Function-structure of protein-polysaccharide cpmplex constructed by protein engineering.

蛋白质工程构建的蛋白质-多糖复合物的功能结构。

基本信息

  • 批准号:
    08660160
  • 负责人:
  • 金额:
    $ 1.73万
  • 依托单位:
  • 依托单位国家:
    日本
  • 项目类别:
    Grant-in-Aid for Scientific Research (C)
  • 财政年份:
    1996
  • 资助国家:
    日本
  • 起止时间:
    1996 至 1997
  • 项目状态:
    已结题

项目摘要

Protein-polysaccharide conjugates showed the excellent functional properties such as heat stability and emulsifying properties. In order to elucidate the molecular mechanism of the improvements of functional properties, we constructed the glycosylated lysozyme using genetic modification. Complementary DNA encoding hen egg white lysozyme (HEWL) was subjected to site-directed mutagenesis to introduce two N-linked glycosylation sites (Asn^<19>-Try^<20>-Tr^<21> and Asn^<49>-Ser^<50>-Thr^<51>) into both positions 19 and 49 by substituting Arg-21 with Thr and Gly-49 with Asn, respectively. The single and double glycosylated lysozymes (G49N,R21T,R21T/G49N) were expressed in Saccharomyces cerevisiae carrying the yeast expression plasmid inserted the single and double mutant HEWL cDNAs. The mutant lysozymes were predominantly expressed a polymannosyl form with a small amout of two oligomannosyl forms. The polymannosyl lysozymes G49N and R21T were glycosylated at positions 49 and 19 with approximately 300 mannose residues, respectively, while the length of the polymannosyl chains attached to R21T/G49N was approximately 272 and 18 mannose residues. The R21T/G49N showed better emulsifying properties than two types of single polymannosyl lysozymes R21T and G49N.In a case of single polymannosyl lysozyme, G49N showed somewhat better emulsifying properties than R21T.The removal of polymannosyl chain from lysoyme with Endo- beta -N-acetylglucosaminidase (Endo-H) resulted in a dramatic decrease in the emulsifying properties of polymannosyl lysozymes. This suggests that the plysaccharide attachment is essential for the functional property.
蛋白-多糖复合物具有良好的热稳定性和乳化性。为了阐明糖基化溶菌酶功能特性改善的分子机制,我们利用基因修饰技术构建了糖基化溶菌酶。对编码鸡蛋白色溶菌酶(HEWL)的互补DNA进行定点诱变,通过分别用Thr取代Arg-21和用Asn取代Gly-49,在19和49位引入两个N-连接的糖基化位点(Asn^<19>-Try^<20>-Tr^<21>和Asn^<49>-Ser^<50>-Thr^<51>)。将单、双糖基化溶菌酶(G49 N、R21 T、R21 T/G49 N)在携带单、双糖基化HEWL cDNA的酵母表达质粒的酿酒酵母中表达。突变体溶菌酶主要以多甘露糖形式表达,少量为两种寡甘露糖形式。多聚甘露糖基溶菌酶G49 N和R21 T分别在位置49和19处被糖基化,具有约300个甘露糖残基,而连接到R21 T/G49 N的多聚甘露糖基链的长度为约272和18个甘露糖残基。结果表明,R21 T/G49 N的乳化性能优于R21 T和G49 N,其中G49 N的乳化性能略优于R21 T;内切-β-N-乙酰氨基葡萄糖苷酶(Endo-H)去除赖氨酸中的多聚甘露糖链后,多聚甘露糖溶菌酶的乳化性能显著下降。这表明多糖的附着对于功能性质是必不可少的。

