Paenibacillus donghaensis JH8에서 세포외 Xylanase의 특성

임채성,오용식,노동현,Lim, Chae-Sung,Oh, Yong-Sik,Roh, Dong-Hyun 한국미생물학회 2011 미생물학회지 Vol.47 No.1

Xylanase is a class of enzymes that hydrolyze the linear polysaccharide ${\beta}$-1,4-xylan into xylose. This enzyme is applied in the process of paper making and may be used for the process of biofuel production in the future. The Paenibacillus donghaensis JH8, isolated from Donghae deepsea sediment and reported as a novel bacterium, was known to degrade xylan and its xylanase was characterized in this study. The enzyme was maximally induced in the presence of 0.1% xylan. The production of xylanase was started at early logarithmic phase and reached about 55 miliunit at stationary phase of growth. The optimal temperature and pH of extracellular xylanase were found to be $40^{\circ}C$ and pH 6.0, respectively. The activity of xylanase was inhibited by the presence of $Ca^{2+}$, $Mn^{2+}$, $Fe^{2+}$, $Cu^{2+}$, $Al^{3+}$ or EDTA, and activated by $K^+$, $Ag^+$ or DTT. This xylanase was stable at $40^{\circ}C$ for 120 min, but lost almost their activity in 30 min at $60^{\circ}C$. Zymography analysis of concentrated culture supernatant revealed one major band at 42 kDa and two faint bands at 68 and 120 kDa. Xylanase는 선형복합다당인 ${\beta}$-1,4-xylan을 xylose로 가수분해하는 효소의 한 종류이며, 종이제조공정에 응용되고 미래에 바이오 연료의 생산에 사용 될 수 있다. 동해 심층 퇴적물로부터 신종세균으로 보고된 Paenibacillus donghaensis JH8은 배지중의 xylan을 분해한다고 알려져 있으며, 여기에서는 이 효소의 특성을 조사하였다. 효소는 0.1% xylan 존재에서 최고로 유도되었으며, xylanase의 생산은 초기 대수성장기에 효소를 생산하기 시작하여, 정지기에서 약 55 miliunit에 도달하였다. 세포외성 xylanase의 최적온도와 pH는 각각 $40^{\circ}C$와 pH 6.0이였다. Xylanase의 활성은 $Ca^{2+}$ 및 $Mn^{2+}$, $Fe^{2+}$, $Cu^{2+}$, $Al^{3+}$, EDTA의 존재에 의해 억제되었고, $K^+$, $Ag^+$, DTT에 의해 활성화되었다. 이 xylanase는 $40^{\circ}C$에서 120분간 활성을 유지하며 안정하였지만, $60^{\circ}C$에서는 30분에서 거의 모든 활성을 잃어버리는 특성을 보여주었다. 농축된 배양 상등액의 zymography 분석시 42 kDa의 주 밴드와 68과 120 kDa에 두 개의 아주 희미한 밴드를 나타내었다.

