Pectobacterium carotovorum subsp. carotovorum LY34에서 Lsoamylase 유전자 클로닝 및 효소 활성의 필수 잔기 확인 Cloning of Isoamylase Gene of Pectobacterium carotovorum subsp. carotovorum LY34 and Identification of Essential Residues of Enzyme원문보기
연부균인 Pectobacterium carotovorum subsp. carotovorum LY34로부터 이소아밀라제 유전자 (glgX)를 클로닝한 후 대장균 숙주에서 발현시켰다. 이 효소는 ${\alpha}-1$,6-글루코시드 결합을 가수분해하였으나 ${\alpha}-1$,4-글루코시드 결합은 가수분해 하지 못하였다. 유전자는 658개의 아미노산을 암호화하는 1,977개의 DNA 염기서열로 이루어져 있었고 이 유전자에 의해 암호화되는 아미노산 서열을 다른 아밀라제 효소들과 비교한 결과 이소아밀라제 유전자와 유사하였으며 4개의 보존 지역을 확인하였다. SDS-PAGE에 의해 확인된 단백질의 크기는 약 74 kDa 이었다. 효소 활성은 pH 7.0, $40^{\circ}C$에서 가장 높은 활성을 나타났으며 $Ca^{2+}$ 첨가로 활성이 증가되었다. 이 효소의 보존되어 있는 아미노산 중에 글루탐산 370번, 아스파르트산 335번 및 442번 잔기를 알라닌으로 치환시킨 결과 활성이 약해졌다. 이 결과로부터 이들 잔기들이 효소활성에 중요한 역할을 하는 것으로 추정된다.
연부균인 Pectobacterium carotovorum subsp. carotovorum LY34로부터 이소아밀라제 유전자 (glgX)를 클로닝한 후 대장균 숙주에서 발현시켰다. 이 효소는 ${\alpha}-1$,6-글루코시드 결합을 가수분해하였으나 ${\alpha}-1$,4-글루코시드 결합은 가수분해 하지 못하였다. 유전자는 658개의 아미노산을 암호화하는 1,977개의 DNA 염기서열로 이루어져 있었고 이 유전자에 의해 암호화되는 아미노산 서열을 다른 아밀라제 효소들과 비교한 결과 이소아밀라제 유전자와 유사하였으며 4개의 보존 지역을 확인하였다. SDS-PAGE에 의해 확인된 단백질의 크기는 약 74 kDa 이었다. 효소 활성은 pH 7.0, $40^{\circ}C$에서 가장 높은 활성을 나타났으며 $Ca^{2+}$ 첨가로 활성이 증가되었다. 이 효소의 보존되어 있는 아미노산 중에 글루탐산 370번, 아스파르트산 335번 및 442번 잔기를 알라닌으로 치환시킨 결과 활성이 약해졌다. 이 결과로부터 이들 잔기들이 효소활성에 중요한 역할을 하는 것으로 추정된다.
The gene encoding for isoamylase of the Pectobacterium carotovorum subsp. carotovorum (Pcc) LY34 was cloned and expressed into Escherichia coli $DH5{\alpha}$. Isoamylase catalyzes the hydrolysis of ${\alpha}-1,6-glycosidic$ linkages specifically in amylopectin, glycogen, and de...
The gene encoding for isoamylase of the Pectobacterium carotovorum subsp. carotovorum (Pcc) LY34 was cloned and expressed into Escherichia coli $DH5{\alpha}$. Isoamylase catalyzes the hydrolysis of ${\alpha}-1,6-glycosidic$ linkages specifically in amylopectin, glycogen, and derived oligosaccharides, while the enzyme did not hydrolyze ${\alpha}-1,4-glycosidic$ linkages of amylose. The isoamylase gene (glgX) had an open reading frame of 1,977 bp encoding 658 amino acid residues with a calculated molecular weight of 74,188 Da. The molecular weight of the enzyme was also estimated to be 74 kDa by activity staining of a SDS-PA gel. The mature GlgX had a calculated pI of 4.91. Isoamylase from Pcc LY34 had 70% amino acid identity with isoamylase from Pectobacterium chrysanthemi and contained the four regions conserved among all amylolytic enzymes. The isoamylase was optimally active at pH 7.0 and $40^{\circ}C$. GlgX was $Ca^{2+}-dependent$. The changes of Asp-335, Glu-370, and Asp-442 into Ala, respectively, using site-directed mutagenesis techniques showed that three residues are essential to isolamyalse (GlgX) activity. The sequences around those residues were highly conserved in isoamylase of different origins and GlgX of the glg operon in glycongen biosynthesis.
