• Journal of Applied Biological Chemistry
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• 제43권4호
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• pp.246-249
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• 2000

We investigated the production of chitin oligosaccharides using food grade papain. A solution of commercial food grade papain (FGP) was dialyzed for 12 h before measuring its chitinolytic activity. The effects of enzyme concentration, reaction temperature, and pH on the endochitinase and $\beta$-N-acetylglucosaminidase activities and the thermostability of these enzymes were investigated. In adddition, the reaction products were analyzed with gel filtration on a Bio-Gel P2. The endochitinase activity was twentyfold higher than that of $\beta$-N-acetylglucosaminidase. The optimal endochitinase activity was at pH 3.0, while the maximal $\beta$-N-acetylglucosaminidase activity was at pH 6.0. The reaction product consisted mainly of the dimer of N -acetylglucosamine, with a small amount of its trimer. Under the experimental conditions, $120{\mu}g$ of chitin oligomers were obtained with 1 mg of FGP protein after an incubation of 2 h.

• 한국동물학회지
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• 제36권1호
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• pp.123-132
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• 1993

곤충의 탈피작용에서 일어나는 키틴 분해대사를 구명하기 위한 목적으로 배추흰나비 (Pieris rupae L.)에서 3종의 서N-acetylglucosaminldase(El, Ell, Elll)를 분리, 정제하였다. Polvacrylamide gel 상에서 이들 효소의 활성은 기질과 triphenvltetrazolium chloride와의 반응 결과 생기는 발색정도로 확인하였다. 각각의 분자량은 76,000, 55,000, 35,000 da, pl 값은 모두 5.8이었으며 후p$\beta$GlcNAc와 각p$\beta$GaINAc에 대해서 다같이 기질특이성을 나타내었다. 또한 El과 Elll는 탈피 시기와 일치하는 전용 말기에 최대 활성을 나타내었으며 Ell는 용화 직후에 최대 활성을 나타내었다.

• 한국동물학회지
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• 제30권2호
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• pp.167-176
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• 1987

배추흰나비의 전용기 혈림프에서 $\beta$-N-Acetylglucosaminidase를 DEAE-cellulose ion exchange 및 Sephadex G-200 column chromatography 법을 이용하여 정제하였다. 정제된 효소는 7% acrylamide gel 전기영동에서 단일 band를 나타내었으며, 당을 함유한 복합단백질이었다. 최적 pH는 5.0, 최적반응온도는 6$0^{\circ}C$이었으며, 각 온도에서 10분간 incubation한 열안정성에 있어서는 6$0^{\circ}C$까지 비교적 높은 활성을 나타내었지만 7$0^{\circ}C$ 이후에는 거의 활성을 나타내지 않았다. 또한 Hg$^2$+ 처리는 심한 효소활성의 저해 현상을 보였지만, Mn$^2$+, $Mg^2$+ 등은 활성의 증가를 나타내었으며, Km 값은 6.67$\times$10-$^3$M, pl 값은 5.8, 분자량은 4.1$\times$105 daltons 이었다.

• 한국잠사곤충학회지
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• 제41권3호
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• pp.147-153
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• 1999

Chitinolytic enzymes such as ${\beta}$-N-acetylglucosaminidase are major hydrolases involved in insect molting. We have isolated, sequenced a CDNA encoding ${\beta}$-N-acetylglucosaminidase from the silkworm, Bombyx mandarina, and compared its sequence with genes encoding chitinolytic enyzmes from other sources. The insert DNA in the clone is 3,284 nucleotides long with an open reading frame of 1,788 nucleotides that encodes a protein of 596 amino acids with a molecular weight of 68.2 kDa. There is a 3’-untranslated region composed with 1.479 nucleotides and are several potential polyadenylation signals. The predicted amino acid sequence apparently contains a leader peptide of 23 amino acids. A search of the amino acids sequence databases for sequences similarities to other ${\beta}$-N-acetylglucosaminidases or ${\beta}$-N-acetylhexosaminidases. The highest similarity matched with the enzyme from B. mori, which has a sequence identity of 95%. On the other hand, the identity between the B. mandarina enzyme and those from M. sexta and human are 70% and 24%, respectively.

• Journal of Microbiology and Biotechnology
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• 제22권2호
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• pp.190-198
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• 2012

RNA interference (RNAi) is rapidly becoming a valuable tool in biological studies, as it allows the selective and transient knockdown of protein expression. The short-interfering RNAs (siRNAs) transiently silence gene expression. By contrast, the expressed short-hairpin RNAs induce long-term, stable knockdown of their target gene. Trichoplusia ni (T. ni) cells are widely used for mammalian cell-derived glycoprotein expression using the baculovirus system. However, a suitable shRNA expression system has not been developed yet. We investigated the potency of shRNA-mediated gene expression inhibition using human and Drosophila U6 promoters in T. ni cells. Luciferase, EGFP, and ${\beta}$-N-acetylglucosaminidase (GlcNAcase) were employed as targets to investigate knockdown of specific genes in T. ni cells. Introduction of the shRNA expression vector under the control of human U6 or Drosophila U6 promoter into T. ni cells exhibited the reduced level of luciferase, EGFP, and ${\beta}$-N-acetylglucosaminidase compared with that of untransfected cells. The shRNA was expressed and processed to siRNA in our vector-transfected T. ni cells. GlcNAcase mRNA levels were down-regulated in T. ni cells transfected with shRNA vectors-targeted GlcNAcase as compared with the control vector-treated cells. It implied that our shRNA expression vectors using human and Drosophila U6 promoters were applied in T. ni cells for the specific gene knockdown.

