Characterization of Barley ${\alpha}$-Amylase Chimeric Enzymes Expressed in Pichia pastoris

Pichia pastoris에서 발현된 보리 알파아밀라제 Chimera 효소들의 특성

  • Kim, Tae-Jip (Department of Food Science and Technology, Chungbuk National University) ;
  • Yuk, Jeong-Bin (Department of Food Science and Technology, Chungbuk National University) ;
  • Choi, Seung-Ho (Department of Food Science and Technology, Chungbuk National University) ;
  • Jang, Myoung-Uoon (Department of Food Science and Technology, Chungbuk National University) ;
  • Svensson, Birte (Department of Systems Biology, Technical University of Denmark)
  • Received : 2010.02.24
  • Accepted : 2010.03.10
  • Published : 2010.03.31

Abstract

Two different ${\alpha}$-amylase isozymes (AMY1 and AMY2) found in barley malt share up to 80% of amino acid sequence identity with each other, but their enzymatic properties differ remarkably. AMY1 shows the highest activity at low concentration of calcium ion, while AMY2 is highly active at high calcium concentration. Meanwhile, BASI (Barley ${\alpha}$-Amylase/Subtilisin Inhibitor) protein specifically inhibits only AMY2. In the present study, three separate regions in AMY genes (I, II, and III) were assigned on the basis of restriction enzyme sites and four kinds of chimeric amylases have been obtained by swapping a part of regions with each other. Each chimera gene was successfully over-expressed in Pichia pastoris. From the results of enzymatic characterization, both AMY211 and AMY122 showed the mixed or intermediate type of calcium-dependent activity between AMY1 and 2. Meanwhile, only AMY221 chimera could be significantly inhibited by BASI protein. As a result, it can be proposed that some amino acid residues in the region I and II, except region III, of barley ${\alpha}$-amylases play very important roles in calcium-dependency and interaction with BASI.

보리 맥아로부터 발견된 서로 다른 알파아밀라제 동질효소(AMY1, AMY2)는 80%에 달하는 높은 아미노산 서열의 상동성을 보이지만, 두 효소의 특성은 서로 달라 AMY1 효소는 낮은 농도의 칼슘 조건에서 최대 활성을 보이는 반면, AMY2 효소는 높은 칼슘이온 농도에서 높은 활성을 나타낸다. 또한 BASI (Barley ${\alpha}$-Amylase/Subtilisin Inhibitor) 단백질은 AMY2 효소만을 특이적으로 저해한다. 따라서 본 연구에서는 AMY1과 AMY2 효소의 유전자를 I, II, III의 세 부위로 나눈 후, 제한효소 처리에 의해 일부 부위를 상호 치환한 4종의 chimera 효소를 추가로 제조하고, Pichia pastoris 균주에서 대량 발현하였다. 이들 효소의 특성을 비교한 결과, 제 I 부위만이 상호치환된 AMY211 및 AMY122 효소의 경우, AMY1과 AMY2의 중간적 칼슘 의존성을 나타내었으며, BASI에 의한 저해효과는 AMY2의 제 I, II 부위를 포함하는 AMY221 효소에서만 관찰되었다. 따라서 보리 아밀라제의 제 I 부위 및 제 II 부위에 존재하는 아미노산 잔기들이 칼슘 의존성 및 BASI와의 결합에 중요한 역할을 담당하는 반면 제 III 부위는 이들 효소의 활성 차이에 영향을 미치지 않음을 확인하였다.

Keywords

References

  1. Bak-Jensen, K.S., G. André, T.E. Gottschalk, G. Paës, 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. https://doi.org/10.1074/jbc.M312825200
  2. 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.
  3. Davies, G. and B. Henrissat. 1995. Structure and mechanism of glycosyl hydrolases. Structure 3, 853-859. https://doi.org/10.1016/S0969-2126(01)00220-9
  4. 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. https://doi.org/10.1006/prep.1996.0093
  5. 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. https://doi.org/10.1016/0378-1119(93)90415-Y
  6. Kadziola, A., J. Abe, B. Svensson, and R. Haser. 1994. Crystal and molecular structure of barley $\alpha$-amylase. J. Mol. Biol. 239, 104-121. https://doi.org/10.1006/jmbi.1994.1354
  7. Kadziola, A., M. Søgaard, 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. https://doi.org/10.1006/jmbi.1998.1683
  8. 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. https://doi.org/10.1007/BF00017982
  9. 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. https://doi.org/10.1074/jbc.272.36.22456
  10. Matsuura, Y., M. Kusuniki, W. Harada, and M. Kakudo. 1984. Structure and possible catalytic residues of Taka-amylase A. J. Biochem. 95, 697-702. https://doi.org/10.1093/oxfordjournals.jbchem.a134659
  11. 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$$\rightarrow$$\alpha$ loop 2 of the catalytic ($\beta$/ $\alpha$)8-barrel and is critical for activity and substrate specificity. Eur. J. Biochem. 269, 5377-5390. https://doi.org/10.1046/j.1432-1033.2002.03185.x
  12. 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. https://doi.org/10.1021/bi020508+
  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. https://doi.org/10.1016/S0969-2126(03)00151-5
  14. 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 isozymespecific properties. Eur. J. Biochem. 221, 277-284. https://doi.org/10.1111/j.1432-1033.1994.tb18739.x
  15. Rogers, J.C. and C. Milliman. 1983. Isolation and sequence analysis of a barley $\alpha$--amylase cDNA clone. J. Biol. Chem. 258, 8169-8174.
  16. Sogaard, 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. https://doi.org/10.1016/0378-1119(90)90384-4
  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 1.9A resolution. Structure 6, 649-659. https://doi.org/10.1016/S0969-2126(98)00066-5
  18. 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.