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

Abstract

The functional and structural significance 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, fluorescence and CD spectrometric properties, substrate-binding and hydrolysis abilities, thermostability, and thermodenaturation kinetics. Kinetic analyses revealed that the overall catalytic efficiency, k (cat)/K (m), was slightly decreased for the truncated enzymes toward the soluble starch or pullulan substrate. Changes to the substrate affinity, K (m), and turnover rate, k (cat), varied in different directions for both types of substrates between TetApuM955 and TetApuR855. TetApuR855 exhibited a higher thermostability than TetApuM955, and retained similar substrate-binding ability and hydrolyzing efficiency 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 TetApuM955 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 fibronectin 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.

Degradation of Granular Starch by the Bacterium Microbacterium aurum Strain B8.A Involves a Modular α-Amylase Enzyme System with FNIII and CBM25 Domains

“…Little is known about the function(s) of FNIII domains in amylases. Published reports about FNIII domains in carbohydrate-acting enzymes do not show a clear common function, with results pointing at possible roles in substrate binding or enzymatic activity (38)(39)(40)(41)(42)(43). So far, there are no reports of FNIII domains in prokaryotes with a function in protein-protein interactions.…”

The evolutionary origin and possible functional roles of FNIII domains in two Microbacterium aurum B8.A granular starch degrading enzymes, and in other carbohydrate acting enzymes

“…In these studies, removal of only the CBM usually affects activity on insoluble substrates [15,16,[34][35][36][37][38][39][40], while the additional removal of the FNIII domains showed no further effects. In a study of the chitinase A1 from Bacillus circulans WL-12a, the single CBM present was re-attached after removal of one or two intermediate FNIII domains, allowing a direct test for the FNIII function [14].…”

Effect of C-terminal truncation on enzyme properties of recombinant amylopullulanase from Thermoanaerobacter pseudoethanolicus

“…Both enzymes produced the same hydrolysis product profile of maltose and maltotriose from these substrates, respectively (Fig. 3, Lin et al 2008). The optimum pH of 6.0 for both amylase and pullulanase activity and the optimum temperature of 75°C were determined for both enzymes (data not shown).…”

“…Previously, truncation mutagenesis analyses of TetApu have revealed that the enzyme active region was within the TetApu R855 molecule Lin and Leu 2002;Lin et al 2008). In this study, further C-terminal truncated molecules such as TetApuQ818, TetApuG809 and TetApuK791, were constructed by PCR mutagenesis techniques and were expressed by the heterologous E.coli-pET expression system.…”

“…The thermostable and enzymatically active region of the apu gene (2.9 kb, TetApuM955) was previously subcloned by PCR from Thermoanaerobacter pseudoethanolicus genomic DNA (Lin and Leu 2002;Lin et al 2008). The C-terminal truncated mutants of TetApuM955 were constructed based on the C-terminal amino acid sequence of FnIII1 (N819-A908) and AamyC motif (E727-Q818).…”