Biologia, Bratislava, 57/Suppl. 11: 229-238, 2002.

ISSN 0006-3088 (Biologia).

 

Full Paper

Studies on the C-terminal region of barley a-amylase 1 with emphasis on raw starch-binding.

 

Brian K. Tibbot, Dominic W. S. Wong* & George H. Robertson

USDA-ARS, Western Regional Research Center, 800 Buchanan Street, Albany, CA 94710 USA; tel.: ++ 1 510 5595877, fax: ++ 1 510 5595777, e-mail: bkt@pw.usda.gov

* corresponding author

Received: October 4, 2001 / Accepted: April 18, 2002

 

Abstract

The mature form of barley seed, low-pI a-amylase (AMY1) is a polypeptide 414 amino acids in length and possesses a catalytic site and a non-catalytic raw starch-binding (SB) site. In our previous work, a truncated AMY1 cDNA encoding the C-terminal region (134 aa) comprising domain C and part of domain A, but excluding the SB site Trp278-Trp279, was expressed in Escherichia coli. The recombinant protein rSB134 bound to cyclohepta-amylose (CHA)-Sepharose and was eluted with free CHA. This work indicated that a second non-catalytic SB site is located in the C-terminal region and binds in the absence of the first SB site and N-terminal region. A shorter truncation rSB62 comprising domain C did not bind, suggesting that domain C alone cannot bind starch and that a portion of domain A is required. In the current study, analysis of a human serum albumin (HSA)-SB fusion rSB68 expressed in the yeast Pichia pastoris further suggests that domain C alone cannot bind starch. We also investigated the role of the conserved amino acids VWEK near the C-terminal end of AMY1. Truncated and site-directed mutant forms of rAMY1 expressed in P. pastoris had greatly reduced activity, suggesting that the conserved region is required. This region likely plays a role in structural stability.

 

Key words: amylase, barley, carbohydrate binding, cyclodextrin, Pichia pastoris, raw starch, recombinant protein.