KSBB Journal

The Korean Society for Biotechnology and Bioengineering (한국생물공학회)
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Action Patterns of Chitinase and Separations of Chitooligosaccharides Produced by Chitinolytic Hydrolysis

키티나제에 의한 키토올리고당의 생성활성 규명과 올리고당의 당별 분리 생산

  • Kim, Kwang (Division of Metallurgical and Chemical Engineering College of Engineering, Dong-A University)
  • 김광 (동아대학교 재료금속화학공학부 화학공학과)
  • Published : 2002.02.01
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Abstract

N-acetyl-D-glucosamine oligosaccharides [(GlcNAc)n] whose degree of polymer-ization is from one to ten (n=1-10) were fractionated by column chromatography on CM-Sephadex. Electro dialysis from a partially deacetylated chitosan hydrolysate prepared crudely with the N-acetyl-D-glucosaminidase(chitinase) and exo-N, N'-diacetylchito-biohydrolase(chitobiase) of Serratia marcescens QM B1466. Reducing sugar compositions and sequences of the N-acetyl-glucosamine oligosaccharides were identified by N-acetylation, randomly cleavage with chitinase and ego-splitting with chitobiase. N-acetyl-glucosamine heterochitooligosaccharides with glucosamine oligosaccharides, (GlcN)n at the reducing end residues together with $(GlcN)_1\sim(GlcN)_4$ were detected. Separation was accomplished by prefractionation with election by 0 to 1.0 M NaCl gradient solution. $(GlcNAc)_1 =4.25%,\; (GlcNAc)_2=4.49%,; (GlcNAc)_3=11.1%,\; (GlcNAc)_4=2.5%,$$ $(GlcNAc)_{5}$=0.64%, $(GlcNAc)_{6}$=2.12% and $(GlcNAc)_{7}$=1.21%, respectively, were crystallized after electrodialysis and lyophilization Each N-acetyl-D-glucosamine oligosaccharides content were detected by HPLC.

중합도 n$\leq$10을 갖는 N-아세틸-D-글루코사민(GlcNAc)을 미생물 Serratia marcesce-ns QM B14f6의 최소배지 회분식 발효를 이용하여 키틴 및 키토산을 분해하는 효소 키티나제(1.5 unit/mL) [키토바이아제(3.48 unit/mL)가 포함됨]를 생성시킨다. 효소 반응에 의한 키토올리고당의 생성 활성을 규명하기 위하여 부분적으로 탈아세틸화된 키토산을 효소적 가수분해 반응시킴으로써 생성된 N-아세틸-D-글루코사민과 D-글루코사민의 키토올리고당의 생성 패턴을 확인 조사한다. 이 혼합 올리고머로부터 CM-Sephadex의 컬럼 크로마토그래피에 의하여 당별로 분획시켜 추출한 헤테로 키토 올리고당들을 각각 N-아세틸화하고 이 최종 생성물을 전기투석 장치로서 정제하여 키토올리고 1-7당을 제조하였다. 부분적으로 탈아세틸화 키토산(환원당으로서 2697 mg/mL)을 효소반응에 의해 생성시킨 키토올리고당은 1당으로서 GlcNAc=4.25%, 2당 $(GlcNAc)_2$=4.49%, 3당 $(GlcNAc)_3$=11.1%, 4당 $(GlcNAc)_4$=2.5%, 5당 $(GlcNAc)_{5}$ =0.64%, 6당$(GlcNAc)_{6}$=2.12%, 7당 $(GlcNAc)_{7}$=1.21%가 각각 제조되었다.

Keywords

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