The DPPH radical scavenging activity of LMP from JHLB (20 mg/mL) was 92.21% and ABTS radical scavenging activity (15 mg/mL) was 99.50%. FRAP activity (30 mg/mL) was $364.72{\mu}M/TE$ and ORAC activity (1 mg/mL) was $101.85{\mu}M/TE$. The anti-wrinkle potential was assessed by evaluating the elastase- and collagenase inhibition potential of these LMP. We found that 200 mg/mL of LMP from JHLB inhibited elastase activity by 41.32%, and 100 mg/mL of LMP from JHLB inhibited collagenase activity by 91.32%. The cell viability of untreated HS68 human dermal fibroblasts was 45% when exposed to a UVB radiation dose of $100mJ/cm^2$. After 24 h of incubation with $500{\mu}g/mL$ LMP from JHLB, the cell viability increased to 60%. These results indicate that LMP from JHLB has potential utility as an anti-oxidant and anti-wrinkle agent in the food and cosmetic industry. Additional in vivo tests should be carried out to further characterize these potential benefits.