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한국미생물·생명공학회지 = Korean journal of microbiology and biotechnology v.38 no.2, 2010년, pp.151 - 157  
본 등재정보는 저널의 등재정보를 참고하여 보여주는 베타서비스로 정확한 논문의 등재여부는 등재기관에 확인하시기 바랍니다.

Staggered Extension Process를 통해 제조한 보리 알파아밀라제 Chimera 효소의 특성
Enzymatic Properties of Barley $\alpha$-Amylase Chimeric Enzymes Produced by Staggered Extension Process

김태집    (충북대학교 식품공학과   ); 최승호    (충북대학교 식품공학과   ); 장명운    (충북대학교 식품공학과   ); 박정미 ;    (충북대학교 식품공학과 ;  );
  • 초록

    보리 맥아로부터 발견된 서로 다른 알파아밀라제 동질효소(AMY1, AMY2)는 80%에 달하는 높은 아미노산 서열의 상동성을 보이지만, 효소적 특성은 서로 매우 다르다. 따라서 본 연구에서는 staggered extension process(StEP) 기술을 이용하여 AMY1과 AMY2 유전자가 조합된 5종의 chimera 효소를 제조하고, 각각의 특성을 비교하여 총 8개 부위(I~VIII)의 영향을 확인하였다. 결과적으로, AMY-D2, D8, E12 chimera 효소의 경우, AMY1과 AMY2의 중간적 칼슘의존성을 보였으며, BASI(barley $\alpha$ -amylase/subtilisin inhibitor) 단백질에 의한 저해효과는 AMY-E10 효소에서만 관찰되었다. 한편 AMY-C6의 경우, AMY1과 유사한 효소특성을 보였으며, AMY-E10은 AMY2 형태의 칼슘의존성을 나타내었다. 따라서 보리 아밀라제의 제 II, III, IV부위가 BASI와의 상호작용에 중요한 역할을 담당하며, 제 III, V, VI, VII부위는 칼슘의존성에 부분적인 영향을 미치는 것으로 판단하였다.


    Barley malt produces two different $\alpha$ -amylase isozymes (AMY1 and AMY2), which share up to 80% of amino acid sequence identity with each other. However, their enzymatic properties differ remarkably. In this study, five chimeric enzymes between AMY1 and 2 were constructed by staggered extension process (StEP) technique, and their enzymatic properties were characterized. According to the results, chimeric AMY-D2, D8, and E12 showed the mixed or intermediate types of calcium-dependent activity between AMY1 and 2. Meanwhile, only AMY-E10 chimera could be significantly inhibited by barley $\alpha$ -amylase/subtilisin inhibitor (BASI) protein. Chimera AMY-C6 showed the same calcium-dependency as AMY1, while AMY-E10 was closely similar to AMY2. As a result, it can be proposed that some amino acid residues in the region II, III, and IV of barley $\alpha$ -amylases can play very important roles in the interaction with BASI, and those in III, V, VI, and VII may partly affect on the calcium-dependent activity.


  • 주제어

    Barley $\alpha$-amylase isozymes .   staggered extension process(StEP) .   chimeric enzymes .   calcium-dependent activity .   Pichia pastoris.  

  • 참고문헌 (24)

