Secreted expression of Azotobacter chroococcum alginate lyase gene(algL) in Pichia pastoris and primary analysis of enzymic properties

YUE Ming; DING Hong-biao; QIAO Yu

Article Contents

Article Navigation > Haiyang Xuebao > 2007 > 29(5): 154-160

YUE Ming, DING Hong-biao, QIAO Yu. Secreted expression of Azotobacter chroococcum alginate lyase gene(algL) in Pichia pastoris and primary analysis of enzymic properties[J]. Haiyang Xuebao, 2007, 29(5): 154-160.

Citation:

YUE Ming, DING Hong-biao, QIAO Yu. Secreted expression of Azotobacter chroococcum alginate lyase gene(algL) in Pichia pastoris and primary analysis of enzymic properties[J]. Haiyang Xuebao, 2007, 29(5): 154-160.

Citation:

PDF( 685 KB)

Secreted expression of Azotobacter chroococcum alginate lyase gene(algL) in Pichia pastoris and primary analysis of enzymic properties

Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China

Received Date: 2006-03-10
Rev Recd Date: 2006-07-16

Abstract

Abstract

An alginate lyase gene of Azotobacter chroococcum, called algL, has been cloned with PCR technique.The sequence encoding the mature peptide of alginate lyase of Azotobacter chroococcum was added with EcoRI and NotI.Then the gene was inserted into the Pichia pastoris vector pPIC9K and introduced into the host Pichia pastoris GS115 by PEG method.After screen, the recombinant Pichia pastoris strain was obtained and induced in 25mL methylot rophic culture medium.Expression of the algL gene in P.pastor is cells resulted in the expression of alginate lyase activity (1 400 U/cm³) and the appearance of a new protein of 43 kDa detected on SDS-PAGE.To characterize the recom binantenzyme, it was found that the optimum temperature is 40℃ at pH 8.5 and that activity of alginate lyase decreased rapidly when analyzed above 60℃.The activity of recom binant AlgL decreased by over 50% in the presence of 10 mmol/L Cu²⁺ or Fe²⁺, furthermore, 10mmol/L Co²⁺, Mn²⁺, Ca²⁺ or 1mmol/L Fe²⁺ decreased it by 20% respectively.Other cations including K⁺, Na⁺, Zn²⁺ did not affect significantly the enzymatic activity.
- alginate lyase,
- Pichia pastoris,
- algao ligosaccharides,
- expression induced

FullText(HTML)

References(27)

References

黄知清,严兴洪.海藻研究开发的发展概述[J].海洋技术,2002,21(2):22-25.

邓富文.酶解技术用于马尾藻提取海藻酸工艺的研究[J].广西化工,1999,28(2):11-13.

海带营养丰富功能多[J].保鲜与加工,2002,3(3):39.

秦国奎,张玉忠,陈秀兰,等.海藻酸盐裂解酶研究进展[J].中国生物工程杂志,2004,24(2):26-33.

ANA P,ALBERTO P,ANYONIO P.Cloning and Expression of the algL Gene,Encoding the Azotobacter chroococcum Alginate Lyase:Purification and Characterization of the Enzyme[J].J Bacteriol,1999,181(5):1409-1414.

HENRY I N,PATRICIA C S.Alginic acid degradation by eliminases from abalone hepatopancreas[J].J Bio Chem,1967,242 (5):845-851.

STEVEN R M,NEAL L S.Alginate Synthesis in Pseudomonasaeruginosa:the Role of AlgL (Alginate Lyase) and AlgX[J].J Bacteriol,1996,178(3):625-632.

HAN Feng,GONG Qian-hong,SONG Kai,et al.Sequence Analysis and Expression of Gene AlyVI Encoding Alginate Lyase from Marine Bacterium Vibrio sp.QY101[J].DNA Sequence,2004:1-8.

TOMOO S,MIWA O,YOSHIO E.Novel alginate lyases from marine bacterium Alteromonas sp.strain H-4[J].Carbohydr Res,1997,304:69-76.

ERTESVAG H,ERLIEN F,SKJAK-BRAEK G,et al.Biochemical properties and substrate specificities of a recombinantly produced Azotobacter vinelandii alginate lyase[J].J Bacteriol,1998,180(15):3 779-3 784.

SHIMIZU E,OJIMA T,NISHITA K.cDNA cloning of an alginate lyase from abalone,Haliotis discus hannai[J].Carbohydr Res,2003,338(24):2 841-2 852.

MADGWICK J,HAUG A,LARSEN B.Alginate lyase in the brown alga Laminaria digitata (Huds.) Lamour[J].Acta Chem Scand,1973,27(2):711-712.

刘万顺,李濒.海洋无脊椎动物消化酶的研究Ⅰ:紫贻贝、日本鲟、滨螺消化酶的初步分析和应用[J].山东海洋学院学报,1988,18(1):54-57.

萨姆布鲁克 J,拉塞尔 D W(黄培堂,等译).分子克隆实验指南上、下册[M].北京:科学出版社,2002.1949.

袁兆慧,韩丽君,林伟,等.褐藻酸降解酶的制备及其性质研究[J].海洋科学,2005,29(2):78-80.

马歇克 D R,门永J T,布格斯R R,等(朱厚础,等译).蛋白质纯化与鉴定实验指南[M].北京,科学出版社,1999.279.

刘岩,江晓路,管华诗.褐藻胶裂解酶研究进展[J].中国水产科学,2001,7(4):99-103.

HONG F,MEINANDER N Q,JONSSON L J.Fermentation strategies for improved heterologous expression of laccase in Pichia pastoris[J].Biotechnol Bioeng,2002,79 (4):438-449.

SREEKRISHNA K,BRANKAMP R G.Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophicy yeast Pichia pasroris[J].Gene,1997,190 (1):55-62.

NAKADA H I,SWEENY P C.Alginic acid degradation by eliminases from abalone hepatopancreas[J].Biol Chem,1967,242:845-851.

favorov vv,vozhova ei,denisenko va,et al.A study of reaction catalysed by alginate lyase Ⅵ from the sea molluse,Littorina sp.[J].Biochim Biophys Acta,1979,569:259-266.

SHIRAIWA Y,ABE K,SASAKI S F,et al.Alginate lyase activities in the extracts from several brown algae[J].Bot Mar,1975,18:97-104.

BROWN B J,PRESTON J F Ⅲ.L-Guluronan-specific alginate lyase from a marine bacterium associated with Sargassum[J].Carbohydr Res,1991,211:91-102.

孙忠保,阎宏,马永军.寡糖在动物生产中的应用[J].宁夏农学院学报,2004,25(4):80-84.

柴栋,王睿.藻酸盐裂解酶对粘液型铜绿假单胞菌生物被膜的影响[J].国外医药抗生素分册,2001,22(4):172-175.

EFTEKHAR F,SPEERT D P.Alginase Treatment of Mucoid Pseudomonas aeruginosa Enhances Phagocytosis by Human MonocyteDerived Macrophages[J].Infect Immun,1988,56(11):2 788-2 793.

TOMOO S,YOSHIO E,TAKAHISA K.Application of an alginate lyase from Alteromonas sp.for isolation of protoplasts from a brown algae L ami naris japonica[J].Nippon Suisan Gakkaishi,1993,59 (4):705-709.

Relative Articles

Supplements(0)

Cited By

Proportional views