Purification and characterization of a novel thermostable 4‐α‐glucanotransferase of Thermotoga maritima cloned in Escherichia coli

Abstract

Maltodextrin glycoslyltransferase (4-a-glucanotransferase) of the extremely thermophilic ancestral bacterium Thermotoga maritima has been purified from an Escherichia coli clone expressing the corresponding T. maritima MSB8 chromosomal gene. T. maritima 4-a-glucanotransferase, an approximately 53-kDa monomeric enzyme, is the most thermophilic glycosyltransferase described to date. It retained more than 90% of its maximum activity at temperatures from 55°C up to 80°C.The proposed action modus is the transfer of 1 ,4-a-glucanosyl chains, thus resulting in the disproportionation of 1 ,Ca-glucans. It converted soluble starch, amylopectin, and amylose, thereby changing the iodine staining properties of these substrates. The addition of low-molecular-mass maltooligosaccharides, which act as glucanosyl acceptor molecules, enhanced the reaction and resulted in the formation of a series of linear maltohomologues from two to more than nine glucose units in size. Use of either of the malto-oligosaccharides maltotetraose, maltopentaose, maltohexaose, or maltoheptaose as sole substrate also yielded linear maltohomologues. On the other hand, maltose and maltotriose were not disproportionated by 4-a-glucanotransferase, although both were good acceptors for glucanosyl transfer. Glucose did not function as an acceptor in transfer reactions. Glucose also never appeared as a reaction product. The chain length of glucanosyl segments transferred ranged from two to probably far more than six glucose residues.Comparison of the N-terminal amino acid sequence of 4-a-glucanotransferase with other published protein sequences revealed significant similarity to sequences near the N-termini of various eucaryotic maltases and bacterial cyclodextrin glycosyltransferases, suggesting its relatedness on the molecular level with other starch-and maltodextrin-converting enzymes.The extreme environment inhabited by Thermotoga maritima (growth range 55-80°C with an optimum at 80°C [l]) has imposed severe selective pressure upon this microorganism's enzymatic outfit which must be thermophilic as well as thermostable enough to guarantee survival at very high temperatures. Therefore, T. maritima seems to be a profitable source for the iolation of highly thermostable enzymes whose biochemical and structural analysis may be extremely helpful in the task of unraveling the molecular basis of thermostability. Additionally, the thermophilic enzymes produced by this organism may prove to be valuable catalysts for industrial applications.We have isolated the gene for an interesting starch-converting enzyme of T. maritima from a gene library of this organism constructed in E. coli and have purified and charac-

Thermotoga maritima maltosyltransferase, a novel type of maltodextrin glycosyltransferase acting on starch and malto‐oligosaccharides

“…Also, perfect complementarity was found between the postulated ribosome-binding sequence (5′ ... AG-GAGG... 3′), preceding the mmtA ORF with a spacing of 7 bp, and the 3′-end of the 16 S rRNAs of both T. maritima and E. coli (3′-UCUUUCCUCCACU... 5′ and 3′-AUUCCUCCACU... 5′, respectively). Despite the presence of consensus-like expression signals, the observed high expression level of MTase was surprising since the codon usage found in mmtA and other Thermotoga genes is quite different in some instances from the codon usage of highly expressed E. coli genes [4,45], a fact which in certain cases was proposed to impair high-level heterologous expression [22]. For example, 85% of all arginines in the MTase polypeptide are encoded by the triplets AGA and AGG, which are strictly avoided in highly expressed E. coli genes.…”

“…None of the MGTases of E. coli, Haemophilus influenzae, Streptococcus pneumoniae (designated as amylomaltases) or potato (D-enzyme) appeared as hits with BLASTP. Likewise, the second MGTase of T. maritima, GTase, which has a similarly broad transfer specificity as the amylomaltases and D-enzyme [4,6], also only shares marginal local similarity, but no significant fulllength similarity with MTase.…”

“…This also holds true for another, previously described MGTase of T. maritima, 4-A-glucanotransferase (GTase). GTase transfers segments as small as maltotriosyl or maltosyl units, but also very large ones, such that the products of transfer to radioactively labelled maltose are too large to be separated by TLC [4,5]. Besides its transfer activity, MTase must also have weak hydrolytic activity releasing maltose from suitable donors, which in our opinion is responsible for the observed production of a spectrum of evennumbered small malto-oligosaccharides after extensive incubation of the enzyme with amylose or amylopectin in the absence of any low molecular mass acceptor.…”

“…more thermostable than another previously identified maltodextrin glycosyltransferase of T. maritima (4-A-glucanotransferase, GTase; [4]). In terms of activity at high temperatures, these two T. maritima enzymes are matched only by one other recently described maltodextrin glycosyltransferase, i.e.…”

Three-way Stabilization of the Covalent Intermediate in Amylomaltase, an α-Amylase-like Transglycosylase

“…Amylomaltases are 4-␣-glucanotransferase enzymes (1)(2)(3)(4) that are structurally and mechanistically related to ␣-amylases (family 13 of the glycoside hydrolases or GH13) (5-7) despite their classification in a separate glycoside hydrolase family (glycoside hydrolase family 77). However, amylomaltases almost exclusively catalyze transglycosylation reactions, whereas ␣-amylase-like enzymes mostly catalyze hydrolysis.…”