The gene encoding for isoamylase of the Pectobacterium carotovorum subsp. carotovorum (Pcc) LY34 was cloned and expressed into Escherichia coli $DH5{\alpha}$. Isoamylase catalyzes the hydrolysis of ${\alpha}-1,6-glycosidic$ linkages specifically in amylopectin, glycogen, and derived oligosaccharides, while the enzyme did not hydrolyze ${\alpha}-1,4-glycosidic$ linkages of amylose. The isoamylase gene (glgX) had an open reading frame of 1,977 bp encoding 658 amino acid residues with a calculated molecular weight of 74,188 Da. The molecular weight of the enzyme was also estimated to be 74 kDa by activity staining of a SDS-PA gel. The mature GlgX had a calculated pI of 4.91. Isoamylase from Pcc LY34 had 70% amino acid identity with isoamylase from Pectobacterium chrysanthemi and contained the four regions conserved among all amylolytic enzymes. The isoamylase was optimally active at pH 7.0 and $40^{\circ}C$. GlgX was $Ca^{2+}-dependent$. The changes of Asp-335, Glu-370, and Asp-442 into Ala, respectively, using site-directed mutagenesis techniques showed that three residues are essential to isolamyalse (GlgX) activity. The sequences around those residues were highly conserved in isoamylase of different origins and GlgX of the glg operon in glycongen biosynthesis.
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문제 정의
As our aim of this study is to understand the relationship between the function and structure of isoamylase, we first sequenced the entire isolated DNA fragment and deduced the primary sequence of isoamylase of Pcc LY34. In this article, we describe the cloning and sequencing of the isoamylase gene from Pcc LY34 and the biochemical characterization of the recombinant enzyme expressed in E. coli DH5a and the catalytic residues of isoamylase clarified by site-directed mutagenesis.
제안 방법
expressed. As our aim of this study is to understand the relationship between the function and structure of isoamylase, we first sequenced the entire isolated DNA fragment and deduced the primary sequence of isoamylase of Pcc LY34. In this article, we describe the cloning and sequencing of the isoamylase gene from Pcc LY34 and the biochemical characterization of the recombinant enzyme expressed in E.
이론/모형
Louis, MO, USA). Nucleotide sequences were determined by the dideoxy-chain termination method using the PRISM Ready Reaction Dye terminator/primer cycle sequencing kit (Perkin-Elmer Corp., Norwalk, CT, USA). Tlic samples were analyzed with an automated DNA sequencer (Model 3100 Applied Biosystems, Foster City, CA, USA).
0), 200 μ 1 of the enzyme solution was added, and the samples were incubated at different temperatures for 20 to 60 min. The amounts of reducing sugars released were determined by the dinitrosalicylic acid method [3, 7]. Blank samples were used to correct the non-enzymatic release of reducing sugars.
The fractions with enzyme activity eluted as a single protein peak and the purity of the enzyme were assessed by SDS-PAGE. The protein concentration was determined by the method of Bradford [5].
성능/효과
coli [24], GlgX of Pasteurella multocida [17], and GlgX of Haemophilus influenzae. GlgX of Pcc LY34 shared 70% amino acid identity with AmyX of P. chrysanthemi, 65% with GlgX of Yersinia pestis, 63% with GlgX of E. coli, 61% with GlgX of S. typhimurium and 51% with GlgX of H. influenzae (Table 1). Also, GlgX of Pcc LY34 has partial homology with isoamylase, pullulanase, a-dextran endo-lz6-a-glucosidasez alkaline amylopullulanase, a-amylase, and branching enzymes (data not shown).
