• 미생물학회지
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• 제52권4호
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• pp.463-470
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• 2016

Xylan의 효소적 가수분해는 고부가가치 기능성 물질 또는 바이오에너지 생산을 위한 발효성 당을 얻는 가장 유용한 방법 중 하나이다. endo-${\beta}$-Xylanase는 xylan 주사슬 내부의 ${\beta}$-1,4-결합을 가수분해하여 xylobiose, xylotriose를 포함한 다양한 XOS를 생산하는 핵심 효소이다. 이들 효소 중에서 glucuronoxylanase GH30은 methylglucuronic acid가 측쇄에 수식된 xylan에 특이적으로 작용한다. 본 연구에서는 Paenibacillus amylolyticus KCTC 3005에서 유래한 2종의 xylan 가수분해효소(PaXN_10과 PaGuXN_30) 유전자를 클로닝하고, Escherichia coli에서 각각 발현시켰다. PaXN_10 (38.7 kDa)은 ${\beta}$-xylanase GH10 계열, PaGuXN_30 (58.5 kDa)은 glucuronoxylanase GH30에 해당하는 효소이며, $50^{\circ}C$와 pH 7.0에서 최대 활성을 나타내었다. 가수분해 특성 연구를 통해 P. amylolyticus가 목질계 glucuronoxylan을 분해하는 효소 시스템을 제안하였다. 세포 외로 분비되는 PaGuXN_30은 glucuroxylan을 가수분해하여 methylglucuronic acid 측쇄를 가지는 다양한 aldouronic acid mixtures를 생성하며, 이러한 분해산물은 세포 내로 이동하여 PaXN_GH10에 의해 xylose, xylobiose와 같은 저분자 XOS로 분해되어 세포 내 대사경로에 이용될 수 있다. 또한 이들 효소의 가수분해특성을 이용하여 다양한 탄수화물 소재 생산이 가능할 것으로 기대한다.

• 한국작물학회:학술대회논문집
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• 한국작물학회 2018년도 추계학술대회
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• pp.204-204
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• 2018

• KSBB Journal
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• 제13권3호
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• pp.312-319
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• 1998

The dilute-acid pretreatment/hydrolysis of hemicellulose in oak wood using a percolation reactor was investigated. The experimental conditions ranged 160∼180$^{\circ}C$ and 0.05∼0.2 wt.% sulfuric acid. XMG(xylan+mannan+galactan) recovery was higher when sulfuric acid was used as leaching solvent than water. Also it was important for high XMG recovery to keep leaching temperature higher after reaction. XMG recovery was decreased as the size of wood chips was increased. At an optimum condition (reaction condition= 170$^{\circ}C$, 0.1% sulfuric acid, 1ml/min, 10min, leaching condition=0.1% sulfuric acid, 2mL/min, 20 min), the product yield and the sugar concentration were about 92% and 2.7%, respectively.

• 농업과학연구
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• 제43권4호
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• pp.641-649
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• 2016

This study was conducted to evaluate the yield of bio-ethanol produced by separate hydrolysis and fermentation (SHF) with the pretreated rapeseed straw (RS) using crude enzyme of Cellulomonas flavigena and Saccharomyces cereviase. Crude enzyme of C. flavigena showed enzymatic activity of 14.02 U/mL for CMC 133.40 U/mL, for xylan 15.21 U/mL, for locust gum and 15.73 U/mL for rapeseed straw at pH 5.0 and $40^{\circ}C$, respectively. The hemicellulose contents of RS was estimated to compromise 36.62% of glucan, 43.20% of XMG (xylan + mannan + galactan), and 2.73% of arabinan by HPLC analysis. The recovering ratio of rapeseed straw were investigated to remain only glucan 75.2% after 1% $H_2SO_4$ pretreatment, glucan 45.44% and XMG 32.13% after NaOH, glucan 44.75% and XMG 5.47% after $NH_4OH$, and glucan 41.29% and XMG 41.04% after hot water. Glucan in the pretreatments of RS was saccharified to glucose of 45.42 - 64.81% by crude enzyme of C. flavigena while XMG was made into to xylose + mannose + galactose of 58.46 - 78.59%. Moreover, about 52.88 - 58.06 % of bio-ethanol were obtained from four kinds of saccharified solutions by SHF using S. cerevisiae. Furthermore, NaOH pretreatment was determined to show the highest mass balance, in which 21.22 g of bio-ethanol was produced from 100 g of RS. Conclusively, the utilization of NaOH pretreatment and crude enzyme of Cellulomonas flavigena was estimated to be the best efficient saccharification process for the production of bio-ethanol with rapeseed straw by SHF.

