• Journal of Microbiology and Biotechnology
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• 제20권3호
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• pp.467-473
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• 2010

To improve the expression efficiency of recombinant endo-$\beta$-1,4-glucanase in P. pastoris, the endo-$\beta$-1,4-glucanase (egI) gene from Aspergillus niger was synthesized using optimized codons. Fourteen pairs of oligonucleotides with 15 bp overlap were designed and the full-length syn-egI gene was generated by two-step PCR-based DNA synthesis. In the synthesized endo-$\beta$-1,4-glucanase gene syn-egI, 193 nucleotides were changed, and the G+C content was decreased from 54% to 44.2%. The syn-egI gene was inserted into pPIC9K and transformed into P. pastoris GS115 by electroporation. The enzyme activity of recombinant P. pastoris stain 2-7# reached 20.3 U/ml with 1% barley $\beta$-glucan and 3.3 U/ml with 1% carboxymethylcellulose (CMC) as substrates in shake flasks versus 1,270.3 U/ml and 220.7 U/ml for the same substrates in 50-1 fermentors. The molecular mass of the recombinant protein was approximately 40 kDa as determined by SDS-PAGE analysis, the optimal temperature for recombinant enzyme activity was $70^{\circ}C$, and the optimal pH was 5.0 when CMC was used as the substrate.

• 미생물학회지
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• 제24권3호
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• pp.295-301
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• 1986

The bacteria, which were capable of producing ${\beta}-1$, 3-glucanase inducibly by utilizing cell wall of Aspergillus fumigatus as a sole carbon source, were isolated from soil in the campus of Kyungpook National University. Among them, the strain which produced the enzyme excellently was selected and identified to be Pseudomonas stutzeri KF 13 by morphological, cultural and physiological examination. The optimal conditions for the enzyme production from Pseudomonas stutzeri KF 13 were investigated. the enzyme production was reached maximum state shen the broth cultured for 72hr at $30^{\circ}C$. And the enzyme showed the highest activity in the medium containing 3.5% cell wall as an inducer, 15% yeast autolysate as a nitrogen source and 0.05% $MnSO_4$ at pH 7.5.

• 미생물학회지
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• 제37권4호
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• pp.253-258
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• 2001

Bacillus circulans KCT3004 기원의 $\beta$-1,3-glucanase 유전자를 함유한 재조합 플라스미드 pLM460과 pUB110을 이용하여 shuttle 플라스미드 pMLS1180을 제작하고 Bacillus 세포에 이동.발현시켰다. pLMS1180으로 형질전환된 B. subtilis와 B. megaterium은 효율적으로 $\beta$-1,3-glucanase를 생산하였고, 이 효소들은 세포의 증식과 비례하여 생산되었다. 형질전환체가 생산하는 $\beta$-1,3-glucanase의 최대 활성을 유전자 공여 균주인 B. circulans와 비교하여 보니, B. subtilis는 14배, B. megaterium은 5배 정도의 높은 활성을 나타내었다. 그리고 대장균 형질전환체는 분비율이 7% 정도인데 반하여 B. subtilis 형질전환체는 생산된 효소를 전부, B. megaterium 형질전환체는 약 97%를 세포 외로 분비하는 것을 알 수 있었다. SDS-PAGE를 통해 대장균과 B. subtilis, B. megaterium에서 발현된 효소의 분자량을 분석해 보니 약 38,000으로 추정되었다. 또한, 이들 형질전환체가 생산하는 $\beta$-1,3-glucanase는 laminarin에 작용하여 주된 산물로서 laminaribiose (G2), laminaritriose (G3) 이상의 다양한 laminarioligosaccharide들을 생산함이 확인되었다. pLMS1180의 각 숙주 내에서이 안정성을 살펴본 결과 B.megaterium에서는 88%, 대장균에서는 75%, B. subtilis에서는 48%로 나타났다.

• 한국가금학회지
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• 제21권3호
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• pp.195-205
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• 1994

Effects of the addition of \beta-glucanase to barley-based layer diets were examined by feeding 200 Leghorn layers with corn-based (Control) and \beta-glucanase supplemented diets (Barley+ Enzyme). The results obtained are sumrrarized as follows. 1. There were no siginificant (P>0.05) differences in hen-day egg production(%) and average egg weight between two treatments, indicating that the \beta-glucanase supplemented barley could successfully replace the commonly used corn in the layer diets. 2. Although there was no statistical difference (P>0.05) between two treatments, the daily feed consumption was numerically high in layers fed the barly diet compared to the corn-based diet. 3. Availabilities of crude fat and crude fiber of the barley diet were significantly poor (P<0.05) as compared to corn diet. 4. The \beta-glucarase supplementation depressed the viscosity of barley diets and excreta from therm. 5. Both serum and egg yolk cholesterol were not significantly affected by the addition of \beta-glucarase in the barley based diet. Our data indicate that the barley grain supplemented with \beta-glucarase can be sucessfully used as an energy source of layer diet when there is a price advantage. Although some possibilities to produce low cholesterol egg were recognized in this study, further studies pertaining to long-term feeding experiment and elucidaton of the metabolic interrelationship between serum and yolk cholesterol, are required to confirm the result.

