Heterologous expression of a deep-sea thermostable amylopullulanase and enzymatic activity analysis of the fusion protein

JIAO Yu-liang; WANG Shu-jun; LU Ming-sheng; FANG Yao-wei; LIU Shu

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JIAO Yu-liang, WANG Shu-jun, LU Ming-sheng, FANG Yao-wei, LIU Shu. Heterologous expression of a deep-sea thermostable amylopullulanase and enzymatic activity analysis of the fusion protein[J]. Haiyang Xuebao, 2011, 33(5): 133-138.

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Heterologous expression of a deep-sea thermostable amylopullulanase and enzymatic activity analysis of the fusion protein

College of Food Science and Technology, Huaihai Institute of Technology, Lianyungang 222005, China

Received Date: 2010-12-30

Abstract

Abstract

Molecular phylogenetic tree analysis on a thermostable amypopullulanase in an Archaeaon strain Thermococcus siculi HJ21 isolated from a deep-sea hydrothermal vents was performed based on experimental enzymatic analysis and amino acid squences of amylopullulanases deposited in GenBank. The N-terminal catalytic region of the amylopullulanase was heterologously expressed in E. coli through pMal-c2x expression system, resulting a fusion protein in which there is a maltose binding protein fused to the N-terminus of the N-terminal catalytic region of the amylopullulanase. Alpha-amylase and pullulanase activities of the fusion protein were experimentally analyzed. The optimal temperatures of the two activities were both at 100 ℃. The optimal pHs of amylase and pullulanase activities were at 5 and 6 respectively. The specific activities of the amylase and pullulanase activities were 6.5 and 11.5 U/mg respectively. The results showed that α-amylase activity was lower than pullulanase activity and the C-terminal region of the thermostable amylopullulanase was non-necessary for the enzymatic activities. The thermostable amylopullulanase fusion protein obtained in this study could be further used for combination with thermostable amylases in the sugar industry.
- thermostable amylopullulanase,
- heterologous expression,
- enzymatic analysis

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References(16)

References

HORVáTHOVáV, GODáNY A, TURDíK E, et al. α-Amylase from Thermococcus hydrothermalis: Re-cloning aimed at the improved expression and hydrolysis of corn starch[J]. Enzyme and Microbial Technology, 2006, 39(6): 1300-1305.

WANG S, LU Z, LU M, et al. Identification of archaeon-producing hyperthermophilic alpha-amylase and characterization of the alpha-amylase[J]. Applied Microbiololy and Biotechnology, 2008, 80(4): 605-614.

BEN-MESSAOUD E, BEN-AMMAR Y, MELLOULI L, et al. Thermostable pullulanase type I from new isolated Bacillus thermoleovorans US105: cloning, sequencing and expression of the gene in E. coli[J]. Enzyme and Microbial Technology, 2002, 31(6): 827-832.

LEVEQUE E, JANECeks MultinationalA CourierDB MultinationalA CourierRCp, HAYE B, et al. Thermophilic archaeal amylolytic enzymes[J]. Enzyme and Microbial Technology, 2000, 26(1): 3-14.

TANIGUCHI H, HONNDA Y. Amylases. Encyclopedia of Microbiology[M].Third Edition . San Diego, USA: Academic Press, 2009: 159-173.

HORVTHOV V, GODNY A,TURDK E, et al. α-amylase from Thermococcus hydrothermalis: Re-cloning aimed at the improved expression and hydrolysis of corn starch[J]. Enzyme and Microbial Technology, 2006, 39(6): 1300-1305.

ERRA-PUJADA M, CHANG-PI-HIN F, DEBEIRE P, et al. Purification and properties of the catalytic domain of the thermostable pullulanase type II from Thermococcus hydrothermalis[J]. Biotechnology Letters, 2001, 23(16): 1273-1277.

HORVATHOVA V, JANEcWP MultinationalA CourierDBpcEK cWP MultinationalA CourierRCp, STURDíK E. Amylolytic Enzymes: Molecular Aspects of Their Properties[J]. General Physiology and Biophysics, 2001, 20(1): 7-32.

ZONA R, CHANG-PI-HIN F, O'DONOHUE M J, et al. Bioinformatics of the glycoside hydrolase family 57 and identification of catalytic residues in amylopullulanase from Thermococcus hydrothermalis[J]. European Journal of Biochemistry, 2004, 271(14): 2863-2872.[ZK)]

王淑军, 吕明生, 李华钟, 等. 深海古菌Thermococcus siculi HJ21高温普鲁兰酶基因的克隆及表达[J]. 食品科学, 2010, 31(19): 309-312

MONGAY F C, MARTIN V C. Preparation d'un tampon universel de force ionique 0,3 M[J]. Talanta,1977, 24(12): 747-748.

KIM JH, SUNAKO M, ONO H. Characterization of Gene Encoding Amylopullulanase from Plant-Originated Lactic Acid Bacterium, Lactobacillus plantarum L137[J]. Journal of Bioscience and Bioengineering, 2008, 106(5): 449-459.

CANTAREL B L, COUTINHO P M, RANCUREL C, et al. The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics[J]. Nucleic Acids Research, 2009, 37(Database issue): D233-238.

[JP3]DONG G, VIEILLE C, ZEIKUS J G. Cloning, sequencing, and expression of the gene encoding amylopullulanase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme[J]. Applied and Environmental Microbiology, 1997, 63(9): 3577-3584.

LIN H Y, CHUANG H H, LIN F P. Biochemical characterization of engineered amylopullulanase from Thermoanaerobacter ethanolicus 39E-implicating the non-necessity of its 100 C-terminal amino acid residues[J]. Extremophiles, 2008, 12(5): 641-650.

KANG S, VIEILLE C, ZEIKUS J G. Identification of Pyrococcus furiosus amylopullulanase catalytic residues[J]. Applied Microbiology and Biotechnology, 2005, 66(4): 408-413.

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