• Journal of Microbiology and Biotechnology
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• 제14권3호
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• pp.474-482
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• 2004

The $\alpha$-L-arabinofuranosidase (Arfase) gene of Bacillus stearothermophilus No. 236 was cloned and sequenced. The ORF of the gene, designated arfA, encoded a 507 -residue polypeptide with calculated molecular mass of 57 kDa. The Arfase produced by a recombinant Escherichia coli strain containing the arfA gene was purified to apparent homogeneity and characterized. The molecular mass of the Arfase determined by SDS-PAGE was 60 kDa. However, according to gel filtration, it was estimated to be approximately 190 kDa. These results indicated that the functional form of the Arfase is trimeric. The optimal pH and temperature for the enzyme activity were pH 6.5 and $55^{\circ}C$, respectively. The half-life of the enzyme at $60^{\circ}C$ was about 6 h. Kinetic experiments at $45^{\circ}C$ with pNPM (p-nitrophenyl $\alpha$-L-arabinofuranoside) as a substrate gave the $K_m and V_{max}$ values of 1.19 mM and 26.1 U/ mg, respectively. When the enzyme was combined with Bacillus stearothermophilus No. 236 endoxylanase and $\beta$-xylosidase, it hydrolyzed arabinoxylan into L-arabinose and xylose more efficiently than Arfase alone. This synergistic effect suggested that the complete hydrolysis of xylan with large amounts of arabinose side chains required Arfase as well as endoxylanase and $\beta$-xylosidase.

• 한국균학회지
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• 제24권4호통권79호
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• pp.255-264
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• 1996

호알카리성 진균 Cephalosporium sp. RYM-202가 생산하는 alkaline xylanase (CX-III)의 작용에 의해 xylan 기질로부터 생성되는 주요 가수분해 산물은 xylobiose와 중합도가 4이상인 xylooligosaccharides이었다. 이 효소는 xylobiose에 대한 분해능을 가지고 있지 않지만 xylotriose로부터는 xylobiose를, xlyotetraose로부터 xylobiose와 xylotriose를 주산물로 형성하였다. 이러한 결과들은 CX-III가 transglycosidase 활성을 소유하는 전형적인 endo-type xylanase임을 보여준다. N-bromosuccinimide에 의한 CX-III의 화학적 변환 실험결과 효소 1분자 당 2개의 tryptophan 잔기가 활성에 관여하는 것으로 나타났다. 그러나 iodoacetamide 및 diethylpyrocarbonate에 의한 효소활성의 저해효과는 나타나지 않음으로써 이 효소의 활성부위에 cysteine과 histidine 잔기가 필수적이지 않음이 확인되었다.

• 한국미생물·생명공학회지
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• 제33권3호
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• pp.178-183
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• 2005

토양으로부터 세포외로 xylanase를 분비 생산하는 방선균 WL-2가 분리되었으며, 분리균의 16S rRNA 염기서열과 형태${\cdot}$배양${\cdot}$생리적 특성을 조사한 결과 Streptomyces속 균주로 확인되었다. 분리균의 배양상등액에 존재하는 xylanase는 pH 6.0과 $60^{\circ}C$의 반응조건에서 반응성이 가장 높았으며, pH $4.5{\~}6.5$ 범위에서 최대활성의 $90{\%}$ 이상을 나타냈다. Xylanase의 생산을 위한 배지를 최적화하기 위해서 G.S.S 배지성분을 여러 종류의 탄수화물로 대체하였다. ${\alpha}-Cellulose$, oat spelt xylan과 엿당과 같은 탄수화물은 Streptomyces sp. WL-2의 xylanase 생산성을 급격히 증가시키는 것으로 확인되었다. ${\alpha}-Cellulose(1\%)$와 엿당($1{\%}$)을 함유한 변형배지에서 xylanase의 최대생산성이 120 U/ml로 확인되었다.