Mutation of one Glu residues (E370) and two Asp residues (D335 and D442) to Ala, resulted in the activity determination of the mutant enzymes clearly indicated that D335, E370, and D442 were essential for Pcc LY34 isoamylase (Table 4), and those residues were well conserved in all the sequences listed in Fig. 3. Recently, the three-dimensional structure of isoamylase from Pseudomonas has been solved by Katsuya et al.
signal peptide. The amino acid sequences of GlgX are very similar to that of AmyX of P. chrysanthemi, with 70%; identity to that of GlgX of Y. pestis, with 65%. Isoamylase belongs to the a-amylase family.
참고문헌 (35)
Abe, J. L, C. Ushijima and S. Hizukuri, 1999. Expression of the isoamylase of Flavobacterium ordoraium KU in Escherichia coli and identification of essential residues of the by site-directed mutagenesis. Appl. Environ. Microbiol. 65, 4163-4170
Beguin, P. 1990. Molecular biology of cellulose degradation, Annu. Rev. Microbiol. 44, 219-248
Bernfeld, P. 1955. Amylases a and $\beta$ . Methods Enzymol. 1, 149-155
Bonafonte, M. A., C. Solano, B. Sesma, M. Alvarez, L. Montuenga, D. Garcia-Ros and C. Gamazo. 2000. The relationship between glycogen synthesis, biofilm formation and virulence in Salmonella enteritidis. FEMS Microbiol. Lett. 191, 31-36
Bradford, M. M. 1976. A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254
Brooker, J. D. and J. M. McCarthy. 1997. Gene knockout of the intracellular amylase gene by homologous recombination in Streptococcus bovis. Curr. Microbiol. 35, 133-138
Hong, S. Y., K. M. Cho, Y. H. Kim, S. J. Hong, S. J. Cho, Y. U. Cho, H. Kim and H. D. Yun. 2006. Cloning and identification of essential residues for thermostable $\beta$ -glucosidase gene from Thermotoga maritima MSB8. J. Life Sci. 16, 1148-1157
Jeanningros, R., N. Creuzet-Sigal, C. Frixon and J. Cattaneo. 1976. Purification and properties of a debranching enzyme from Escherichia coli. Biochim. Biophys. Acta 438, 186-199
Katsuya, Y., Y. Metaki, M. Kubota and Y. Matsusuura. 1998. Three-dimensional structure of Pseudomonas isoamylase at 2.2 ${\AA}$ resolution. J. Mol. Biol. 281, 885-897
Keen, N. T., C. Boyd and B. Henrissat. 1996. Cloning and characterization of a xylanase gene from corn strains of Erwinia chrysanthemi. Mol. Plant Microbe Interact 9, 651-657
Krohn, B. M., G. F. Barry and G. M. Kishore. 1997. An isoamylase with neutral pH optimum from a Flavobacterium species: Cloning, characterization and expression of the iam gene. Mol. Gen. Genet. 254, 469-478
Lim, W. J., S. R. Park, S. J. Cho, M. K. Kim, S. K. Ryu, S. Y. Hong, W. T. Seo, H. Kim and H. D. Yun. 2001. Cloning and characterization of an intracellular isoamylase gene from Pectooacierium chrysanthemi PY35. Biochem. Biophys. Res. Commun. 287, 348-54
Lim, W. J., S. R. Park, C. L. An, J. Y. Lee, S. Y. Hong, E. C. Shin, E. J. Kim, J. O. Kim, H. Kim and H. D. Yun. 2003. Cloning and characterization of a thermostable intracellular $\alpha$ -amylase gene from the hyperthermophilic bacterium Thermotoga maritima MSB8. Res. Microbiol. 154, 681-687
Lim, W. J., S. K. Ryu, S. R. Park, M. K. Kim, C. L. An, S. Y. Hong, E. C. Shin, J. Y. Lee, Y. P. Lim and H. D. Yun, 2005. Cloning of celC, third cellulase gene, from Pectobacierium carotovorum subsp. carotovorum LY34 and its comparison to those of Pectooacierium sp. J. Microbiol. Biotechnol. 15, 302-309
May, B. J., Q. Zhang, L. L. Li, M. L. Paustian, T. S. Whittam and V. Kapur. 2001. Complete genomic sequence of Pasteurella multocida, Pm70. Proc. Natl. Acad. Sci. 98, 3460-3465
