• 한국식품과학회지
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• 제35권6호
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• pp.1188-1192
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• 2003

새우나 게 껍질로부터 chitin을 유일한 탄소원으로 하는 집적배양에 의하여 chitinase 활성이 우수한 균주를 2종 분리하였다. 분리균주는 형태학적, 그람염색, 16s rDNA 서열분석을 통하여 Arthrobacter nicotianae로 동정되어, 각각 Arthrobacter nicotianae CH4와 Arthrobacter nicotianae CH13으로 명명하였다. 두 종의 분리균주로부터의 chitinase는 모두 pH $3.0{\sim}9.0$에서 90% 이상의 효소활성을 유지하여 높은 pH안정성을 나타내었다. 온도의 영향은 $20{\sim}60^{\circ}C$ 구간에서 최적 효소 활성의 $70{\sim}90%$를 유지하여 열안정성도 뛰어났다. A. nicotianae CH4와 A. nicotianae CH13이 분비하는 chitinase 조효소를 0.1% colloidal chitin 기질에 반응시켜서 반응산물로 생산되는 chitin 올리고당을 HPLC를 사용하여 분석하였다. A. nicotianae CH4 조효소에 의한 효소반응산물로는 올리고머인 $(GlcNAc)_4$이, A. nicotianae CH13는 단량체인 $(GlcNAc)_1$이 전체 반응산물의 각각 98% 이상 생성되었다.

• 한국미생물·생명공학회지
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• 제47권1호
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• pp.78-86
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• 2019

대사공학을 이용하여 N-아세틸글루코사민(GlcNAc)을 생산하는 재조합 Corynebacterium glutamicum을 개발하였다. 먼저 GlcNAc를 생산하는 기반균주를 제작하기 위하여, N-acetylglucosamine-6-phosphate deacetylase와 glucosamine-6-phosphate deaminase를 암호화하는 nagAB와 N-acetylmannosamine-6-phosphate epimerase를 암호화하는 nanE를 C. glutamicum ATCC 13032에서 순차적으로 결손하여, 최종적으로 KG208 균주를 제작하였다. 또한, glucosamine-6-phosphate synthase를 암호화하는 C. glutamicum 유래의 glmS와 glucosamine-6-phosphate N-acetyltransferase를 암호화하는 Saccharomyces cerevisiae 유래의 gna1을 각각 여러 발현벡터에 클로닝하였다. 여러 발현 조합의 플라스미드들 중에서 pCXI40-glmS와 pCEI40-gna1을 함유한 제조합균주 KG440은 삼각플라스크 발효에서 1.77 g/l의 GlcNAc와 0.63 g/l의 글루코사민을 생산하였다.

• Applied Microscopy
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• 제31권1호
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• pp.49-57
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• 2001

섬유아세포 표면의 미세구조적 특성과 세포표면에 존재하는 당 단백질 말단 GlcNAc(N-acetylglucosamine)와 NeuNAc(N-acetylneuraminic acid)는 섬유세포의 이동과 세포의 인식에 중요한 역할을 하는 것으로 알려졌다. 따라서 섬유세포의 미세구조적 특성을 전자현미경을 사용하여 관찰하였으며, 당 단백질 말단 GlcNAc와 NeuNAc의 분포를 확인하기 위하여 WGA 황금입자 복합체를 반응시켜 전자현미경으로 관찰하였다. 그 결과 배양섬유세포의 표면에 미세구조적 특성은 세포의 분화 정도와 세포의 부위에 따라 다양한 형태를 형성하며, 세포의 배양시간에 따라 정도의 차이는 있으나 일반적인 미세융모의 분포와 세포질 돌기의 분화는 세포의 주위 환경에 따라 다양한 형태로 분화하는 특성이 있는 것으로 확인되었다. 섬유세포의 세포표면에 분포하는 당 단백질 말단의 일종으로 섬유세포의 이동과 세포인식에 관여하는 lectin WGA 수용체 인 sialic acid (GlcNAc; N-acetylgalactosamine, NeuNAc; N-acetyl neuraminic acid)는 세포질의 조면소포체에서 생성되어 액포상태로 이동되어 섬유세포의 외로 분비되고 분비된 sialic acid는 세포의 표면과 돌기의 표면에 당 단백질 말단으로 분화하여 섬유세포의 이동과 세포인식 등의 섬유세포 기능에 관여하는 것으로 규명되었다.