• 미생물학회지
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• 제31권1호
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• pp.63-71
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• 1993

A xylanase (xylanase I) was purified 11.9-fold from the culture filtrate of Trichoderma koningii ATCC 26113 by the column chromatography on Sephadex G-75, SP-Sephadex C-50, DEAE-Sephadex A-50 and Sephadex G-50 with an overall yield of 8.2%. The molecular mass determined by gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis was found to be a monomeric polypeptide of ca. 35 kDa. The isoelectric point of the enzyme was estimated to be 9.3. The optimal reaction pH and temperature are 5.8 and 55.deg.C, respectively. The enzyme is stable up to 60.deg.C, while 78% of its activity is lost after the incubation for 10 min at 70.deg.C. The enzyme hydrolyzes sylan with relatively high activity, as well as carboxymethyl cellulose and avicel. The $K_{m}$ values of the enzyme for oat-spelf sylan, larchwood xylan and Avicel were 3.5, 1.6 and 10. 1 mg/ml, respectively. The enzyme hydrolyzed oat-spelt sylan to sylose, sylobiose, sylotriose and arabionoxylobiose, while it degraded larchwood xylan to xylose, xylobiose, xylotriose and arabionoxylobiose, while it degraded larchwood xylan to xylose, xylobiose and xylotriose as the major products. The hydrolysis patterns indicate that xylanase I is endo-enzyme.e.

• Journal of Microbiology and Biotechnology
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• 제24권11호
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• pp.1559-1565
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• 2014

Cellulase and xylanase are main hydrolysis enzymes for the degradation of cellulosic and hemicellulosic biomass, respectively. In this study, our aim was to develop and test the efficacy of a rapid, high-throughput method to screen hydrolytic-enzyme-producing microbes. To accomplish this, we modified the 3,5-dinitrosalicylic acid (DNS) method for microwell plate-based screening. Targeted microbial samples were initially cultured on agar plates with both cellulose and xylan as substrates. Then, isolated colonies were subcultured in broth media containing yeast extract and either cellulose or xylan. The supernatants of the culture broth were tested with our modified DNS screening method in a 96-microwell plate, with a $200{\mu}l$ total reaction volume. In addition, the stability and reliability of glucose and xylose standards, which were used to determine the enzymatic activity, were studied at $100^{\circ}C$ for different time intervals in a dry oven. It was concluded that the minimum incubation time required for stable color development of the standard solution is 20 min. With this technique, we successfully screened 21 and 31 cellulase- and xylanase-producing strains, respectively, in a single experimental trial. Among the identified strains, 19 showed both cellulose and xylan hydrolyzing activities. These microbes can be applied to bioethanol production from cellulosic and hemicellulosic biomass.

• Journal of Microbiology and Biotechnology
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• 제23권3호
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• pp.397-404
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• 2013

Paenibacillus xylanilyticus KJ-03 was isolated from soil samples obtained from a field with Amorphophallus konjac plants. A gene encoding xylanase was isolated from KJ-03 and cloned using a fosmid library. The xynA gene encodes xylanase; it consists of 1,035 bp and encodes 345 amino acids. The amino acid sequence deduced from the P. xylanilyticus KJ-03 xylanase showed 81% and 69% identities with those deduced from the P. polymyxa E681 and Paenibacillus sp. HPL-001 xylanases, respectively. The xynA gene comprises a single domain, consisting of a catalytic domain of the glycosyl hydrolase (GH) 10 family. The xynA gene was expressed in Escherichia coli BL21 (trxB), and the recombinant xylanase was purified by Niaffinity chromatography. The purified xylanase showed optimum activity with birchwood xylan as a substrate at $40^{\circ}C$ and pH 7.4. Treatment with $Mg^{2+}$ and $Li^+$ showed a slight decrease in XynA activity; however, treatment with 5 mM $Cu^{2+}$ completely inhibited its activity. The results of the thin layer chromatography analysis indicated that the major hydrolysis product was xylobiose and small amounts of xylose and xylotriose. XynA showed increased activity with oat spelt xylan and birchwood xylan, but showed only slight activity with locust bean gum.