Improving the thermal stability of cellobiohydrolase Cel7A from Hypocrea jecorina by directed evolution


Abstract

Secreted mixtures of Hypocrea jecorina cellulases are able to efficiently degrade cellulosic biomass to fermentable sugars at large, commercially relevant scales. H. jecorina Cel7A, cellobiohydrolase I, from glycoside hydrolase family 7, is the workhorse enzyme of the process. However, the thermal stability of Cel7A limits its use to processes where temperatures are no higher than 50 °C. Enhanced thermal stability is desirable to enable the use of higher processing temperatures and to improve the economic feasibility of industrial biomass conversion. Here, we enhanced the thermal stability of Cel7A through directed evolution. Sites with increased thermal stability properties were combined, and a Cel7A variant (FCA398) was obtained, which exhibited a 10.4 °C increase in Tm and a 44-fold greater half-life compared with the wild-type enzyme. This Cel7A variant contains 18 mutated sites and is active under application conditions up to at least 75 °C. The X-ray crystal structure of the catalytic domain was determined at 2.1 Å resolution and showed that the effects of the mutations are local and do not introduce major backbone conformational changes. Molecular dynamics simulations revealed that the catalytic domain of wild-type Cel7A and the FCA398 variant exhibit similar behavior at 300 K, whereas at elevated temperature (475 and 525 K), the FCA398 variant fluctuates less and maintains more native contacts over time. Combining the structural and dynamic investigations, rationales were developed for the stabilizing effect at many of the mutated sites.

Submission Details

ID: UQuoyfyA

Submitter: Shu-Ching Ou

Submission Date: Oct. 11, 2018, 10:32 a.m.

Version: 1

Publication Details
Goedegebuur F;Dankmeyer L;Gualfetti P;Karkehabadi S;Hansson H;Jana S;Huynh V;Kelemen BR;Kruithof P;Larenas EA;Teunissen PJM;Ståhlberg J;Payne CM;Mitchinson C;Sandgren M,J Biol Chem (2017) Improving the thermal stability of cellobiohydrolase Cel7A from <i>Hypocrea jecorina</i> by directed evolution. PMID:28860192
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1AZ6 1997-11-25T00:00:00+0000 0 THREE-DIMENSIONAL STRUCTURES OF THREE ENGINEERED CELLULOSE-BINDING DOMAINS OF CELLOBIOHYDROLASE I FROM TRICHODERMA REESEI, NMR, 23 STRUCTURES
1AZH 1997-11-18T00:00:00+0000 0 THREE-DIMENSIONAL STRUCTURES OF THREE ENGINEERED CELLULOSE-BINDING DOMAINS OF CELLOBIOHYDROLASE I FROM TRICHODERMA REESEI, NMR, 14 STRUCTURES
1AZJ 1997-11-18T00:00:00+0000 0 THREE-DIMENSIONAL STRUCTURES OF THREE ENGINEERED CELLULOSE-BINDING DOMAINS OF CELLOBIOHYDROLASE I FROM TRICHODERMA REESEI, NMR, 18 STRUCTURES
1AZK 1997-11-18T00:00:00+0000 0 THREE-DIMENSIONAL STRUCTURES OF THREE ENGINEERED CELLULOSE-BINDING DOMAINS OF CELLOBIOHYDROLASE I FROM TRICHODERMA REESEI, NMR, 19 STRUCTURES
1CBH 1989-05-30T00:00:00+0000 0 DETERMINATION OF THE THREE-DIMENSIONAL STRUCTURE OF THE C-TERMINAL DOMAIN OF CELLOBIOHYDROLASE I FROM TRICHODERMA REESEI. A STUDY USING NUCLEAR MAGNETIC RESONANCE AND HYBRID DISTANCE GEOMETRY-DYNAMICAL SIMULATED ANNEALING
1CEL 1994-05-17T00:00:00+0000 1.8 THE THREE-DIMENSIONAL CRYSTAL STRUCTURE OF THE CATALYTIC CORE OF CELLOBIOHYDROLASE I FROM TRICHODERMA REESEI
1DY4 2000-01-26T00:00:00+0000 1.9 CBH1 IN COMPLEX WITH S-PROPRANOLOL
1EGN 2000-02-16T00:00:00+0000 1.6 CELLOBIOHYDROLASE CEL7A (E223S, A224H, L225V, T226A, D262G) MUTANT
1Q2B 2003-07-24T00:00:00+0000 1.6 CELLOBIOHYDROLASE CEL7A WITH DISULPHIDE BRIDGE ADDED ACROSS EXO-LOOP BY MUTATIONS D241C AND D249C
1Q2E 2003-07-24T00:00:00+0000 1.75 CELLOBIOHYDROLASE CEL7A WITH LOOP DELETION 245-252 AND BOUND NON-HYDROLYSABLE CELLOTETRAOSE

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
91.1 Exoglucanase 1 P19355 GUX1_HYPRU
97.7 Exoglucanase 1 A0A024RXP8 GUX1_HYPJR
100.0 Exoglucanase 1 P62695 GUX1_TRIKO
100.0 Exoglucanase 1 P62694 GUX1_HYPJE