고온성 Bacillus sp.의 Xylanases 특성 비교

윤기홍 우송대학교 1997 우송대학교 논문집 Vol.2 No.-

토양에서 분리된 cellulase-free xylanase 생산균인 고온성 Bacillus sp. KK-1의 배양상등액으로 부터 내열성 xylanase S를 정제하였다. Bacillus sp. KK-1의 xylanase 유전자를 확보하기 위해 총 염색체 DNA를 이용하여 xylanase 유전자를 대장균에 크로닝한 결과 xylanase 활성을 보이는 30개 대장균 형질전환주를 얻어 이들이 생산하는 xylanases의 열안정성을 조사한 결과 모두 xylanase S보다 낮았다. 또한 형질전환주에서 분리된 plasmids를 여러가지 제한효소로 절단하여 분석한 결과 동일한 부분의 DNA조각을 공유하고 있었다. 이로써 이들 형질전환주는 동일한 xylanase를 생산하고 있다고 판단되며 이를 xylanase Y라 명명하고 대장균 형질전환주 균체로 부터 xylanase Y를 정제하였다. 정제된 xylanase S와 xylanase Y의 성질을 비교 분석하였는데 이들은 전혀 다른 성질을 보였으므로 Bacillus sp. KK-1이 최소한 두 종류의 xylanases를 생산하는 것을 알 수 있다. 특히 xylanase S는 열안정성이 매우 높아 65C에서도 약 10시간 방치하여도 효소 활성이 75% 이상 유지 되었으나 xylanase Y는 50C에서 30분간 방치하여도 활성을 약 25% 정도 상실하였다. Xylanase Y가 열안정성이 xylanase S에 비해 매우 낮으므로 Bacillus sp. KK-1를 50C에서 약 16시간 동안 배양하여 얻은 배양상등액에서는 xylanase S와는 달리 xylanase Y의 활성이 거의 상실되어 정제될 수 없었던 것으로 여겨진다. A thermostable xylanase, designated xylanase S, was purified from the culture supernatant of Bacillus sp. KK-1 which was isolated from natural soil. In order to obtain Bacillus sp. KK-1 xylanase gene. xylanase gene was cloned in Escherichia coli from Bacillus sp. KK-1. From approximately 20,000 transformants. 30 E, coli colonies showing xylanase activity was obtained. It was found that almost all xylanases from the E, coil clones was less stable than xylanase S at 60℃. Issert DNAs of recombinant plasmids, isolated from E. coil clones, shared the common DNA fragment of Bacillus sp. KK-1, suggesting that E coli clones produced an identical xylanase. Xylanase, named xylanse Y, was therefore purified from cell free extract of Em coli, The physical and chemical properties fo purified xylanase Y were identified to be different from those of xylanase S, resulting that Bacillus sp. KK-1 produced at least two xylanses. Especcially ,xylanase S retained over 75% io its maximum activity at 65℃ for 10 h. But , xylanase Y lost 25% of its activity at 50℃ within 30 min. From this result. it was supposed that xylanase Y could not purified from the culture supernatant of Banillus sp. KK-1 growing for 16 h at 50℃ because of inactivation of the xylanase Y unlike xylanase S.

Exo-xylanase 생산균의 분리 및 동정

하재석,이영남,임재윤 한국산업미생물학회 1992 한국미생물·생명공학회지 Vol.20 No.1

부패한 나무, 퇴비, 제지공장의 폐지 및 폐수 등으로부터 분리한 300여 종류의 섬유소 분해균 중 xylanase 활성이 다른 균주에 비해 비교적 높았던 33번 균주를 균의 형태학적, 생화학적 특성과, 균체 지방산 조성에 의하여 Pseudomonas sp.로 동정하였다. Xylanase 활성의 최적 온도와 최적 pH는 각각 50℃와 5.5이었고, 효소의 안정성은 45℃ 이하의 온도와 pH 5.0에서 7.0 사이에서 유지되었다. 이 균주가 생산하는 xylanase는 효소반응분해물의 paper chromatography에 의하여 주로 exo-type의 xylanase임이 밝혀졌다. 자연계에 존재하는 천연 섬유질 biomass를 탄소원으로 이용하여 균을 배양한 다음 xylanase의 생산성을 검토한 결과 볏짚을 탄소원으로 이용할 때 xylanase의 생산성이 가장 좋은 것으로 나타났다. The xylanase producing microorganisms occurring on rotten woods were selectively isolated on the modified Czapek-Dox medium supplemented with 0.5% xylan as a sole carbon source. Among more than three-hundred isolates of xylanase producing microorganisms, only two bacterial isolates were turned out to be more potent xylanase producer than the reference strain of xylanase producer, Aureobasidium pullulans NRRL Y-2311. The exo-xylanase producer, bacterial isolate No.33 was identified as a strain of Pseudomonas sp. on the basis of morphological and biochemical characterizations as well as cellular fatty acid composition. Optima of pH and of temperature for enzyme reactions of xylanase were 5.5 and 50℃, respectively. The enzyme was stable in a range of pH 5.O∼7.0 and below 45℃. Among the number of carbohydrate substrates, xylose was turned out to be a potent inducer of Pseudomonas sp. No.33 exo-xylanase. Among the raw materials tested, rice straw was the best material for xylanase production by Pseudomonas sp. strain No.33.

