Multiple Proline Substitutions Cumulatively Thermostabilize Bacillus Cereus ATCC7064 Oligo‐1,6‐Glucosidase

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Abstract
Nine residues of Bacillus cereus ATCC7064 oligo-1,6-glucosidase were replaced stepwise with proline residues. Of the nine residues, Lys121, Glu208 and Glu290 were at second sites of β turns; Asn109, Glu175 and Thr261 were at N-caps of α helices; Glu216, Glu270 and Glu378 were in coils within loops. The replacements were carried out in the order, Lys121→Pro, Glu175→Pro, Glu290–Pro, Glu208→Pro, Glu270→Pro, Glu378→Pro, Thr261→Pro, Glu216→Pro and Asn109 →Pro. The resultant nine active mutant enzymes contained 1–9 more proline residues than B. cereus oligo-1,6-glucosidase. The thermostability of these mutants was additively enhanced with the increase in the number of proline residues introduced. The increase in the thermostability was most remarkable when proline residues were introduced at second sites of β turns or at N-caps of α helices. The above results afforded irrefragable proof for the proline rule as an effective principle for increasing protein thermostability [Suzuki, Y., Oishi, K., Nakano, H. & Nagayama, T. (1987) Appl. Microbiol Biotechnol. 26, 546–551].