Improved Thermostability of Clostridium thermocellum Endoglucanase Cel8A by Using Consensus-Guided Mutagenesis

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>The use of thermostable cellulases is advantageous for the breakdown of lignocellulosic biomass toward the commercial production of biofuels. Previously, we have demonstrated the engineering of an enhanced thermostable family 8 cellulosomal endoglucanase (EC 3.2.1.4), Cel8A, from<jats:named-content content-type="genus-species">Clostridium thermocellum</jats:named-content>, using random error-prone PCR and a combination of three beneficial mutations, dominated by an intriguing serine-to-glycine substitution (M. Anbar, R. Lamed, E. A. Bayer, ChemCatChem<jats:bold>2:</jats:bold>997–1003, 2010). In the present study, we used a bioinformatics-based approach involving sequence alignment of homologous family 8 glycoside hydrolases to create a library of consensus mutations in which residues of the catalytic module are replaced at specific positions with the most prevalent amino acids in the family. One of the mutants (G283P) displayed a higher thermal stability than the wild-type enzyme. Introducing this mutation into the previously engineered Cel8A triple mutant resulted in an optimized enzyme, increasing the half-life of activity by 14-fold at 85°C. Remarkably, no loss of catalytic activity was observed compared to that of the wild-type endoglucanase. The structural changes were simulated by molecular dynamics analysis, and specific regions were identified that contributed to the observed thermostability. Intriguingly, most of the proteins used for sequence alignment in determining the consensus residues were derived from mesophilic bacteria, with optimal temperatures well below that of<jats:named-content content-type="genus-species">C. thermocellum</jats:named-content>Cel8A.</jats:p>

Efficient synthesis of a statin precursor in high space-time yield by a new aldehyde-tolerant aldolase identified from Lactobacillus brevis

“…Generally, DERA was inactivated by aldehyde through reaction of aldehyde with the surface lysine residues of protein. 30,31 Ten published DERA sequences were selected with amino acid identities ranging from 27 to 52% with respect to LbDERA, and thirteen (T29L, T38F, S45P, I48V, E78K, E101A, L128V, K139D, S166G, K169T, H193R, M209I and T214G) out of 229 positions showed distinct differences (50% consensus cut-off) from the consensus sequence. 19 Here, we describe a simple way to improve the aldehyde tolerability of LbDERA through improving the global stability of LbDERA by consensus sequence approach.…”

Polarity Alteration of a Calcium Site Induces a Hydrophobic Interaction Network and Enhances Cel9A Endoglucanase Thermostability

“…The D468A or D468V mutation efficiently increased the AaCel9A halflife 13 times at 75°C (Fig. 3A) compared to that of other singlebase mutations that have been reported to enhance the thermostability of endoglucanases (31)(32)(33)(34). Endoglucanases have been designed to improve thermostability through such methods as error-prone PCR and the introduction of glycine or proline onto the enzyme surface.…”

Advances in consolidated bioprocessing systems for bioethanol and butanol production from biomass: a comprehensive review

“…(Deng and Fong, 2011). The recombinant technology focuses on directed mutagenesis, genetic and metabolic engineering of cellulolytic microbes to be alcohologenic or alcohologenic microbes to be cellulolytic (Lynd et al, 2005;Anbar et al, 2012;Parisutham et al, 2014). Industrial bioethanol producing microbes do not commonly possess cellulolytic ability, and do not metabolize other sugars in the presence of glucose due to carbon catabolite repression either.…”