Xylanase를 생산하는 Streptomyces sp. YB914의 특성과 효소 생산성

윤기홍,Yoon, Ki-Hong 한국미생물·생명공학회 2009 한국미생물·생명공학회지 Vol.25 No.4

토양으로부터 세포외로 xylanase를 분비 생산하는 방선균 YB-914가 분리되었으며, 형태, 배양, 생화학적 특성을 조사한 결과 Streptomyces 속 균주로 확인되었다. 분리균의 배양상등액에 존재하는 xylanase는 pH 5.5과 $55^{\circ}C$의 반응조건에서 반응성이 가장 높았으며, pH 4.5~7.0 범위에서 최대활성의 80% 이상을 나타냈다. Xylanase의 생산을 위한 배지를 최적화하기 위해서 G.S.S 배지성분을 여러 종류의 탄수화물로 대체하였다. Oat spelt xylan, corn cob xylan, 밀기울 및 유당과 같은 탄수화물은 Streptomyces sp. YB914의 xylanase 생산성을 증가시키는 것으로 확인되었으며, galactose와 arabinose는 효소 생산을 크게 억제하였다. Oat spelt xylan(1%)와 유당(1.5%)을 함유한 변형배지에서 xylanase의 최대생산성이 48 U/mL로 확인되었다. A strain YB914 was isolated from soil as a producer of the extracellular xylanase, which catalyzes the hydrolysis of oat spelt xylan. The strain YB914 was identified as Streptomyces sp. on the basis of its morphological, cultural and biochemical properties. The xylanase of culture filtrate was the most active at $55^{\circ}C$ and pH 5.5, and retained 80% of its maximum activity at the range of pH 4.5~7.0. In order to optimize the culture medium for xylanase production, ingredients of G.S.S medium were replaced by several carbohydrates. The carbohydrates such as oat spelt xylan, corn cob xylan, wheat bran and lactose increased the xylanase productivity of Streptomyces sp. YB914. However, xylanase production was greatly repressed by galactose or arabinose. The maximum xylanase productivity was reached to 48 U/mL in the modified medium containing 1% oat spelt xylan and 1.5% lactose.

Bacillus alcalophilus AX2000 유래 xylanase 유전자 (XynT)의 Cloning과 염기서열 분석

박영서,Park Young-Seo 한국생명과학회 2005 생명과학회지 Vol.15 No.5

Xylanase를 생산하는 알칼리 내성 Bacillus alcalophilus AX2000의 chromosomal DNA로부터 xylanase 유전자를 cloning하여 그 염기배열 순서를 결정한 다음 이로부터 유전자 발현에 관련된 구조를 분석하였다. Xylanase 유전자의 cloning을 위해 제한효소 PstI으로 절단한 B. alcalophilus AX2000의 chromosomal DNA와 pUC19을 ligation 시켜 E. coli $DH5\alpha$에 형질전환시킨 후 형질전환체 중에서 xylanase 활성을 나타내는 재조합 plasmid pXTY99를 분리하였다. 재조합 plasmid pXTY99은 pUC19의 PstI 부위 내에 7kb의 외래 DNA가 삽입 되 었다. Cloning된 xylanase 유전자(xynT)의 염기배열을 분석한 결과 유전자의 크기는 1,020 bp이었고 이는 340개의 아미노산으로 구성된 분자량 40 kDa의 poly-peptide를 coding하고 있었다. 이 염기배열은 AUG 개시 codon으로부터 각각 259와 282 base상류에 TACAAT의 -10 box와 GTTCACA인 -35 box로 추정되는 염기배열이 존재하였으며 ribosome 결합부위가 존재하였다. B. alcalophilus AX2000의 xylanase와 아미노산배열의 유사성이 가장 높은 xylanase는 Bacillus sp. N137과 B. stearothemophilus 21 유래의 xylanase로 각각 $61\%$와 $59\%$의 유사성을 나타내었다. A gene coding for xylanase from alkali-tolerant Bacillus alcalophilus AX2000 was cloned into Escherichia coli $DH5\alpha$ using pUC19. Among 2,000 transformants, one transformant showed clear zone on the detection agar plate containing oat-spells xylan. Its recombinant plasmid, named pXTY99, was found to carry 7.0 kb insert DNA fragment. When the nucleotide sequence of the cloned xylanase gene (xynT) was determined, xynT gene was found to consist of 1,020 base-pair open reading frame coding for a poly-peptide of 340 amino acids with a deduced molecular weight of 40 kDa. The coding sequence was preceded by a putative ribosome binding site, and the transcription initiation signals. The deduced amino acid sequence of xylanase is similar to those of the xylanases from Bacillus sp. Nl37 and B. stearothermophilus 21 with $61\%$ and $59\%$ identical residues, respectively.

