Cloning of the gene encoding a novel thermostable alpha-galactosidase from Thermus brockianus ITI360

Abstract

An alpha-galactosidase gene from Thermus brockianus ITI360 was cloned, sequenced, and expressed in Escherichia coli, and the recombinant protein was purified. The gene, designated agaT, codes for a 476-residue polypeptide with a calculated molecular mass of 53, 810 Da. The native structure of the recombinant enzyme (AgaT) was estimated to be a tetramer. AgaT displays amino acid sequence similarity to the alpha-galactosidases of Thermotoga neapolitana and Thermotoga maritima and a low-level sequence similarity to alpha-galactosidases of family 36 in the classification of glycosyl hydrolases. The enzyme is thermostable, with a temperature optimum of activity at 93 degrees C with para-nitrophenyl-alpha-galactopyranoside as a substrate. Half-lives of inactivation at 92 and 80 degrees C are 100 min and 17 h, respectively. The pH optimum is between 5.5 and 6.5. The enzyme displayed high affinity for oligomeric substrates. The K(m)s for melibiose and raffinose at 80 degrees C were determined as 4.1 and 11.0 mM, respectively. The alpha-galactosidase gene in T. brockianus ITI360 was inactivated by integrational mutagenesis. Consequently, no alpha-galactosidase activity was detectable in crude extracts of the mutant strain, and it was unable to use melibiose or raffinose as a single carbohydrate source.

FIG. 1

The insert of the gene library plasmid pOF932 and structure of subclones. α-Galactosidase active and inactive clones are indicated by + and −, respectively. The activity was determined by using pNP-α-galactoside as a substrate. One of the two BglII sites used for the cloning of the fragment in pOF1031 comes from the vector region (pRESIII) of pOF932.

FIG. 2

Alignment of T. brockianus ITI360 AgaT (partial amino acid sequence) with the α-galactosidases of T. maritima, GalA (35), accession no. 2660642; T. neapolitana, Agl1 (31), accession no. AF011400; B. stearothermophilus NUB3621, AgaN (16), accession no. AF130985; S. mutans, Aga (in the alignment designated Aga1) (1), accession no. P27756; E. coli, RafA (4), accession no. P16551; P. pentosaceus, AgaR and AgaS, accession no. L32093; and T. reesei, AglII (38), accession no. Z69254. Hyphens indicate gaps. Alignment was achieved by using CLUSTAL W, version 1.60 (53). Identical residues are indicated by shaded boxes. The consensus pattern of eucaryotic and bacterial α-galactosidase is indicated. A conserved cysteine residue is marked with a solid triangle.

FIG. 3

SDS–10% polyacrylamide gel of crude extract before and after thermal precipitation and purified recombinant T. brockianus ITI360 α-galactosidase. Lanes: 1, molecular mass markers; 2, crude extract of JM109/pOF1037 before thermal precipitation; 3, crude extract of JM109/pOF1037 after thermal precipitation; 4, column fraction of purified AgaT, corresponding to the peak of activity. The sizes of the marker proteins in kilodaltons are indicated.

FIG. 4

Production of AgaT in E. coli RM448. (A) Exchange of rare E. coli codons within the first 21 bp of agaT after the start codon. The upper sequence shows the first 21 bp of agaT in pOF3822. The lower sequence shows the first 21 bp of agaT with exchanged codons as they occur in pTR4. The exchanged codons are marked by shaded boxes. The codon usage (fraction) according to the codon usage table for enteric bacterial highly expressed genes (Wisconsin Sequence Analysis Package, Genetics Computer Group, Inc.) (14) is indicated below the corresponding codons. The EcoRI sites, used for the ligation in pBTac1, are underlined. (B) SDS-PAGE of crude extracts of RM448 containing the plasmids pOF3822 and pTR4 after thermal precipitation. Lanes: 1, molecular mass markers; 2, RM448 without plasmid; 3, RM448 with pOF3822; 4, RM448 with pTR4. The sizes of the marker proteins (in kilodaltons) are shown. AgaT is indicated by an arrow.

FIG. 5

Effect of temperature and pH on the activity of AgaT. (A) Effect of temperature on pNP-α-galactoside hydrolysis (⧫) and raffinose hydrolysis (■). Standard assays at pH 6.5 with purified AgaT were performed. The raffinose concentration was 100 mM. (B) Thermoinactivation of recombinant AgaT. After thermal precipitation of crude extract (1 mg ml⁻¹) as described in the text, the enzyme was preincubated in 100 mM phosphate buffer (pH 6.5) at 92°C (×), 86°C (▴), 80°C (■), and 75°C (⧫) for different periods of time and then assayed for residual activity at 37°C. All activity tests were done in triplicate. The maximum variation from the mean values (shown) was less than 5%.

FIG. 6

Southern blot analysis of T. brockianus ITI360 (wild type) and OF642 (ΔagaT::kan) chromosomal DNA. DNA was digested with SacI, electrophoresed on 1% agarose gel, transferred to a nylon membrane, and hybridized with an agaT gene fragment (A) and kan gene fragment (B) as probes. Furthermore, the DNA was digested with BamHI, electrophoresed and blotted as before, and hybridized with an agaT gene fragment as a probe (C). In each case, lane 1 contains ITI360 and lane 2 contains OF642. The positions of size markers are shown as horizontal lines, and their sizes are given in base pairs. (A and B) λ-EcoRI-HindIII. (C) λ-HindIII. Sizes of detected fragments are given in kilobase pairs. Restriction map of the strains based on sequence analysis and the Southern blot analysis (D). Striped bars indicates DNA fragments used as probes in the hybridization reactions. Dashed lines indicate regions which flank the sequences homologous to the Thermus sequences in pOF642 (integration cassette).

FIG. 7

Phylogenetic tree (dendrogram) showing the evolutionary relationships of α-galactosidase amino sequences according to the alignment in Fig. 2 and the amino sequence of an enzyme belonging to family 27 (AglA of Aspergillus niger), which was used as an outgroup. The tree was constructed as described in Materials and Methods.