• BMB Reports
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• 제35권3호
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• pp.313-319
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• 2002

N-Acetyl-$\beta$-D-hexosaminidase ($\beta$-HexNAc'ase) (EC 3.2.1.52) was purified from rice seeds (Oryza sative L. var. Dongjin) using ammonium sulfate (80%) precipitation, Sephadex G-150, CM-Sephadex, and DEAE-Sephadex chromatography, sequentially. The activities were separated into 7 fractions($F_1-F_7$) by CM-Sephadex chromatography. Among them, F6 was further purified to homogeneity with a 13.0% yield and 123.3 purification-fold. The molecular mass was estimated to be about 52 kDa on SDS-PAGE and 37.4 kDa on Sephacryl S-300 gel filtration. The enzyme catalyzed the hydrolysis of both p-nitrophenyl-N-acetyl-$\beta$-D-hexosaminide (pNP-GlcNAc) and p-nitrophenyl-N-acetyl-$\beta$-D-hexosaminide (pNP-GalNAc) as substrates, which are typical properties of $\beta$-HexNAc'ase. The ratio of the pNP-GlcNAc'ase activity to the pNP-GalNAc'ase activity was 4.0. However, it could not hydrolyze chitin, chitosan, pNP-$\beta$-glucopyranoside, or pNP-$\beta$-glucopyranoside. The enzyme showed $K_m$, $V_{max}$ and $K_{cat}$ for pNP-GlcNAc of 1.65 mM, $79.49\;mM\;min^{-1}$, and $4.79{\times}10^6\;min^{-1}$, respectively. The comparison of kinetic values for pNP-GlcNAc and pNP-GalNAc revealed that the two enzyme activities are associated with a single binding site. The purified enzyme exhibited optimum pH and temperature for pNP-GlcNAc of 5.0 and $50^{\circ}C$, respectively. The enzyme activity for pNP-GlcNAc was stable at pH 5.0-5.5 and $20-40^{\circ}C$. The enzyme activity was completely inhibited at a concentration of 0.1 mM $HgCl_$ and $AgNO_3$, suggesting that the intact thiol group is essential for activity. Chloramine T completely inhibited the activity, indicating the possible involvement of methionines in the mechanism of the enzyme.

• Molecules and Cells
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• 제38권5호
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• pp.402-408
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• 2015

Protein O-GlcNAcylation, dictated by cellular UDP-N-acetylglucosamine (UDP-GlcNAc) levels, plays a crucial role in posttranslational modifications. The enzyme GlcNAc kinase (NAGK, E.C. 2.7.1.59) catalyzes the formation of GlcNAc-6-phosphate, which is a major substrate for the biosynthesis of UDP-GlcNAc. Recent studies have revealed the expression of NAGK in different types of cells especially in neuronal dendrites. Here, by immunocytochemistry (ICC) and immunonucleochemistry (INC) of cultured rat hippocampal neurons, HEK293T and GT1-7 cells, we have showed that NAGK immuno-reactive punctae being present in the nucleoplasm colocalized with small nuclear ribonucleoprotein-associated protein N (snRNPN) and p54NRB, which are speckle and paraspeckle markers, respectively. Furthermore, NAGK IR cluster was also found to be colocalized with GTF2H5 (general transcription factor IIH, polypeptide 5) immuno reactive punctae. In addition, relative localization to the ring of nuclear lamin matrix and to GlcNAc, which is highly enriched in nuclear pore complexes, showed that NAGK surrounds the nucleus at the cytoplasmic face of the nuclear outer membrane. By in situ proximity ligation assay (PLA) we confirmed the colocalization of NAGK with snRNPN in the nucleus and in dendrites, while we also verified the interactions of NAGK with p54NRB, and with GTF2H5 in the nucleus. These associations between NAGK with speckle, paraspeckle and general transcription factor suggest its regulatory roles in gene expression.

• Parasites, Hosts and Diseases
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• 제32권2호
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• pp.101-110
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• 1994

심장사상충 자충이 합성한 당단백의 분지(분지) 역할을 하는 N-linked high mannose 타입 올리고당의 구조에 대한 조사를 수행하였다. 사상충 자충을 방사선 표식 2-[$^3H$] mmannose를 함유한 배지에서 24시간 배양하였다. 자충으로부터 분리 정제한 당단백을 pronate로 소화시킨 다음. concanavalin A-Sepharose 크로마토그라피하여 분획하였다. 37%의 mannose가 자충 대사에 이용되었으며 pectin chromatography를 사용하여 high mannose 타입의 올리고당을 회수하였다. 이 올리고당을 $endo-{\beta}-N-acetylglucosaminidase$ H 효소로 소화시킨 후 HPLC를 사용하여 highmannose 타입 올리고당의 구성을 분석하였다. 심장사상충 자충이 합성한 high mannose 타입 올리고당의 형태는 $Man_5GIcNAc_2,{\;}Man_6GIcNAc_2,{\;}Man_7GIcNAc_2과{\;}Man_8GlcNAc_2$로 확인되었다. 이와 같이 심장사상충 자충이 체내 합성한 high mannose 타입 올리고당이 일반 척추동물이 체내 합성한 high mannose 타입 올리고당과 구조적으로 일치하였다.