Bacillus pumilus TX703 유래 Xylanase 유전자(xynK)의 Cloning과 염기서열 분석

박영서 한국생명과학회 2002 생명과학회지 Vol.12 No.2

A gene coding for xylanase from thermo-tolerant Bacillus pumilus TX703 was cloned into Escherichia coli DH5 $\alpha$ using pUC19. Among 7,400 transformants, four transformants showed clear zones on the detection agar plates containing oat-spells xylan. One of them which showed highest xylanase activity was selected and its recombinant plasmid, named pXES106, was found to carry 2.24 kb insert DNA fragment. When the nucleotide sequence of the cloned xylanase gene (xynK) was determined, xynK gene was found to consist of 1,227 base-pair open reading frame coding for a polypeptide of 409 amino acids with a deduced molecular weight of 48 kDa. The coding sequence was preceded by a putative ribosome binding site, the transcription initiation signals, and cia-acting catabolite responsive element. The deduced amino acids sequence of xylanase is similar to those of the xylanases from Hordeum vulgare (barley) and Clostridium thermocellum, with 39 and 31% identical residues, respectively. The amino acids sequence of this xylanase was quite different from those of the xylanases from other Bacillus species. Xylanase를 생산하는 내열성 Bacillus pumilus TX703의 chromosomal DNA로부터 xylanase 유전자를 cloning하여 그 염기배열 순서를 결정한 다음 이로부터 유전자 발현에 관련된 구조를 분석하였다. Xylanase 유전자의 cloning을 위해 제한효소 HindIII로 절단한 B. pumilus TX703의 chromosomal DNA와 pUC19을 ligation시켜 E. coli DH5 $\alpha$에 형질전환시킨 후 형질전환체 중에서 xylanase 활성을 나타내는 재조합 plasmid pXES106을 분리하였다. 재조합 plasmid pXES106은 pUC19의 HindIII 부위 내에 2.24 kb의 외래 DNA가 삽입되었고, 이 plasmid DNA를 분리하여 E. coli DH5 $\alpha$에 재형질전환시킨 결과 vector 내에 xylanase 유전자가 cloning되었음을 확인하였다. Cloning된 유전자의 염기배열을 분석한 결과 이 유전자의 총 크기는 2,187 bp였고 이는 409개기 아미노산을 coding 하는 open reading frame 1,227 bp를 포함하고 있었다. 이 염기배열은 ATG개시 codon으로부터 각각 193과 216 base 상류에 TTTAAT의 -10 box와 TCGAAA인 -35 box로 추정되는 염기배열이 존재하였고 -10 box로부터 7 bp하류에 전사개시점인 A가 위치하고 있었다. 또한, 개시 codon으로부터 432 bp 상류에 공통염기배열과 14개의 염기 중 11개의 염기가 일치하는 TGATGGCGTCGGCA의 catabolite responsive element (CRE)가 존재하였다. B. pumilus TX703의 xylanase와 아미노산배열의 유사성이 가장 높은 xylanase는 Hordeum vulgare의 isozyme X-I이었고 본 xylanase는 208번째와 322번째에 glutamic acid 잔기를 가지고 있어 Clostridium thermocellum, Dictyoglomus thermophilum, Thermotoga neapolitana 등에서 밝혀진 바와 같이 glutamic acid 부위가 xylanase의 활성부위라 여겨진다.

