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Enzyme engineering for nonaqueous solvents. II. Additive effects of mutations on the stability and activity of subtilisin E in polar organic media

Chen, Keqin and Robinson, Amy C. and Van Dam, Mariana E. and Martinez, Pascal and Economou, Chariklia and Arnold, Frances H. (1991) Enzyme engineering for nonaqueous solvents. II. Additive effects of mutations on the stability and activity of subtilisin E in polar organic media. Biotechnology Progress, 7 (2). pp. 125-129. ISSN 8756-7938. doi:10.1021/bp00008a007.

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Single amino acid substitutions increase the activity and stability of subtilisin E in mixtures of organic solvents and water, and the effects of these mutations are additive. A variant of subtilisin E that exhibits higher activity in mixtures of dimethylformamide (DMF) and water (Q103R) was created by random mutagenesis combined with screening for improved activity (K. Chen and F. H. Arnold, in preparation). Another mutation, N218S, known to improve both the activity and stability of subtilisin BPN′, also improves the activity and stability of subtilisin E in the presence of DMF. The effects of the two substitutions on transition-state stabilization are additive. Furthermore, the Q103R mutation that improves activity has no deleterious effect on subtilisin stability. The double mutant Q103R+N218S is 10 times more active than the wild-type enzyme in 20% (v/v) DMF and twice as stable in 40% DMF. Although the effects of single mutations can be impressive, a practical strategy for engineering enzymes that function in nonaqueous solvents will most likely require multiple changes in the amino acid sequence. These results demonstrate the excellent potential for engineering nonaqueoussolvent-compatible enzymes.

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Arnold, Frances H.0000-0002-4027-364X
Additional Information:© 1991 American Institute of Chemical Engineers (AIChE). Issue online: 5 September 2008; Version of record online: 5 September 2008; Manuscript Accepted: 19 February 1991. This research is supported by the Energy Conservation and Utilization Technologies program of the Department of Energy and the Beckman Institute of the California Institute of Technology. F.H.A. is the recipient of an NSF Presidential Young Investigator Award and a David and Lucile Packard Foundation Fellowship. A.C.R. is supported by an NIH postdoctoral fellowship, GM13953-02.
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Department of Energy (DOE)UNSPECIFIED
Caltech Beckman InstituteUNSPECIFIED
David and Lucile Packard FoundationUNSPECIFIED
NIH Postdoctoral FellowshipGM13953-02
Issue or Number:2
Record Number:CaltechAUTHORS:20171121-065828114
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Official Citation:Chen, K., Robinson, A. C., Van Dam, M. E., Martinez, P., Economou, C. and Arnold, F. H. (1991), Enzyme Engineering for Nonaqueous Solvents. II. Additive Effects of Mutations on the Stability and Activity of Subtilisin E in Polar Organic Media. Biotechnol Progress, 7: 125–129. doi:10.1021/bp00008a007
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:83380
Deposited By: Tony Diaz
Deposited On:21 Nov 2017 17:12
Last Modified:15 Nov 2021 19:57

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