Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 1998 Mar 3;95(5):2056-60.
doi: 10.1073/pnas.95.5.2056.

Engineering an enzyme to resist boiling

Affiliations

Engineering an enzyme to resist boiling

B Van den Burg et al. Proc Natl Acad Sci U S A. .

Abstract

In recent years, many efforts have been made to isolate enzymes from extremophilic organisms in the hope to unravel the structural basis for hyperstability and to obtain hyperstable biocatalysts. Here we show how a moderately stable enzyme (a thermolysin-like protease from Bacillus stearothermophilus, TLP-ste) can be made hyperstable by a limited number of mutations. The mutational strategy included replacing residues in TLP-ste by residues found at equivalent positions in naturally occurring, more thermostable variants, as well as rationally designed mutations. Thus, an extremely stable 8-fold mutant enzyme was obtained that was able to function at 100 degrees C and in the presence of denaturing agents. This 8-fold mutant contained a relatively large number of mutations whose stabilizing effect is generally considered to result from a reduction of the entropy of the unfolded state ("rigidifying" mutations such as Gly --> Ala, Ala --> Pro, and the introduction of a disulfide bridge). Remarkably, whereas hyperstable enzymes isolated from natural sources often have reduced activity at low temperatures, the 8-fold mutant displayed wild-type-like activity at 37 degrees C.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Ribbon drawing of the three-dimensional model of the 8-fold mutated TLP-ste variant. The 55–69 region is shown in blue. The side chains for the amino acids that were mutated in this study (A4T, T56A, G58A, T63F, S65P, A69P) are shown in red and the disulfide bridge cross-linking residue 60 (N60C) in the critical region with residue 8 (G8C) in the underlying β-hairpin is shown in yellow. The light-blue sphere indicates the catalytic zinc ion.
Figure 2
Figure 2
First-order inactivation of TLPs. Stabilities of TLPs were determined at different temperatures. Shown are the first-order inactivation curves for TLP-ste incubated at 80°C (▴) and 90°C (+), thermolysin at 90°C (▵), and the 8-fold mutant at 100°C (•).
Figure 3
Figure 3
Proteolytic specificity of TLP-ste and the 8-fold mutant. β-Casein was incubated with TLP-ste or the 8-fold mutant at a molar ratio of 1,000:1 for 1 hr at 60°C and 100°C, respectively. Proteolysis products were analyzed by reversed-phase HPLC.
Figure 4
Figure 4
Hydrolysis of protease-resistant α-amylase from Bacillus licheniformis by the 8-fold mutant. B. licheniformis α-amylase was incubated with purified TLP-ste, the 8-fold mutant, or without protease for 60 min at the temperature indicated. After incubation the samples were cooled on ice, which resulted in aggregation of the substrate in the samples that had been incubated at 100°C. Precipitates (observed only in the 100°C samples) were collected by centrifugation and redissolved in 6 M urea. Both supernatants (Upper) and redissolved precipitates (Lower) were subjected to standard SDS/PAGE. No significant degradation of α-amylase occurred at temperatures of 80°C and lower, irrespective of the enzyme used. In cases where the samples were incubated at 100°C without added protease or with TLP-ste the aggregate formed after cooling contained mature α-amylase (Lower), indicating that no hydrolysis had occurred. The B. licheniformis α-amylase that was incubated with the 8-fold mutant at 100°C was completely hydrolyzed, and no aggregate was formed.

Comment in

  • Enzyme engineering reaches the boiling point.
    Arnold FH. Arnold FH. Proc Natl Acad Sci U S A. 1998 Mar 3;95(5):2035-6. doi: 10.1073/pnas.95.5.2035. Proc Natl Acad Sci U S A. 1998. PMID: 9482832 Free PMC article. Review. No abstract available.

References

    1. Agard D A. Science. 1993;260:1903–1904. - PubMed
    1. Lomas D A, Evans D L, Finch J T, Carrell R W. Nature (London) 1992;357:605–607. - PubMed
    1. Pepys M B, Hawkins P N, Booth D R, Vigushin D M, Tennent G A, Soutar A K, Totty N, Nguyen O, Blake C C F. Nature (London) 1993;362:553–557. - PubMed
    1. Chrunyk B A, Wetzel R. Protein Eng. 1993;6:733–738. - PubMed
    1. Takagi M, Imanaka T, Aiba S. J Bacteriol. 1985;163:824–831. - PMC - PubMed