Random exchanges of non-conserved amino acid residues among four parental termite cellulases by family shuffling improved thermostability.
There have been two major problems preventing applications of termite cellulases; one was difficulty for their hetelologous overexpression, and another is their low thermostability. We previously achieved adaptation of termite cellulase genes to an overexpression system of Escherichia coli by family shuffling of four orthologous cDNAs (Biosci. Biotechnol. Biochem., 2005; 69: 1711-1720). Using the adapted mutant cDNAs as parental genes combined with native-form cDNAs, we performed further family shuffling and obtained mutant cDNAs, which gave enzymes with improved thermostability. The best-evolved clone (PA68) was improved by 10 degrees C in maximum stability (retaining 90% original activity for 30 min incubation) from the parental enzymes, and kept 54% of its original activity for 150 min at 50 degrees C, whereas the most thermostable enzyme amongst the parents (A18) retained 30% of its original activity. PA68 showed 889 (micromoles of reducing sugars/min/mg of protein) in V(max) and 560 (micromoles of reducing sugars/min/mg of protein) in the specific activity against carboxymethylcellulose, which corresponds to 9.8 and 13.1 times of those of one of the ancestral enzymes rRsEG. In summary, we improved thermostability of the termite cellulase and increased the V(max) value and specific activity by combining only cDNAs encoding enzymes adapted for normal temperatures.
Ni
Jinfeng
J
National Institute of Agrobiological Sciences, 1-2 Owashi, Tsukuba, Ibaraki 305-8634, Japan.
Takehara
Motomi
M
Miyazawa
Mitsuhiro
M
Watanabe
Hirofumi
H
D000468:Alkalies; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D044602:Cellulases / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D017124:Conserved Sequence; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D020049:Isoptera / Q000201:enzymology*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D013379:Substrate Specificity; D013696:Temperature
D000468:Alkalies; D000596:Amino Acids; D044602:Cellulases