Paenibacillus sp. DG-22로부터 xylanase 생산의 최적화

Lee, Yong-Eok Korean Society of Life Science 2003 생명과학회지 Vol.13 No.5

목재 저장소의 토양에서 분리된 호열성 세균인 Paenibacillus sp. DG-22로부터 xylanase를 생산하기 위한 배양조건을 최적화시키기 위해 연구를 수행하였다. Xylanase생산은 세포의 생장과 연관된 양상을 나타내었다. Xylanase 활성은 배양상청액에서만 발견된 반면 $\beta-xylosidase$활성은 주로 세포와 결합되어 있었다. Xylanase활성의 형성은 자일란에 의해 유도되었고 포도당과 자일로스에 의해서 억제되었다. 여러 상업적 자일란을 이용하여 xylanase의 생산양상을 조사한 결과 0.1-0.5%의 birchwood xylan에서 가장 높은 생산율을 나타내었다. 조사된 여러 질소 원들 중 효모추출물이 xylanase생산을 위하여 최적이었다. xylanase의 활성은 $Co^{2+},\; Cu^{2+},\; Fe^{3+},\; Hg^{2+}\;$ 와$\; Mn^{2+}$ 이온들에 의하여 억제된 반면 $Ca^{2+},\; Mg^{2+},\; Ni^{2+},\; Zn^{2+}$ 이온들과 DTT에 의해서는 촉진되었다. 수은은 5 mM의 농도에서 xylanase 활성을 완전히 파괴하였다. 자일란 가수분해의 주된 산물은 자일로바이오스, 자일로트라이오스 그리고 자일로 올리고당이었고 이것은 이 효소가 endoxylanase라는 것을 나타낸다. Investigations were carried out to optimize the culture conditions for the production of xylanase by Paenibacillus sp. DG-22, a moderately thermophilic bacterium isolated from timber yard soil. Xylanase production showed a cell growth associated profile. Xylanase activity was found only in the culture supernatant, while $\beta-xylosidase$ activity was mainly associated with the cells. The formation of xylanase activity was induced by xylan and repressed by glucose and xylose. The production profile of xylanase was examined with various commercial xylan and maximum yield was achieved with 0.1∼ 0.5% birchwood xylan. Among various nitrogen sources tested, yeast extract was optimal for the production of xylanase. The xylanase activity was inhibited by $Co^{2+},\; Cu^{2+},\; Fe^{3+},\; Hg^{2+}\;$ and$\;Mn^{2+}$ ions while $Ca^{2+},\; Mg^{2+},\; Ni^{2+},\; Zn^{2+}$ions and DTT stimulated xylanase activity Mercury (II) ion at 5 mM concentration abolished all the xylanase activity. The predominant products of xylan-hydrolysate were xylobiose, xylotriose, and higher xylooligo-saccharides, indicating that the enzyme was an endoxylanase.

