Isolation of a thermostable enzyme variant by cloning and selection in a thermophile.

Abstract

We developed a method for rapidly generating thermostable enzyme variants. Our strategy is to introduce the gene coding for a given enzyme from a mesophilic organism into a thermophile, Bacillus stearothermophilus. Variants that retain the enzymatic activity at the higher growth temperatures of the thermophile are then selected. This strategy was applied to kanamycin nucleotidyltransferase, which confers resistance to the antibiotic kanamycin. B. stearothermophilus carrying the wild-type enzyme is resistant to the antibiotic at 47C but not at 55C and above. Variants that were kanamycin resistant at 63°C were obtained by selection of spontaneous mutants, by passage ofa shuttle plasmid through theEscherichia colimutD5 mutator strain and introduction into B. stearothermophilus by transformation, and by growing the thermophile in a chemostat. The kanamycin nucleotidyltransferases purified from these variants were all more resistant to irreversible thermal inactivation than is the wild-type enzyme, and all have the same single amino acid replacement, aspartate to tyrosine at position 80. Mutants that are even more heat stable were derived from the frt variant by selecting for kanamycin resistance at 70C, and these carry the additional change of threonine to lysine at position 130. This strategy is applicable to other enzymatic activities that are selectable in thermophiles or that can be screened for by plate assays.The ability of some microorganisms to grow at extreme temperatures (1) implies that their enzymes are stable and active at these temperatures. This is largely borne out when enzymes from thermophilic sources are studied in vitro; such enzymes are indeed more thermostable than the equivalents isolated from phylogenetically related mesophilic organisms (2). Correlations between an increase in the proportion of hydrophobic residues and the degree of thermostability have been observed (3-5). Internal electrostatic interactions (6) and disulfide linkages (7) have also been proposed as features that stabilize proteins. However, the role of individual amino acid residues in enhancing the resistance to thermal denaturation of an enzyme from a thermophile is not known.We wish to understand the contributions of individual amino acids to the overall stability of a protein's structure. Comparisons between enzymes from mesophiles and thermophiles are complicated because these proteins, although homologous, usually differ in more than one residue. More precise inferences can be based on comparisons of temperature-sensitive mutations which encode, as a result of single amino acid changes, proteins that retain activity but are less resistant to heat denaturation than the wild-type counterpart. Thus, for example, many temperature-sensitive mutations of phage T4 lysozyme have been studied with the aim of correlating the changes in stability with changes in the protein structure (8). However, an x-ray crystallographic study of one of these mutant enzymes (9) revealed that a variant in which a histidine residue repla...

The denaturation and degradation of stable enzymes at high temperatures

“…A variety of studies [27][28][29][30][31] have shown that the majority of point mutations have little effect on stability. For example, of over 2000 mutations made to T4 lysozyme, 91 % had no significant effect on thermal stability [24,25].…”

“…However, for those mutations that alter stability, destabilization is a more common effect. For a number of enzymes where enough data have been gathered [28][29][30][31], including lysozyme [28] and subtilisin [31], the occurrence of random point mutations which cause stabilization is of the order of 1 in 10 000. The destabilizing mutations in lysozyme (about 9 % of the total) all occurred at amino acids which have restricted mobility [24,25,32].…”

Characterization of a lysK gene as an argE homolog in Thermus thermophilus HB27

“…Ampli¢ed fragment was digested with SphI and EcoRI and ligated with pUC18 previously digested with the same enzymes. Resulting plasmid named pDFRE was digested with Aor51HI, treated with calf intestine alkaline phosphatase, and ligated with blunt-ended HindIII fragment of pUC39-442KmR [13] that contains heat-stabilized kanamycin nucleotidyltransferase gene [14]. The resulting plasmid was named pDFREKmR and used for knockout of the lysK gene of T. thermophilus HB27 as described previously [7].…”

Development of an in vivo method to identify mutants of phage T4 lysozyme of enhanced thermostability

“…Methods to identify thermostable variants have previously been developed for kanamycin nucleotidyltransferase (Matsumuratk Aiba, 1985;Liao et al, 1986), subtilisin (Bryan et al, 1986), and glucose dehydrogenase (Makino et al, 1989).…”