• 한국작물학회지
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• 제43권1호
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• pp.19-22
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• 1998

Effects of acute anoxia on carbohydrate hydrolytic enzyme activities and free amino acid contents in malt were examined. Malts were prepared with barley grains germinated for 7 days which contained the highest levels of amylolytic and(1-3,1-4)-$\beta$-glucanase activities. $\alpha$-Amylase and $\beta$-amylase activities in malts were not significantly affected by anoxia for 5 or 10 h.(1-3,1-4)-$\beta$-Glucanase activity, however, decreased about 7 to 10% by anoxia for 5 or 10 h. Alanine and $\gamma$-aminobutyric acid content changed drastically. Alanine contents in malts increased by 2.2- and 2-fold, and $\gamma$-aminobutyric acid contents by 1.4- and 1.9-fold under anoxia for 5 and 10 h, respectively.

• Applied Biological Chemistry
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• 제37권1호
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• pp.43-48
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• 1994

벼 세포 현탁배양여액으로부터 chitooligosaccharides에 의해 유도된 염기성 단백질 ICG를 순수분리하였다. 본 단백질은 chitin과 laminarin을 가수분해하므로 chitinase와 ${\beta}-1,3-glucanase$ 활성을 함께 보유하고 있는 것으로 나타났으며, 벼잎 단백질 RCG-2와는 달리 lysozyme 활성을 가지고 있지 않았다. 분자량이 52.53 kd인 본 효소의 chitinase 활성은 pH 5.0, $60^{\circ}C$, ${\beta}-1,3-glucanase$ 활성을 pH 4.0, $40^{\circ}C$의 조건에서 최대로 나타났다.$NAG_5$에 대한 $K_M$ 및 $V_{max}$ 수치는 각각 0.474 mM, 2.997 nM/min., laminarin에 대한 것은 각각 1.004 mM, 0.739 nM/min.로 나타나, ICG는 RCC-2보다 높은 기질친화성을 가지고 있으며 chitin을 chitooligosaccharide로 분해하는 endo형 chitinase로 판명되었다.

• Journal of Applied Biological Chemistry
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• 제52권2호
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• pp.51-57
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• 2009

식물병의 생물학적 방제에 관련한 chitinase, ${\beta}$-1,3-glucanase, peroxidase의 발현 패턴을 살펴보기 위하여 3 품종(Capitol, Pollen 및 Saturnin)의 유로용 유채를 조사하였다. 유채 old leaf에서 병발생관련 단백질의 활성 중에서 chitinase의 경우 단백질 mg당 9.7${\sim}$11.8 unit, ${\beta}$-1,3-glucanase의 경우 단백질 mg당 11.1${\sim}$17.3 unit, peroxidase의 경우 단백질 mg당 0.6${\sim}$1.7 unit를 나타내었다. 유채 뿌리내 효소의 활성 중에서 chitinase의 경우 단백질 mg당 39.2${\sim}$49.0 unit, ${\beta}$-1,3-glucanase의 경우 단백질 mg당 49.9${\sim}$62.0 unit, peroxidase의 경우 단백질 mg당 2.4${\sim}$3.8 unIt를 나타내었다. Chitinase와 ${\beta}$-1,3-glucanase 활성은 Saturnin 잎과 Capitol 뿌리내에서 가장 높았고, 반면 Capitol 잎에서 가장 낮은 수준을 보였다. 또한, chitinase, ${\beta}$-1,3-glucanase 및 peroxidase 활성은 Saturnin 뿌리내에서 가장 낮은 수준을 보였다. Chitinase 동위효소가 잎(73, 51, 40, 34, 29 kDa)과 뿌리 (100, 57 34, 29 kDa)의 SDS-PAGE 겔 상에서 보였다. ${\beta}$-1,3-glucanase 동위효소가 잎과 뿌리 (75, 55 kDa)의 SDS-PAGE 겔 상에서 보였다. Peroxidase 활성염색은 Pollen의 잎과 뿌리내에서 가장 강하게 나타났다. Peroxidase 동위효소는 잎(122, 114, 93 kDa)과 뿌리(135, 122, 114, 93 kDa)의 Native-PAGE 겔 상에서 보였다. 이상의 결과로 볼 때 유채 조직내 효소 발현 패턴의 확립은 유채 생육기간 동안 식물병에 대한 저항성과 관련하여 중요한 자료가 될 것으로 사료된다.