Properties of Aspergillar Xylanase and the Effects of Xylanase Supplementation in Wheat-based Diets on Growth Performance and the Blood Biochemical Values in Broilers

Wu, Yubo,Lai, Changhua,Qiao, Shiyan,Gong, Limin,Lu, Wenqing,Li, Defa Asian Australasian Association of Animal Productio 2005 Animal Bioscience Vol.18 No.1

Three experiments were conducted to study the property of xylanase and the effects of xylanase in wheat-based diets on growth performance of broilers, respectively. Experiment 1 was performed in vitro to evaluate the effect of different pH and temperature on xylanase activity, and to evaluate the enzymic stability under different conditions. The results indicated that the optimum temperature and pH for xylanase activity were $50^{\circ}C$ and 4.5, respectively. The activity of enzyme solution was reduced rapidly after the treatment of water bath above $60^{\circ}C$ for 10 min. The enzyme was relatively stable at pH 3.5 to 8.0 and deteriorated when incubated at pH below 3.5. In Experiment 2, a total of 378 d-old male Arbor Acres broilers were randomly distributed to 7 different treatments with 6 replicates (9 birds) in each treatment. The treatments were as follows: (1) corn based diet (CS), (2) wheat based diet (WS), (3) WS+ 0.05% xylanase, (4) WS+0.15% xylanase, (5) WS+0.25% xylanase, (6) WS+0.35% xylanase, (7) WS+0.45% xylanase. The results showed that the body weight and feed/gain ratio of the broilers fed wheat-based diets have been significantly improved (p<0.05) compared to that fed corn-based diet in the first 3 wk. With regard to the wheat-based diets, the xylanase supplementation had a tendency to improve the growth performance in first 3 wk. After 3 wk, no significant difference (p>0.05) was found among all these different treatments. The supplementation of xylanase and the type of diets did not affect the feed intake but increased the concentration of triglyceride in serum. In Experiment 3, a total of 360 d-old male Arbor Acres broilers were assigned to 30 groups with 12 birds in each group randomly. These groups were then randomly distributed to 5 different treatments with 6 replicates within each treatment. The broilers of each treatment were fed one of the diets as follows: (1) Corn based diet, (2) White wheat based diet (WW) (3) White wheat based diet+0.25% xylanase, (4) Red wheat based diet, (5) Red wheat based diet+0.25% xylanase. The results showed that the body weight and feed/gain ratio had been significantly improved (p<0.05) by xylanase supplementation in the first 2 or 3 wk. The effect of xylanase in red wheat diet is a little higher than that used in white wheat diet. From the results of the present experiments, it can be concluded that the supplementation of Aspergillar xylanase can improve the performance of the broilers fed the wheat-based diet.

고지탈묵용 Cellulase 및 Xylanase 생산

김욱한,손광희,복성해,오세균 한국산업미생물학회 1992 한국미생물·생명공학회지 Vol.20 No.5

효소탈묵용 cellulase와 xylanase 생산을 위한 조건을 조사한 결과 배지초기 pH, Tween 80, 포자접종량, 질소원의 종류 및 탄소원의 종류에 따라 효소 생산량이 크게 변하였다. Cellulase 생산을 위한 최적조건으로는 pH 5.O∼6.5, Tween 80 0.02%, 포자현탄액(1×10^7㎖) 접종량 0.5∼l.0%, 질소원으로 면실박, 탄소원으로 옥분을 사용하였을 때 가장 효과적이었다. 또한, xylanase 생산을 위한 최적조건으로는 pH 6.5, Tween 80 0.Ol%, 질소원으로 corn steep liquor, 탄소원으로는 신문고지를 사용하였을 때 효과적이었으며, 이때 포자현탁액의 접종량은 cellulase 생산의 경우와 동일하였다. 한편, cellulase와 xylanase의 동시생산을 위해서는 플라스크 규모에서 질소원으로 면실박 0.5%, 탄소원으로 신문고지 1.0%와 옥분 2.0%를 첨가하였을 때 효과적이었으며, 이때 생산된 배양액의 cellulase의 활성은 6.11∼7.22IU/㎖, xylanase의 활성은 97.7IU/㎖이었다. 그러나 fermentor 규모에서는 동일 배지조성에서 효소생산이 다소 감소하였으며, 특히 xylanase 경우 배양액의 효소활성이 낮아서 이에 대한 보완이 요구된다. The optimal conditions for cellulase and xylanase production by Trichoderma reesei 28217 were studied for enzymatic deinking of old newspaper. The amounts of cellulase and xylanase from the strain was varied by initial medium pH, Tween 80, inoculum size of spore suspension, and carbon and nitrogen sources. The optimal conditions for cellulase production were pH 5.O∼6.5, 0.02% of Tween 80, 0.5∼1.O% of inoculum size of spore suspension (1×10^7/㎖), cotton-seed meal as nitrogen source, and corn flour as carbon source. On the other hand, the optimal conditions for xylanase production were pH 6.5, 0.01% of Tween 80, corn steep liquor as nitrogen source, and disintegrated old newspaper as carbon source. The inoculum size for xylanase production was the same as for cellulase production. The concomitant production of cellulase and xylanase in shake flask culture was efficiently induced in the medium containing 0.5% cottonseed meal as nitrogen source and 1.O% old newspaper and 2.O% corn flour as carbon sources. In this case the activities of cellulase and xylanase produced were 6.11∼7.22 IU/㎖ and 97.7 IU/㎖, respectively. However, the cellulase production in 5ℓ fermentor scale was slightly decreased compared with that in flask scale. Moreover, xylanase production was severely reduced in a fermentor scale. The study for the reason of decreased enzyme production in fermentor is further needed.