    1. Bush, D. S., L. Sticher, R. Van Huystee, D. wagner, and R. L. Jones. 1989. The calcium requirement for stability and enzymatic activity of two isoforms of barley aleuron ${\alpha}-amylase$. J. Biol. Chem. 264: 19392-19398. 
    2. Kadziola, A., M. Sogaard, B. Svensson, and R. Haser. 1998. Molecular structure of an ${\alpha}-amylase-inhibitor$ complex: implications for starch binding and catalysis. J. Mol. Biol. 278: 205-217. 
    3. Knox, C. A. P., B. Sonthayanon, G. R. Chandra, and S. Muthukrishnan. 1987. Structure and organization of two divergent ${\alpha}-amylase$ genes from barley. Plant Mol. Biol. 9: 3-17. 
    4. Matsui, I. and B. Svensson. 1997. Improved activity and modulated action pattern obtained by random mutagenesis at the fourth ${\beta}-{\alpha}$ loop involved in substrate binding to the catalytic $({\beta}/{\alpha})_8-barrel$ domain of barley ${\alpha}-amylase$ 1. J. Biol. Chem. 272: 22456-22463. 
    5. Rodenburg, K. W., N. Juge, X. J. Guo, M. Sogaard, J. C. Chaix, and B. Svensson. 1994. Domain B protruding at the third ${\beta}$ strand of the ${\alpha}/{\beta}$ barrel in barley ${\alpha}-amylase$ confers distinct isozyme-specific properties. Eur. J. Biochem. 221: 277-284. 
    6. Rogers, J. C. and C. Milliman. 1983. Isolation and sequence analysis of a barley ${\alpha}-amylase$ cDNA clone. J. Biol. Chem. 258: 8169-8174. 
    7. Svendsen, I., J. Hejgaard, and J. Mundy. 1986. Complete amino acid sequence of the ${\alpha}-amylase$ inhibitor of endogenous ${\alpha}-amylase$ and subtilisin. FEBS Lett. 167: 210-214. 
    8. Yuk, J. B., S. H. Choi, T. H. Lee, M. U. Jang, J. M. Park, A. R. Yi, B. Svensson, and T. J. Kim. 2008. Effect of calcium ion concentration on starch hydrolysis of barley ${\alpha}-amylase$ isozymes. J. Microbiol. Biotechnol. 18: 730-734.     
    9. Bak-Jensen, K. S., G. Andre, T. E. Gottschalk, G. Paes, V. Tran, and B. Svensson. 2004. Tyrosine 105 and threonine 212 at outermost substrate binding subsites -6 and +4 control substrate specificity, oligosaccharide cleavage patterns, and multiple binding modes of barley ${\alpha}-amylase$ 1. J. Biol. Chem. 279: 10093-10102. 
    10. Bozonnet, S., T. J. Kim, B. C. Bonsager, B. Kramhoft, P. K. Nielsen, K. S. Bak-Jensen, and B. Svensson. 2003. Engineering of barley ${\alpha}-amylase$. Biocatal. Biotransfor. 21: 209-214. 
    11. Juge, N., J. S. Andersen, D. Tull, P. Roepstorff, and B. Svensson. 1996. Overexpression, purification, and characterization of recombinant barley ${\alpha}-amylases$ 1 and 2 secreted by the methylotrophic yeast Pichia pastoris. Protein Expr. Purif. 8: 204-214. 
    12. Rodenburg, K. W., F. Vallee, N. Juge, N. Aghajari, X. J. Guo, R. Haser, and B. Svensson. 2000. Specific inhibition of barley ${\alpha}-amylase$ 2 by barley ${\alpha}-amylase/subtilisin$ inhibitor depends on charge interactions and can be conferred isozyme 1 by mutation. Eur. J. Biochem. 267: 1019-1029. 
    13. Robert, X., R. Haser, T. E. Gottschalk, F. Ratajczak, H. Driguez, B. Svensson, and N. Aghajari. 2003. The structure of barley ${\alpha}-amylase$ isozyme 1 reveals a novel role of domain C in substrate recognition and binding: a pair of sugar tongs. Structure 11: 973-984. 
    14. Stemmer, W. P. 1994. Rapid evolution of a protein in vitro by DNA shuffling. Nature 370: 389-391. 
    15. Mori, H., K. S. Bak-Jensen, and B. Svensson. 2002. Barley ${\alpha}-amylase$ Met53 situated at the high-affinity subsite -2 belongs to a substrate binding motif in the ${\beta}{\to}{\alpha}$ loop 2 of the catalytic $({\beta}/{\alpha})_8-barrel$ and is critical for activity and substrate specificity. Eur. J. Biochem. 269: 5377-5390. 
    16. Soaard, M. and B. Svensson. 1990. Expression of cDNAs encoding barley ${\alpha}-amylase$ 1 and 2 in yeast and characterization of the secreted proteins. Gene 94: 173-179. 
    17. Vallee, F., A. Kadziola, Y. Bourne, M. Juy, K. W. Rodenburg, R. Haser, and B. Svensson. 1998. Barley ${\alpha}-amylase$ bound to its endogenous protein inhibitor BASI: Crystal structure of the complex at $1.9{\AA}$ resolution. Structure 6: 649-659. 
    18. Kadziola, A., J. Abe, B. Svensson, and R. Haser. 1994. Crystal and molecular structure of barley ${\alpha}-amylase$. J. Mol. Biol. 239: 104-121. 
    19. Juge, N., M. Sogaard, J. C. Chaix, M. F. Martin-Eauclaire, B. Svensson, G. Marchis-Mouren, and X. J. Guo. 1993. Comparison of barley malt ${\alpha}-amylase$ isozyme 1 and 2: Construction of cDNA hybrids by in vivo recombination, characterization and expression in yeast. Gene 130: 159-166. 
    20. Nielsen, P. K., B. C. Bonsager, C. R. Berland, B. W. Sigurskjold, and B. Svensson. 2003. Kinetics and energetics of the binding between ${\alpha}-amylase/subtilisin$ inhibitor and barley ${\alpha}-amylase$ 2 analyzed by surface plasmon resonance and isothermal titration calorimetry. Biochemistry 42: 1478-1487. 
    21. Davies, G. and B. Henrissat. 1995. Structure and mechanism of glycosyl hydrolases. Structure 3: 853-859. 
    22. Zhao, H., L. Giver, Z. Shao, J. A. Affholter, and F. H. Arnold. 1998. Molecular evolution by staggered extension process (StEP) in vitro recombination. Nat. Biotechnol. 16: 258-261. 
    23. Gibbs, M. D., K. M. H. Nevalainen, and P. L. Bergquist. 2001. Degenerate oligonucleotide gene shuffling (DOGS): a method for enhancing the frequency of recombination with family shuffling. Gene 271: 13-20. 
    24. Bonsager, B. C., M. Prætorius-Ibba, P. K. Nielsen, and B. Svensson. 2003. Purification and characterization of the beta-trefoil fold protein barley alpha-amylase/subtilisin inhibitor overexpressed in Escherichia coli. Protein Expr. Purif. 30: 185-193. 

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