Biochemical characterization of engineered amylopullulanase from Thermoanaerobacter ethanolicus 39E-implicating the non-necessity of its 100 C-terminal amino acid residues

Extremophiles (2008) 12:641650 DOI 10.1007/s00792-008-0168-4

ORIGINAL PAPER

Biochemical characterization of engineered amylopullulanase from Thermoanaerobacter ethanolicus 39E-implicatingthe non-necessity of its 100 C-terminal amino acid residues

Hsu-Yang Lin Hsu-Han Chuang Fu-Pang Lin

Received: 19 December 2007 / Accepted: 22 April 2008 / Published online: 24 May 2008 Springer 2008

Abstract The functional and structural signicance of the C-terminal region of Thermoanaerobacter ethanolicus 39E amylopullulanase (TetApu) was explored using C-terminal truncation mutagenesis. Comparative studies between the engineered full-length (TetApuM955) and its truncated mutant (TetApuR855) included initial rate kinetics, uorescence and CD spectrometric properties, substrate-binding and hydrolysis abilities, thermostability, and thermodenaturation kinetics. Kinetic analyses revealed that the overall catalytic efciency, kcat/Km, was slightly decreased

for the truncated enzymes toward the soluble starch or pullulan substrate. Changes to the substrate afnity, Km,

and turnover rate, kcat, varied in different directions for both types of substrates between TetApuM955 and TetA-puR855. TetApuR855 exhibited a higher thermostability than TetApuM955, and retained similar substrate-binding ability and hydrolyzing efciency against the raw starch substrate as TetApuM955 did. Fluorescence spectroscopy indicated that TetApuR855 retained an active folding conformation similar to TetApuM955. A CD-melting unfolding study was able to distinguish between TetA-puM955 and TetApuR855 by the higher apparent transition temperature in TetApuR855. These results indicate that up to 100 amino acid residues, including most of the C-terminal bronectin typeIII (FnIII) motif of TetApuM955,

could be further removed without causing a seriously aberrant change in structure and a dramatic decrease in soluble starch and pullulan hydrolysis.

Keywords Thermoanaerobacter ethanolicus 39E

Amylopullulanase C-terminal truncation

Thermostability Circular dichroism

Introduction

Glycosyl hydrolases (GHases) constitute one large group of diversied saccharides metabolizing enzymes existed on Earth. A classication of GHases based on amino-acid sequence similarities was originally utilized from the analysis of several hundred sequences and was grouped into 35 families (Henrissat 1991). Nowadays, the CAZy web-system was available for individual enzymes to form their own families according to their sequence-structural similarities and catalytic machineries. Almost 100 of different glycosyl hydrolase families were currently classied (Coutinho and Henrissat 1999). Amylopullulanase (Apu, pullulanase type II, E.C.3.2.1.41) cleaves both a-1,4- and a-1,6-glycosidic linkages in starch, pullulan, amylopectin, and related oligosaccharides. Maltotriose and small oligosaccharides are the major products from pullulan or starch, respectively, in the Apu catalyzed reactions. A large number of amylopullulanases have...