• 미생물학회지
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• 제44권3호
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• pp.258-263
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• 2008

대표적인 식물병원성 곰팡이 인 Phytophthora infestans와 Fusarium oxysporum의 생장을 저해하는 근권세균들을 토양에서 분리하여 동정하였으며 이 균주들이 분비하는 항진균성 물질인 siderophore, $\beta-1$,3-glucanase, hydrogen cyanide와 chitinase의 생성능을 조사하였다. 분리균주 중 Bacilus sp. RFO41은 F. oxysporum의 생장을 가장 효율적으로 억제하였으며, siderophore 생성능과 $\beta-1$,3-glucanase의 활성이 가장 우수하였다. 또 다른 분리균주인 Bacilus sp. PS2는 P. infestans의 생장을 가장 많이 억제하였으며, chitinase 활성과 hydrogen cyanide 생성능이 가장 우수하였다. F. oxysporum과 P. infestans에 대한 항진균 효과는 근권세균이 생산하는 siderophore, $\beta-1$,3-glucanase, hydrogen cyanide와 chitinase의 활성에 따라 차이가 있음을 알 수 있었다.

• Journal of Microbiology and Biotechnology
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• 제17권1호
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• pp.58-66
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• 2007

Isolation, expression, and characterization of a novel $endo-{\beta}-1,3(4)-D-glucanase$ with high specific activity and homology to Bacillus lichenases is described. One clone was screened from a genomic library of Paenibacillus sp. F-40, using lichenan-containing plates. The nucleotide sequence of the clone contains an ORF consisting of 717 nucleotides, encoding a ${\beta}-glucanase$ protein of 238 amino acids and 26 residues of a putative signal peptide at its N-terminus. The amino acid sequence showed the highest similarity of 87% to other ${\beta}-1,3-1,4-glucanases$ of Bacillus. The gene fragment Bg1 containing the mature glucanase protein was expressed in Pichia pastoris at high expression level in a 3-1 high-cell-density fermenter. The purified recombinant enzyme Bg1 showed activity against barley ${\beta}-glucan$, lichenan, and laminarin. The gene encodes an $endo-{\beta}-1,3(4)-D-glucanase$ (E. C. 3.2.1.6). When lichenan was used as substrate, the optimal pH was 6.5, and the optimal temperature was $60^{\circ}C$. The $K_m,\;V_{max},\;and\;k_{cat}$ values for lichenan are 2.96mg/ml, $6,951{\mu}mol/min{\cdot}mg,\;and\;3,131s^{-1}$, respectively. For barley ${\beta}-glucan$ the values are 3.73mg/ml, $8,939{\mu}mol/min{\cdot}mg,\;and\;4,026s^{-1}$, respectively. The recombinant Bg1 had resistance to pepsin and trypsin. Other features of recombinant Bg1 including temperature and pH stability, and sensitivity to some metal ions and chemical reagents were also characterized.

• 미생물학회지
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• 제25권4호
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• pp.339-345
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• 1987

A bacterium K-4-3, producing $\beta$-glucan hydrolyzing enzyme, was isolated from soil and identified to be Bacillus subtilis by its morpholohical and physiological characteristics. $\beta$-glucan was coloured using cibacron blue 3G-A and cross linded by the addition of 1, 4-butanedioldiglycidyl ether. This substrate was used for the isolation of $\beta$-glucanase producing microorganism. The $\beta$-glucan hydrolyzing enzyme actibity from isolated K-4-3 strain was also measured using the modified substrate. Bacillus subtilis K-4-3 produced the highest extracellular $\beta$-glucan hydrolyzing activity in the basal medium containing $\beta$-glucan as a carbon source, peptone and tryptone as a nitrogen source, and magnesium sulfate as an inorganic salt. The optimum temperature and initial pH for $\beta$-glucanase production by Bacillus subtilis K-4-3 were $37^{\circ}C$ and pH6. The highest enzyme activity was obtained at the culture age of 54 hrs with rotary shaking at $37^{\circ}C$. The crude enzyme showed the highest activity at pH 7.5-8.0 and $65^{\circ}C$.