• 한국미생물·생명공학회지
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• 제20권5호
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• pp.574-582
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• 1992

Bacillus stearothermophilus exo-xylanase 유전자 DNA가 삽입된 재조합 plasmid pMG1을 가지고 있는 E.coli JM109 exo-xylanase 생산 최적 배양 조건, 생산 효소의 정제 및 정제 효소의 특성 등을 조사 연구하였다. 상기 재조합 E.coli 균주는 0.5 fructose, 0.5 yeast extract, 1.0 tryptone 및 1.0 sodium chloride가 함유된 배지에서 약 10시간 배양했을 때 최대량의 효소를 생산하였으며 생산효소의 94는 세포내에 존재하는 것으로 분석되었다. 생산 효소는 ammonium sulfate 분획, ion exchange chromatography 및 gel filtration 등의 과정을 거쳐 단일 단백질로 정제하였으며 정제 효소는 pH 6.0과 $45^{\circ}C$에서 가장 높은 효소 활성을 나타내었다.또한 1mM $Ca^{2+}$과 $Co^{2+}$ 이온의 첨가는 각각 약 25% 정도의 활성화 효과를 나타내는 반면, 본 효소의 pNPX에 대한 $K_{m}$은 2.75mM, pl값을 4.7, 그리고 분자량은 gel-filtration 법으로는 약 200,000dal., SDS-polyacrylamide gel 전기영동법으로는 약 66,000dal 으로 측정되어 세 개의 동일한 subunit로 구성된 효소 단백질인 것으로 추정되었다. 본 정제 효소는 xylobiose, xylotrioxe 및 xylotetraose 등의 xylo-oligosaccharide를 효과적으로 분해함은 물론이고, 분해율은 낮으나 birchwood xylan, larchwood xylan 및 oatspelt xylan 등의 xyland에도 작용, xylose 생산을 확인함으로써 본 효소는 그 예가 극히 드문 bacterial exo-xylanase인 것으로 분류되었다.

• Journal of Microbiology and Biotechnology
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• 제8권4호
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• pp.378-387
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• 1998

The DNA sequence immediately following the xynA gene of Bacillus stearothermophilus 236 ［about l-kb region downstream from the translational termination codon (TAA) of the xynA gene］was found to have an ability to enhance the xylanase activity of the upstream xynA gene. An 849-bp ORF was identified in the downstream region, and the ORF was confirmed to encode a novel protein of 283 amino acids designated as XaiF (xylanase-activity-increasing factor). From the nucleotide sequence of the xaiF gene, the molecular mass and pI of XaiF were deduced to be 32,006 Da and 4.46, respectively. XaiF was overproduced in the E. coli cells from the cloned xaiF gene by using the T7 expression system. The transcriptional initiation site was determined by primer extension analysis and the putative promoter and ribosome binding regions were also identified. Blast search showed that the xaiF and its protein product had no homology with any gene nor any protein reported so far. Also, in B. subtilis, the xaiF trans-activated the xylanase activity at the same rate as in E. coli. In contrast, xaiF had no activating effect on the co-expressed ${\beta}-xylosidase$ of the xylA gene derived from the same strain of B. stearothermophilus. In addition, the intracellular and extracellular fractions from the E. coli cells carrying the plasmid-borne xaiF gene did not increase the isolated xylanase activity, indicating that the protein-protein interaction between XynA and XaiF was not a causative event for the xylanase activating effect of the xaiF gene.

• 한국식품영양학회:학술대회논문집
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• 한국식품영양학회 2000년도 춘계학술심포지엄
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• pp.1-3
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• 2000

A kind of traditional herbal prescription, Sip-Jeon-Dae-Bo-Tang (TJ-48), has been reported to improve the general condition of cancer patients receiving chemotherapy and /or radiation therapy, and to accelerate hematopoietic recovery from bone marrow injury by mitomycin C. In the present studies, we found that hot-water extract from Atractylodes lancea DC. rhizomes contributed mainly to intestinal immune modulating activity of TJ-48 on Peyer's patch cells mediated-hematopoietic response. After the fractionation, ALR-5 II a-1-1, 5 II b-2-2 and 5 II c-3-1 were further purified from crude polysaccharide fraction. Chemical analyses of each fraction indicated that ALR-5 II a-1-1 mainly contained arabinogalactan fraction whereas ALR-5 II b-2-2 and 5 II c-3-1 mostly comprised pectic polysaccharide fractions as the active polysaccharide ingredients. In order to analyze the essential structure of the activity, ALR-5 II a-1-1 was treated by sequential enzymatic digestion using exo-${\alpha}$-L-arabinofuranosidase and exo-${\beta}$-D-(1\longrightarrow3)-galactanase. Based upon the results of chemical and MALDI-TOF-MS analyses and activity on the digested fractions, the galactosyl side chains consisting of 6-linked Galf and Galp over tetrasaccharide in ALR-5 II a-1-1 might be responsible for the potent intestinal immune modulating activity. To characterize moiety of ALR-5 II c-3-1 for the expression of activity, endo-${\alpha}$-D-(1\longrightarrow4)-polygal acturonase (GL-PGase) purified from dried leaves of Panax ginseng digested ALR-5 II c-3-1. The results of structural analyses and activity on the digested fractions showed that PG-2, which structurally resembles to rhamnogalacturonan II (RG II), and PG-3 (galacturono-oligosaccharides) contained potent intestinal immune modulating activity. Further purification of the other acidic fraction (ALR-5 II b-2-2) indicated that ALR-5 II b-2-2Bb showed that the most potent activity. ALR-5 II b-2-2Bb also contained the unusual component sugars characteristics in RG- II as well as PG-2 derived from ALR-5 II c-3-1, but it could not be digested with GL-PGase. The present studies of relationship between structures and intestinal immune modulating activity of the active polysaccharides purified from A. lancea DC. rhizomes suggested that neutral galactosyl chains consisting mainly of (1\longrightarrow6)-linked Galf and Galp, and RG- II -like moiety with unique component sugars, such as 2-Me-Xyl, 2-Me-Fuc, Api, AceA, Kdo and Dha should play an important role in the potent intestinal immune modulating action of A. lancea DC. rhizomes.