Comparative study of the structure/function relationship of wild‐type and structurally modified maltopentaose‐producing amylase

Abstract

Amylase A-180, which is secreted by a new alkaliphilic organism, isolate 163-26, consists of a single type of polypeptide chain of 186.5 kDa and hydrolyses starch by exo-attack releasing malto-pentaose as preferential product. The structure/function relationship of this unusual starch-degrading enzyme was analysed by introducing 3' deletions into the structural gene. It was found that removal of up to a 110-kDa portion from the C-terminus leaving 563 N-terminal amino acids still led to the formation of a fully active enzyme. The part of the structural gene coding for these 563 N-terminal amino acids was fused with the signal peptide-encoding segment of the cyclodextrin glucanotransferase gene from Klebsiella oxytoca and was cloned into an expression vector. The resulting truncated A-I 80 derivative, A-180/21, was efficiently transported through the cytoplasmic membrane and released into the medium by an Escherichia coli strain which 'leaks' periplasmatic components. A-1 80/21 was purified and its catalytic properties, i.e. specific activity and product specificity, proved to be identical to those of the wild-type enzyme; however, in contrast to the wild-type enzyme, it was unable to bind to raw starch and it displayed an altered temperature and pH dependence of activity.In a previous communication we have reported the genetic and the biochemical properties of a starch-degrading enzyme, amylase A-1 80, which is secreted by an alkaliphilic organism, isolate 126-63, and which preferentially produces linear maltopentaose 111. Amylase A-180 has a size of 186.5 kDa which is unusually large for this type of enzyme. Analysis of the amino acid sequence revealed that it is made up of an N-terminal domain of about 70 kDa which exhibits the sequence characteristics of a-amylases (EC 3.2.1.1) and of a C-terminal 110-kDa segment which contains sequence repeats also found in several bacterial P-amylases (EC 3.2.1.2) [l].The size and the peculiar subdomain structure of A-180 are unique for a starch-degrading enzyme. To analyse whether any of these properties are involved in the product specificity, i.e. malto-pentaose production, and in the exo-hydrolytic attack on starch, which is unusual for an cr-amylase, or whether they are correlated with the catalysis in an alkaline environment, we have undertaken a more detailed structure/function analysis of this enzyme and present our findings here. MATERIALS AND METHODS Materials Determination of amylase activityAmylase activity was assayed by measurement of the amount of total products formed from Noredux 150B (30% mass/vol.) or from pure malto-oligosaccharides (1 YO mass/ vol.) as described previously [I]. Determination of the pH dependence of the activity of amylase A-180/21 was performed in one of the following buffers: 20 mM sodium acetate, 20 mM Pipes/NaOH, 20 mM Tris/HCl or 20 mM sodium borate/ NaOH. All buffers were supplemented with 5 mM CaCl,.