Thermoadaptation of alpha-galactosidase AgaB1 in Thermus thermophilus

Abstract

The evolutionary potential of a thermostable alpha-galactosidase, with regard to improved catalytic activity at high temperatures, was investigated by employing an in vivo selection system based on thermophilic bacteria. For this purpose, hybrid alpha-galactosidase genes of agaA and agaB from Bacillus stearothermophilus KVE39, designated agaA1 and agaB1, were cloned into an autonomously replicating Thermus vector and introduced into Thermus thermophilus OF1053GD (DeltaagaT) by transformation. This selector strain is unable to metabolize melibiose (alpha-galactoside) without recombinant alpha-galactosidases, because the native alpha-galactosidase gene, agaT, has been deleted. Growth conditions were established under which the strain was able to utilize melibiose as a single carbohydrate source when harboring a plasmid-encoded agaA1 gene but unable when harboring a plasmid-encoded agaB1 gene. With incubation of the agaB1 plasmid-harboring strain under selective pressure at a restrictive temperature (67 degrees C) in a minimal melibiose medium, spontaneous mutants as well as N-methyl-N'-nitro-N-nitrosoguanidine-induced mutants able to grow on the selective medium were isolated. The mutant alpha-galactosidase genes were amplified by PCR, cloned in Escherichia coli, and sequenced. A single-base substitution that replaces glutamic acid residue 355 with glycine or valine was found in the mutant agaB1 genes. The mutant enzymes displayed the optimum hydrolyzing activity at higher temperatures together with improved catalytic capacity compared to the wild-type enzyme and furthermore showed an enhanced thermal stability. To our knowledge, this is the first report of an in vivo evolution of glycoside-hydrolyzing enzyme and selection within a thermophilic host cell.

FIG. 1.

The phenotypic characteristics of the isoenzymes AgaA and AgaB and their hybrid enzymes, AgaA1 and AgaB1. The corresponding gene structures are shown on the left. The locations of the BsmI and the HindIII restriction sites in agaA are indicated with nucleotide numbers.

FIG. 2.

The last step in the construction of the T. thermophilus plasmid pOF2812, containing agaB1. The progenitor plasmid pOF288 (shuttle vector) was established in E. coli as described in Materials and Methods. Deletion of the pIC region in pOF288 to produce pOF2812 before transformation of T. thermophilus was supposed to improve plasmid stability in strain OF1053GD, according to previous results (8).

FIG. 3.

Growth of T. thermophilus OF1053GD at 67°C, in minimal medium containing 0.4% melibiose as a single carbohydrate source. ▪, OF1053GD/pOF2811 (agaA1); ⧫, OF1053GD/pOF2812 (agaB1); •, OF1053GD without plasmid.

FIG. 4.

Effect of temperature on the pNP-α-galactopyranoside hydrolyzing activity (A) and stability (B) of AgaA (▪), AgaB1 (⧫), AgaM4 (▴), and AgaM5 (•). Standard pNP-α-galactosidase assays at pH 6.5 were performed using enzymes in E. coli crude extracts of protein concentration 1 mg ml⁻¹. Temperature stability was determined by measuring the residual α-galactosidase activity after incubation of enzymes at a temperature range of 25 to 80°C as described in Materials and Methods. T[1/2], the temperature where the remaining activity is 50% following 30 min of incubation, is indicated by arrows. All activity tests were done in triplicate. The maximum variation from the mean values (shown) was less than 5%.

FIG. 5.

Alignment of α-galactosidase partial amino acid sequences, residues 326 to 387 according to AgaB1 amino acid signature. The sequence region corresponds approximately to the BsmI-HindIII fragment of AgaA and AgaB. Amino acid sequences of bacterial α-galactosidases belonging to family 36 and with a subunit size of about 80 kDa (7) were retrieved from protein databases according to their accession numbers and aligned by using CLUSTALX version 1.8 (30). The sequences are as follows: AgaB1, (the sequence region is identical to the corresponding region in AgaB from B. stearothermophilus [B. stearotherm] KVE39, accession no. AY013287); AgaA1 (the sequence region is identical to the corresponding region in AgaA from B. stearothermophilus KVE39, accession no. AY013286); AgaN from B. stearothermophilus NUB3621 (accession no. AF130985); RafA from E. coli, accession no. P16551; Aga from Streptococcus mutans, accession no. P27756; MelA from Thermoanaerobacter ethanolicus, accession no. CAA69852; AgaR from Pediococcus pentosaceus, accession no. L32093; α-galactosidase from Yersinia pestis, accession no. NP_405164; α-galactosidase from Streptococcus pneumoniae, accession no. NP_346329; α-galactosidase from B. halodurans, accession no. NP_243089. Identical residues are indicated with black boxes (shade threshold, 100%) and gray boxes (shade threshold, 80%). The residue corresponding to amino acid 355 in AgaB1 is marked with a solid triangle.