• 한국습지학회지
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• 제14권1호
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• pp.47-57
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• 2012

하변에 침입하는 외래종 식물은 하변 토양의 생태구조 및 기능에 영향을 미칠 수 있기 때문에 관심의 대상이다. 이에 본 연구에서는 1년간 국내 하변 총 네 지점에서 유기물질 분해율을 대표할 수 있는 미생물 체외효소 활성(${\beta}$-glucosidase, N-acetylglucosaminidase, phosphatase, arylsulfatase)을 외래종 유무에 따라 측정하였다. 하변 침입종인 환삼덩굴과 가시박 생장구 및 외래종 제거구의 토양을 분석한 결과 일부 침입성 덩굴식물 실험구에서 효소활성이 높았으나 계절 및 효소별로 그 특성이 달라 어떤 경향을 밝혀낼 수는 없었다. 허나, 교란이 발생한 하변 생태계에서는 침입성 덩굴 식물이 토양의 유기물질 분해를 가속화시키는 것으로 판단된다.

• 생태와환경
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• 제35권4호통권100호
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• pp.326-330
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• 2002

습지토양을 두 가지 방법으로 멸균한 후, 미생물 효소활성도의 변화를 알아보았다. 단기 멸균의 효과는 톨루엔을 가하여 알아 보았고, 장기적인 효과는 감마선 조사를 이용하였다. 처리된 시료에서 ${\beta}$-glucosidase, ${\beta}$-xylosidase, cellobiohydrolase, phosphatase, arylsulfatase, N -acetylglucosaminidase 활성도를 methylumbelliferyl 계열의 인공기질을 사용하여 측정한 후 공시료와 비교하였다. 톨루엔을 가한 경우 효소에 따라 다른 반응을 보였다. 예를 들어, 처리에 의하여phosphatase의 활성은 증가하였으나 반대로 ${\beta}$-glucosidase와 arylsulfatasem의 활성도는 감소하였다. 감마선 조사는 모든 효소의 활성을 40-80%정도 감소시켰다. 본 연구의 전반적인 결과에 의하면, 적어도 단기적으로는 다량의 효소가 미생물 체외에서 안정적으로 존재하나, 장기적으로는de-novo 합성이 중요함을 알 수 있었다.

• BMB Reports
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• 제43권11호
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• pp.726-731
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• 2010

We report the tissue-specific distribution of chitinolytic activity in Korean ginseng root and characterize two 31-kDa chitinolytic enzymes. These two enzymes (SBF1 and SBF2) were purified 70- and 81-fold with yields of 0.75 and 1.25%, respectively, and exhibited optimal pH and temperature ranges of 5.0-5.5 and 40-$50^{\circ}C$. With [$^3H$]-chitin as a substrate, $K_m$ and $V_{max}$ values of SBF1 were 4.6 mM and 220 mmol/mg-protein/h, respectively, while those of SBF2 were 7.14 mM and 287 mmol/mg-protein/h. The purified enzymes showed markedly less activity with p-nitrophenyl-N-acetylglucosaminide and fluorescent 4-methylumbelliferyl glycosides of D-N-acetylglucosamine oligomers than with [$^3H$]-chitin. End-product inhibition of both enzymes demonstrated that both are endochitinases with different N-acetylglucosaminidase activity. Furthermore, the $NH_2$-terminal sequence of SBF1 showed a high degree of homology with other plant chitinases whereas the $NH_2$-terminal amino acid of SBF2 was blocked.

• Macromolecular Research
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• 제16권1호
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• pp.1-5
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• 2008

The enzymatic hydrolysis of chitin has been studied for almost a century, and early work established that at least two enzymes are required, a chitinase that mainly yields the disaccharide N,N'-diacetylchitobiose, or $(GlcNAc)_2$, and a "chitobiase", or ${\beta}$-N-acetylglucosaminidase, which gives the final product G1cNAc. This pathway has not been completely identified but has remained the central concept for the chitin catabolism through the $20^{th}$ century1 including in marine bacteria. However, the chitin catabolic cascade is quite complex, as described in this review. This report describes three biologically functional genes involved in the chitin catabolic cascade of Vibrios in an attempt to better understand the metabolic pathway of chitin.

• 생명과학회지
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• 제9권4호
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• pp.341-347
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• 1999

Insects use chitinolytic enzyme to digest chitin in the exoskelton during the molting process. We have isolated and sequenced a chitinase-encoding cDNA from the silkworm, Bombyx mandarina, compared its sequenced with genes encoding chitinolytic enzymes from other sources. The insert DNA in the clone is 2,675 nucleotides long with an open reading frame of 1,695 uncletides that encodes a protein of 565 amino acids with a molecuar weight of 63.4 kDa. The 3' -untranslated region of 889 nucleotides is AT-rich and contains two putative polyadenylation signals. The N-terminal sequence of the encoded protein contains numerous hydrophobic residues characteristic of a leader peptide. The amino acid alignment revealed that the endo-$\beta$-N-acetylglucosaminidase had 83% and 97% homology with M. sexta and B. mori, respectively. The deduced amino acid had two highly conserved region at the amino acid residues 97-111 and 139-148 that were related to the existing chitinase.