Bacillus stearothermophilus로부터 Endo-xylanase 유전자의 클로닝 및 Escherichia coli에서의 발현

조쌍구,박성수,박영인,최용진 한국산업미생물학회 1992 한국미생물·생명공학회지 Vol.20 No.3

내알카리성 및 내열성 xylanase를 생산하는 토양 분리균인 Bacillus stearothermophilus의 chromosomal DNA와 pBR322 plasmid DNA의 HindⅢ 절단 DNA 단편을 ligation시켜 E. coli HB101을 형질전환, 약 5천개의 형질전환체를 얻었으며 이들 중에서 세개의 xylanase 양성 형질전환체를 분리하였다. 상기 세 xylanase 양성 형질전환체로부터 분리한 제조합 plasmid(pMG11, pMG12 및 PMG13)는 다같이 xylanase활성과 관계되는 B. stearothermophilus 유래의 동일 4kb 외래 DNA을 가지고 있었으나, pMG13은 외래 DNA의 삽입 방향만이 다름을 확인하였다. B. stearothermophilus 균주는 최소한 세개 이상의 xylanase 활성단백질을 생산하는 것으로 관찰되었으며, xylanase 양성 형질전환체는 그 중 분자량이 가장 큰 효소단백질을 생산하는 것으로 판단되었다. 한편, 형질전환체 xylanase는 xylotetraose 이상의 xylo-oligosaccharide와 xylan 기질에 작용하여 최종 산물로서 xylobiose와 xylotriose을 생산하는 endo-acting 효소로서, trans-xylosidase 활성도 가지고 있는 것으로 추측되었다. Genomic DNA of Bacillus stearothermophilus, which expressed alkalophilic and thermophilic xylanases, was partially digested with HindⅢ, cloned into pBR322, and subsequently transferred into the Escherichia coli HB101 cells. Three among 5,000 transformants screened formed clear zones around their colonies. From the functional clones, three recombinant plasmids(pMG11, pMG12 and pMG13) had been isolated, and they were identified to carry the same 4 kb HindⅢ fragment originated from B. stearothermophilus which was responsible for the xylanase activity. pMG13, however, had the foreign DNA of opposite orientation compared to the other two recombinant plasmids. This recombinant plasmid gave much lower xylanase activity. B. stearothermophilus was observed to produce at least three xylanase activities as evidenced by the PAGE-xylan zymogram. The xylanase from E. coli HB101/pMG12 was judged to correspond to the largest among the three B. stearothermophilus xylanases observed in the zymogrom. The enzyme hydrolyzed xylooligosaccharides larger than xylotriose and degraded xylan to produce xylobiose and xylotriose as major products. The xylanase was considered to have trans-xylosidase activity, too.