Divalent metal ion requirements of a thermostable multimetal beta-galactosidase from Saccharopolyspora rectivirgula.

To understand the roles of metal ions on the catalytic properties and thermostability of the thermostable beta-galactosidase of Saccharopolyspora rectivirgula, a thermophilic actinomycete, we have investigated the binding kinetics and requirements of divalent metal ions by equilibrium dialysis, titration, and metal ion buffer techniques. We found that the monomeric multimetal enzyme (M(r) 136,977) had eight specific binding sites for divalent metal ions. These sites were classified as follows: a very tight (class I) site for Ca2+, three tight (class II) sites consisting of two Ca(2+)-specific sites (class IICa) and one Mn(2+)-specific site (class IIMn; Kd for Mn2+, 2.0 nM), and four loose (class III) sites for Mn2+ (Kd, 1.2 microM) and Mg2+ (Kd, 2 microM). Removal of metal ions bound to class II and III sites of the holoenzyme (Ca3Mn5 species; relative Vmax (Vrel), 100%) by a chelating resin at 4 degrees C yielded a less thermostable Ca1 species (Vrel, 1.7%) with a class I Ca2+ ion, removal of which by a chelating resin at 50 degrees C caused a complete irreversible inactivation of the enzyme. Titration studies revealed that stoichiometric binding of Mn2+ to a class IIMn site of the Ca1 species caused a 33-fold activation whereas binding of Ca2+ to class IICa sites had no effect on enzyme activity. Ca1 species could be also activated 8-fold by heating at 60 degrees C for 20 min, suggesting that the catalytically important class II Mn2+ plays important roles in maintaining the native structure essential for activity. Occupation of class III sites by Mg2+ or Mn2+ was of physiological importance to attain sufficient thermostability by which this extracellular beta-galactosidase remained active for a prolonged time at elevated temperatures as was observed during growth of S. rectivirgula.

Purification and characterization of a Bacillus sp. SAM1606 thermostable alpha-glucosidase with transglucosylation activity.

We purified a novel alpha-glucosidase to homogeneity from an Escherichia coli recombinant transformed with the alpha-glucosidase gene from thermophilic Bacillus sp. SAM1606. The enzyme existed as mono- and multimeric forms of a promoter protein with a relative molecular weight of 64,000 and isoelectric point of 4.6. We isolated a monomeric form of the enzyme and characterized it. The enzyme was unique among the known alpha-glucosidases in both broad substrate specificity and high thermostability. The enzyme hydrolysed a variety of O-alpha-D-glucopyranosides such as nigerose, maltose, isomaltose, sucrose, and trehalose efficiently. The molecular activity (k0) and the Michaelis constant (Km) values at 55 degrees C and pH 6.0 for sucrose were 54.6 s-1 and 5.3 mM, respectively. The optimum pH and temperature for hydrolysis were pH 5.5 and 75 degrees C, respectively. The enzyme exhibited a high transglucosylation activity: it reacted with 1.8 M sucrose at 60 degrees C for 70 h to yield oligosaccharides containing theanderose in a maximum yield of 35% (w/w). High thermostability of the enzyme (stable up to 65 degrees C at pH 7.2 for 10 min) permits the transglucosylation reaction at high temperatures, which would be beneficial for continuous production of oligosaccharides from sucrose.

Structure and expression of a gene coding for thermostable alpha-glucosidase with a broad substrate specificity from Bacillus sp. SAM1606.

We cloned an alpha-glucosidase gene from thermophilic Bacillus sp. SAM1606 to overexpress it in Escherichia coli transformants. Deletion of the 5'-noncoding region as well as expression of the alpha-glucosidase gene under the control of the icp promotor of the insecticidal crystal protein gene from Bacillus thuringiensis subsp. sotto enhanced the enzyme productivity to 23.5 U/ml, which was 12,000-fold higher than that obtained by the strain SAM1606. The open reading frame corresponding to the alpha-glucosidase encoded 587 amino acid residues including a residue coded by the initiation codon TTG, and the molecular mass of the alpha-glucosidase from N-terminal serine was calculated to be 68,886 Da. Sequence analysis revealed that the SAM1606 alpha-glucosidase belonged to the alpha-amylase family. The SAM1606 alpha-glucosidase showed extremely high sequence identity (62-65%) to the Bacillus cereus and Bacillus thermoglucosidasius oligo-1,6-glucosidases, which were 72% identical to each other. Sequence identity in the suggested active site regions were essentially the same (80-82%) among these three enzymes. However, the substrate specificity of the SAM1606 alpha-glucosidase was significantly different from those of the oligo-1,6-glucosidases. The thermostability of these three alpha-glucosidases could be correlated with the increase in the number of proline residues, whose occurrence was predicted at beta turns and coils in the enzymes.