Nakajima, R., T. Imanaka and S. Aiba. 1986. Comparison of amino acid sequences of eleven different $\alpha$ -amylases. Appl. Microbiol. Biotechnol. 23, 355-360
Park, S. R., S. J. Cho and H. D. Yun. 2000. Cloning and sequencing of pel gene responsible for CMCase activity from Erwinia chrysanthemi PY35. Biosci. Biotechnol. Biochem. 64, 820-830
Park, Y. W., S. T. Lim and H. D. Yun. 1998. Cloning and characterization of a CMCase gene, celB, of Erwinia carotovra subsp. carotovora LY34 and its comparison to celA, Mol. Cells 8, 280-285
Park, Y. W., S. T. Lim, S. J. Cho and H. D. Yun, 1997. Characterization of Erwinia caroiovora subsp. carotovora LY34 endo-1,4- $\beta$ -glucanase genes and rapid identification of their gene products. Biochem. Biophysic. Res. Commun. 241, 636-641
Raha, M., I. Kawagishi, V. Muller, M. Kihara and R. M. Macnab. 1992. Escherichia coli produces a cytoplasmic alpha-amylase, AmyA. J. Bacteriol. 174, 6644-6652
Robert, Y. I. and C. R. Meyer. 2000. Cloning and sequencing of glycogen metabolism genes from Rhodobacier sphaeroides 2.4.1. expression and characterization of recomibinant ADP-glucose pyrophosphorylase. Arch. Biochem. Biophys. 376, 47-58
Romeo, T., A. Kumar and J. Preiss. 1988. Analysis of the Escherichia coli glycogen gene cluster suggests that catabolic enzymes are encoded among the biosynthetic genes. Gene 70, 363-376
Rouanet, C., K. Nemura, S. Tsuyumu and W. Nasser. 1999. Regulation of pelD and pelE, encoding major alkaline pectate lyases in Erwinia chrysanthemi: Involvement of the main transcriptional factors. J. Bacteriol. 181, 5948-5957
Sambrook, J. and D. W. Russell. 2001. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor
Satoh, E., T. Uchimura, T. Kudo and K. Komagata. 1997. Purification, characterization, and nucleotide sequence of an intracellular maltotriose-producing alpha-amylase from Streptococcus bovis 148. Appl. Environ. Microbiol. 63, 4941-4944
Simpsen, H. D. and F. Barras. 1999. Functional analysis of the carbohydrate-binding domains of Erwinia chrysanthemi Cel5 (Endoglucanase Z) and an Escherichia coli putative chitinase. J. Bacteriol. 181, 4011-4616
Svenssion, B. 1994. Protein engineering in the $\alpha$ -amylase family: catalytic mechanism, substrate specificity, and stability. Plant. Mol. Biol. 25, 141-157
Takase, K. 1992. Interaction of catalytic-site mutants of Bacillus subtilis $\alpha$ -amylase with substrates and acarbose. Biocheim. Biophys. Acta 1122, 278-282
Takata, H., T. Kuriki, S. Okada, Y. Takesada, M. Lizuka, N. Minamiura and T. Imanaka. 1992. Action of neopullulanase: neopullulanase catalyzes both hydrolysis and transglycosylation at $\alpha$ -(1 $\rightarrow$ 4)-and $\alpha$ -(1 $\rightarrow$ 6)-glucosidic linkages. J. Biol. Chem. 267, 18447-18452
Urlaub, Hand G. Woeber. 1975. Identification of isoamylase, a glycogen-debranching enzyme, from Bacillus amyloliquefaciens. FEBS Lett. 57, 1-4
Yang, H, M. Y. Liu and T. Romeo. 1996. Coordinate genetic regulation of glycogen catabolism and biosynthesis in Escherichia coli via the CsrA gene product. J. Bacteriol. 178, 1012-1017
Zhou, S., F. C. Davis and L. O. Ingram. 2001. Gene integration and expression and extracellular secretion of Erwinia chrysanthemi endoglucanase CelY (celY) and CelZ (celZ) in ethanologenic Klebsiella oxytoca P2. Appl. Environ. Miorobiol. 67, 6-14
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