Enhancement of the alcoholytic activity of alpha-amylase AmyA from Thermotoga maritima MSB8 (DSM 3109) by site-directed mutagenesis

Abstract

AmyA, an alpha-amylase from the hyperthermophilic bacterium Thermotoga maritima, is able to hydrolyze internal alpha-1,4-glycosidic bonds in various alpha-glucans at 85 degrees C as the optimal temperature. Like other glycoside hydrolases, AmyA also catalyzes transglycosylation reactions, particularly when oligosaccharides are used as substrates. It was found that when methanol or butanol was used as the nucleophile instead of water, AmyA was able to catalyze alcoholysis reactions. This capability has been evaluated in the past for some alpha-amylases, with the finding that only the saccharifying fungal amylases from Aspergillus niger and from Aspergillus oryzae present measurable alcoholysis activity (R. I. Santamaria, G. Del Rio, G. Saab, M. E. Rodriguez, X. Soberon, and A. Lopez, FEBS Lett. 452:346-350, 1999). In the present work, we found that AmyA generates larger quantities of alkyl glycosides than any amylase reported so far. In order to increase the alcoholytic activity observed in AmyA, several residues were identified and mutated based on previous analogous positions in amylases, defining the polarity and geometry of the active site. Replacement of residue His222 by glutamine generated an increase in the alkyl glucoside yield as a consequence of a higher alcoholysis/hydrolysis ratio. The same change in specificity was observed for the mutants H222E and H222D, but instability of these mutants toward alcohols decreased the yield of alkyl glucoside.

FIG. 1.

(a) TLC product profile of the hydrolysis reaction of a 30-mg/ml starch solution with 10 U/ml of the wild-type and mutant α-amylases from T. maritima after 24 h of reaction at 85°C. G1, glucose; G2, maltose; G3, maltotriose; G4, maltotetraose; G5, maltopentaose; G6, maltohexaose; G7, maltoheptaose. Lane 1, malto-oligosaccharides; lane 2, wild type; mutants, lanes 3, H222Q; 4, H222E; 5, H222D; 6, W177V; 7, Y178V; 8, F179V; and 9, V259W. (b) Quantification of G1, G2, and G3 for the different variants.

FIG. 2.

(a) Alcoholysis reaction products obtained from 6% starch-20% methanol reaction medium with 20 U/ml of the respective enzymes after 48 h of incubation at 85°C. Lane 1, standards: MG, methyl glucoside; G1, glucose. Lanes 2 to 9, methanolysis reaction with the wild type and H222Q, H222D, H222E, W177V, Y178V, F179V, and V259W mutants, respectively; lanes 10 to 17, subsequent treatment of methanolysis reaction mixtures with A. niger glucoamylases of the wild type and H222Q, H222D, H222E, W177V, Y178V, F179V, and V259W mutants, respectively. (b) Yields of methyl glucoside after 48 h of incubation at 85°C and their subsequent treatment with A. niger glucoamylase of the wild type and variants at position 222. (c) Alcoholysis/hydrolysis ratios for the wild type and variants at position 222. The error bars indicate standard deviations.

FIG. 3.

(A) Homology model obtained for AmyA in which the three characteristic amylase domains are depicted in different colors. The catalytic domain A is shown in gray; domain B and domain C are shown in green and blue, respectively. (B) The acarbose inhibitor (orange) is located between domains A and B and is surrounded by various residues located within 4 Å, among them the catalytic residue Glu258 (red). Possible residues involved in substrate binding are indicated in stick format; some of the amino acids subjected to site-directed mutagenesis were His222 and Phe179, shown in blue, and Val259, shown in yellow.

FIG. 4.

Multiple structural alignment around the four characteristic regions observed in members of glycoside hydrolase family 13. The catalytic residues conserved in all the sequences are marked with asterisks. Aromatic residues involved in transglycosylation activity and the residues that structurally interact with them are highlighted in gray. Two of the residues mutagenized are shown in boldface and underlined. From the top: T. maritima, T. maritima α-amylase (GenBank accession number CAA72194) (37); A. oryzae, A. oryzae α-amylase (PDB accession number 2TAA) (41); A. niger, A. niger α-amylase (PDB accession number 2AAA) (6); H. sapiens sal, Homo sapiens salivary α-amylase (PDB accession number 1JXK) (49); H. sapiens pan, H. sapiens pancreatic α-amylase (PDB accession number 1HNY) (53); Pig panc, Sus scrofa pancreatic α-amylase (PDB accession number 1HX0) (48); Tenebrio, Tenebrio molitor α-amylase (PDB accession number 1JAE) (58); Alteromonas, Pseudoalteromonas haloplanktis α-amylase (PDB accession number 1G94) (1, 2); Barley, Hordeun vulgare α-amylase (PDB accession number 1AMY) (27); B. lichen, B. licheniformis α-amylase (PDB accession number 1VJS) (22); B. amylo, Bacillus amyloliquefaciens chimera α-amylase (PDB accession number 1E43) (8); B. stearo, B. stearothermophilus α-amylase (PDB accession number 1HVX) (60); B. sub, Bacillus subtilis 2633 α-amylase (PDB accession number 1BAG) (17), B. circ1, Bacillus circulans 251 cyclodextrin glycosyltransferase (CGTase) (PDB accession number 1CDG) (35); B. circ2, B. circulans 8 CGTase (PDB accession number 1CGT) (31); B. specie, Bacillus sp. strain 1011 CGTase (PDB accession number 1D7F) (23); B. stearo, B. stearothermophilus CGTase (PDB accession number 1CYG); Termo sulfu, Thermoanaerobacter thermosulfurogenes CGTase (PDB accession number 1A46) (68); Thermus sp., Thermus sp. maltogenic amylase (PDB accession number 1SMA) (29); B. lichen, B. licheniformis maltogenic amylase (GenBank accession number CAA47612) (28); B. stearo, B. stearothermopilus maltogenic amylase (PDB accession number 1QHO) (13).

FIG. 5.

(a) Alcoholysis reaction products obtained from 6% starch-8% butanol saturated with buffer (50 mM Tris, 150 mM NaCl, 2 mM CaCl₂ buffer, pH 7) with 20 U/ml of the respective enzymes after 48 h of incubation at 85°C. Lane 1, standards: BG (butyl glucoside), G1, glucose. Lanes 2 to 5, butanolysis reaction with the wild type and H222Q, H222D, and H222E mutants, respectively; lanes 6 to 9, subsequent treatment of butanolysis reaction mixtures with A. niger glucoamylase of the wild type and H222Q, H222D, and H222E mutants, respectively. (b) Yields of butyl glucoside after 48 h of incubation at 85°C and their subsequent treatment with A. niger glucoamylase from the different variants. (c) Alcoholysis/hydrolysis ratios with the different variants. The error bars indicate standard deviations.

FIG. 6.

Transglycosylation products obtained from maltotriose (a) and maltotetraose (b) using 4 U/ml of the wild type and mutants after 0 and 10 min and 1 and 12 h of incubation at 85°C. Lane 1 is the molecular marker, a mixture of oligosaccharides from glucose to maltoheptaose.

FIG. 7.

Thermal inactivation of the wild type and mutants at His222 of α-amylase from T. maritima after incubation at 85°C.