• BMB Reports
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• 제47권10호
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• pp.593-598
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• 2014

RNA polymerase II carboxyl-terminal domain (RNAPII CTD) phosphatases are responsible for the dephosphorylation of the C-terminal domain of the small subunit of RNAPII in eukaryotes. Recently, we demonstrated the identification of several interacting partners with human small CTD phosphatase1 (hSCP1) and the substrate specificity to delineate an appearance of the dephosphorylation catalyzed by SCP1. In this study, using the established cells for inducibly expressing hSCP1 proteins, we monitored the modification of ${\beta}$-O-linked N-acetylglucosamine (O-GlcNAc). O-GlcNAcylation is one of the most common post-translational modifications (PTMs). To gain insight into the PTM of hSCP1, we used the Western blot, immunoprecipitation, succinylayed wheat germ agglutinin-precipitation, liquid chromatography-mass spectrometry analyses, and site-directed mutagenesis and identified the $Ser^{41}$ residue of hSCP1 as the O-GlcNAc modification site. These results suggest that hSCP1 may be an O-GlcNAcylated protein in vivo, and its N-terminus may function a possible role in the PTM, providing a scaffold for binding the protein(s).

• Journal of Microbiology and Biotechnology
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• 제10권3호
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• pp.307-311
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• 2000

Abstract Extracellular chitinase was purified from the culture liquid of the marine bacterium, Bacillus sp. LJ-25 , and its enzymatic properties were examined. The purified chitinase exhibited a single band on SDS-PAGE and the molecular weight was estimated to be approximately 50 kDa. The optimum pH and temperature for the enzymatic activity were 7.0 and $35^{\circ}C$, respectively. The activity of the chitinase was strongly inhibited by $Zn^{2+}$ and slightly inhibited by $Ba^{2+},{\;}Co^{2+},{\;}Mn^{2+},{\;}and{\;}Cu^{2+}$. The purified chitinase did not hydrolyze $p-nitrophenolN-acetyl-{\bata}-D-glucosaminide{\;}(GlcNAc)_2$ and Micrococcus lysodeikticus cells, which are known to be the substrates for exo-type chitinase. Among the hydrolyzates of colloidal chitin, $(GlcNAc)_2$ was in the highest concentration with small amounts of GlcNAc and $(GlcNAc)_3$..

• Journal of mucopolysaccharidosis and rare diseases
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• 제2권1호
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• pp.13-16
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• 2016

Mucolipidosis (ML) II/III are autosomal recessive diseases caused by deficiency of post-translational modification of lysosomal enzymes. The mannose-6-phosphate (M6P) residue in lysosomal enzymes synthesized by N-acetylglucosamine 1-phosphotransferase (GlcNAc-phosphotransferase) serves as recognition marker for trafficking in lysosomes. GlcNAc-phosphotransferase is encoded by GNPTAB and GNPTG. Mutations in GNPTAB cause severe ML II alpha/beta and the attenuated ML III alpha/beta. Whereas mutations in GNPTG cause the ML III gamma, the attenuated type of ML III variant. For the diagnostic approaches, increased urinary oligosaccharides excretion could be a screening test in clinically suspicious patients. To confirm the diagnosis, instead of measuring the activity of GlcNAc phosphotransferase, measuring the enzymatic activities of different lysosomal hydrolases are useful for diagnosis. The activities of several lysosomal hydrolases are decreased in fibroblasts but increased in serum of the patients. In addition, the sequence analysis of causative gene is warranted. Therefore, the confirmatory diagnosis requires a combination of clinical evaluation, biochemical and molecular genetic testing. ML II/III show complex disease manifestations with lysosomal storage as the prime cellular defect that initiates consequential organic dysfunctions. As there are no specific therapy for ML to date, understanding the molecular pathogenesis can contribute to develop new therapeutic approaches ultimately.

• Biotechnology and Bioprocess Engineering:BBE
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• 제5권3호
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• pp.215-218
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• 2000

Sialytrisaccharides based on $\beta$-galactosyldisaccharides were synthesized using $\beta$-galactosidase and trans-sialidase in one pot. Using $\beta$-galactosidase from Bacillus Ciculans and trans-sialidase from Trypanosoma cruzi simulaneously, 6mM sialyltrisaccharides composed of about 95% NeuAc$\alpha$(2,3)Gal$\beta$(1,4)GlcNAc and 5% NeuAc$\alpha$(2,3)Gal$\beta$(1,6)GlcNAc were produced from a reaction mixture containing 25mM o-nitropheny1-$\beta$-D-galsctolneuraminic acid. One beauty of this reaction was that a secondary hydrolysis of the disaccharide intermediate occurring between the activated galactopyranoside and N-acetylgucosamine was prevented. Using $\beta$-galactosidase from Escherichia cloi and the same trans-sialidase, 15mM sialyltrisaccharides composed of about 90% NeuAc$\alpha$(2,3)Gal$\beta$(1,6)GlcNac and 10% NeuAc$\alpha$(2,3)Gal$\beta$(1,4)GlcNAc were produced from a reaction misture containing 400nM galactose, 800nM N-acetylglucosylation rection between galactose and N-actylgucosamine was diminant since the disaccharide intermediate mainly resulted sreulted in the silylated product.