Identification of thermostabilizing residues in a Bacillus alkaline cellulase by construction of chimeras from mesophilic and thermostable enzymes and site-directed mutagenesis

Abstract

An alkaliphilic Bacillus sp. strain, KSM-64, produces a mesophilic alkaline endo-1,4-L-glucanase that is suitable for use in detergents. The deduced amino acid sequence of the enzyme showed very high homology to that of a thermostable alkaline enzyme from alkaliphilic Bacillus sp. strain KSM-S237. Analysis of chimeric enzymes produced from the genes encoding the mesophilic and thermostable enzymes suggested that the lysine residues at positions 137, 179, and 194 are responsible for their thermal stabilization. Replacing the corresponding Glu137, Asn179, and/or Asp194 with lysine by site-directed mutagenesis made the mesophilic enzyme more thermostable. Analyses of the hydrophilicity of deduced amino acid sequences and isoelectric focusing of the modified enzymes suggested that these three specific lysine residues and their replacements are all located on the surface of the enzyme molecule. This fact further suggested that specific ionic interaction is involved in the thermal stabilization of the enzyme. ß

Effects of mutations in the helix G region of horseradish peroxidase

“…It is perplexing that mutations at these sites, which clearly have a bearing on HRP stability, should yield such modest stability gains. Nonetheless, the present work mirrors previous studies in which amino acid substitutions, not necessarily located in the protein core, can modestly affect the thermostability of recombinant proteins [51,52,53,54]. …”

Combined molecular docking, homology modelling and density functional theory studies to modify dioxygenase to efficiently degrade aromatic hydrocarbons

“…Hakamada et al mutated the Asn179, Asp194 and Glu137 residues of bacterial alkaline cellulase to Lys, and the thermal stability of the enzyme was signicantly improved aer the modication. 47 Zhang et al studied the effects of the amino acid residues at the Cel6A binding site of Thermobida fusca cellulase on its catalytic activity, substrate specicity and ligand affinity and found that aer the modication of the amino acid residues Arg237, Glu263, Lys259 and His159, the mutant had improved hydrolytic activity towards carboxymethyl cellulose. 28 Based on the hydrophilic or hydrophobic characteristics of 20 amino acid residues (Table A1 †), the selected hydrophilic amino acid residues Asn201, Asp205, His208, His213, His295 and Asn297 at the binding site of the NDO enzyme (PDB ID: 1O7G) were replaced by the hydrophobic amino acid residues Ile, Phe, Val, Leu, Trp, Met, Ala, Tyr and Pro, respectively, and 54 kinds of modication schemes for individual amino acid residues of the NDO enzyme (PDB ID: 1O7G) were obtained (shown in Table 1).…”

Thermostabilization by replacement of specific residues with lysine in a Bacillus alkaline cellulase: building a structural model and implications of newly formed double intrahelical salt bridges

“…strain KSM-64 of our stock cultures was used as the source of the gene encoding Egl-64 (Sumitomo et al, 1992). Constructed plasmids were used individually for transformation of B.subtilis ISW1214 to exoproduce wild-type, NK, DK or NK/DK mutant enzyme in the medium described previously (Sumitomo et al, 1995;Hakamada et al, 2001). The N-and C-termini of Egl-Kt correspond to Ala228 and Leu584, respectively, of mature Egl-K (Ozaki et al, 1995).…”

“…Based on the difference of a few amino acids, we constructed several chimeric genes from Egl-64 and Egl-237, and Lys179 and Lys194 residues in Egl-237, not conserved in Egl-64, were found to contribute to thermostability after site-directed mutagenesis. As a result, the double mutation Asn179→Lys (NK)/ Asp194→Lys (DK) (NK/DK) significantly improved thermostability, the level of which was as high as that of the wildtype Egl-237 (Hakamada et al, 2001). One of the most striking features of thermostable enzymes, when compared with mesophilic enzymes, is the decrease in the number of Lys residues, which are mainly replaced with Arg and Glu residues (Argos et al, 1979;Davies et al, 1993;Shirai et al, 1997a).…”