项目成果

期刊论文数量(18)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
Y.Shu, S.Nakamura and A.Kato: "The role of polysaccharide-chain attachment to lysozyme in the excellent emulsifying properties of polymannosyl lysozyme. Nahrung 42,67-69 (1998)" Nahrung. 42. 67-69 (1998)
Y.Shu、S.Nakamura 和 A.Kato:“多糖链附着在溶菌酶上对聚甘露糖基溶菌酶优异乳化特性的作用。Nahrung 42,67-69 (1998)”Nahrung。
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    0
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加藤昭夫: "多糖修飾によるタンパク質の機能改変" 日本農芸化学会誌. 71. 608-611 (1997)
加藤昭夫:“通过多糖修饰对蛋白质进行功能修饰”,日本农业化学学会杂志 71. 608-611 (1997)。
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加藤 昭夫: "多糖修飾によるタンパク質の機能改変" 農化. 71. 608-611 (1997)
Akio Kato:“通过多糖修饰对蛋白质进行功能修饰”Noka。71. 608-611 (1997)
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    0
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A.Kato, S.Nakamura: "New Functional Properties of Glycosylated Lysozymes Constructed by Chemical and Genetic Modifications" ACS Symposium Series. 650. 243-256 (1996)
A.Kato、S.Nakamura:“化学和基因修饰构建的糖基化溶菌酶的新功能特性”ACS 研讨会系列。
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  • 影响因子:
    0
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  • 通讯作者:
A.Kato: "Preparation and Functional Properties of Protein-Polysaccharide Conjugates。in "Surface Activity of Proteins" ed. by S.Magdassi" Marcel Dekker Inc., 21 (1996)
A. Kato:“蛋白质-多糖缀合物的制备和功能特性。S. Magdassi 编辑的“蛋白质的表面活性””Marcel Dekker Inc.,21 (1996)
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KATO Akio其他文献

KATO Akio的其他文献

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{{ truncateString('KATO Akio', 18)}}的其他基金

Molecular Designs for Functional Food Proteins by Genetic Modification
通过基因修饰进行功能性食品蛋白质的分子设计
  • 批准号:
    14360077
  • 财政年份:
    2002
  • 资助金额:
    $ 1.73万
  • 项目类别:
    Grant-in-Aid for Scientific Research (B)
Reduction of antigenicity of allergen proteins by the attachment of polysaccjarides and induction of immune tolerance
通过附着多糖降低过敏原蛋白的抗原性并诱导免疫耐受
  • 批准号:
    13556019
  • 财政年份:
    2001
  • 资助金额:
    $ 1.73万
  • 项目类别:
    Grant-in-Aid for Scientific Research (B)
Molecular design of lysozyme for switching the antimicrobial action
用于切换抗菌作用的溶菌酶的分子设计
  • 批准号:
    12660115
  • 财政年份:
    2000
  • 资助金额:
    $ 1.73万
  • 项目类别:
    Grant-in-Aid for Scientific Research (C)
Posttranslational Modifications of Lysozyme
溶菌酶的翻译后修饰
  • 批准号:
    10460058
  • 财政年份:
    1998
  • 资助金额:
    $ 1.73万
  • 项目类别:
    Grant-in-Aid for Scientific Research (B)
ADVANCED APPLICATION OF LIME
石灰的高级应用
  • 批准号:
    07555671
  • 财政年份:
    1995
  • 资助金额:
    $ 1.73万
  • 项目类别:
    Grant-in-Aid for Scientific Research (A)
Production of Al_2TiO_5 sintered body with ultrafine microstructure.
超细微观结构Al_2TiO_5烧结体的制备
  • 批准号:
    06650968
  • 财政年份:
    1994
  • 资助金额:
    $ 1.73万
  • 项目类别:
    Grant-in-Aid for General Scientific Research (C)
Improvements of Antimicrobial Activity of Lysozyme by Protein Engineering
通过蛋白质工程改进溶菌酶的抗菌活性
  • 批准号:
    05660142
  • 财政年份:
    1993
  • 资助金额:
    $ 1.73万
  • 项目类别:
    Grant-in-Aid for General Scientific Research (C)
Structure-Functionality of Egg-White Lysozyme by Protein Engineering
通过蛋白质工程研究蛋清溶菌酶的结构-功能
  • 批准号:
    02660143
  • 财政年份:
    1990
  • 资助金额:
    $ 1.73万
  • 项目类别:
    Grant-in-Aid for General Scientific Research (C)
Synthesis of Spinel Powder by the Homogeneous Precipitation Method from Inorganic Salts and Their Sintering.
无机盐均相沉淀法合成尖晶石粉及其烧结。
  • 批准号:
    01550604
  • 财政年份:
    1989
  • 资助金额:
    $ 1.73万
  • 项目类别:
    Grant-in-Aid for General Scientific Research (C)
Preparation and Properties of Fine Particles of Noble Metals by Spray-pyrolysis Technique
喷雾热解技术制备贵金属细颗粒及其性能
  • 批准号:
    62550569
  • 财政年份:
    1987
  • 资助金额:
    $ 1.73万
  • 项目类别:
    Grant-in-Aid for General Scientific Research (C)
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