Jones, A., Lamsa, M., Frandsen, T.P., Spendler, T., Harris, P., Sloma, A., Xu, F., Nielsen, J.B., Cherry, J.R.
"Directed evolution of a maltogenic α-amylase from Bacillus sp. TS-25"
Journal of Biotechnology, 134 (3-4), pp. 325-333. (2008)
Directed evolution coupled with a high-throughput robotic screen was employed to broaden the industrial use of the maltogenic α-amylase Novamyl from Bacillus sp. TS-25. Wild-type Novamyl is currently used in the baking industry as an anti-staling agent in breads baked at neutral or near neutral pH. However, the enzyme is rapidly inactivated during the baking process of bread made with low pH recipes and Novamyl thus has very limited beneficial effect for this particular application. In an effort to improve the performance of Novamyl for low pH bread applications such as sourdough and rye, two error-prone PCR libraries were generated, expressed in Bacillus subtilis and screened for variants with improved thermal stability and activity under low pH conditions. Variants exhibiting improved performance were iteratively recombined using DNA shuffling to create two generations of libraries. Relative to wild-type Novamyl, a number of the resulting variants exhibited more than 10 °C increase in thermal stability at pH 4.5, one of which demonstrated substantial anti-staling properties in low pH breads. © 2008 Elsevier B.V. All rights reserved.
Blanchard, S., Armand, S., Couthino, P., Patkar, S., Vind, J., Samain, E., Driguez, H., Cottaz, S.
"Unexpected regioselectivity of Humicola insolens Cel7B glycosynthase mutants"
Carbohydrate Research, 342 (5), pp. 710-716. (2007)
Four Humicola insolens Cel7B glycoside hydrolase mutants have been evaluated for the coupling of lactosyl fluoride on O-allyl NI-acetyl-2II-azido-β-chitobioside. Double mutants Cel7B E197A H209A and Cel7B E197A H209G preferentially catalyze the formation of a β-(1→4) linkage between the two disaccharides, while single mutant Cel7B E197A and triple mutant Cel7B E197A H209A A211T produce predominantly the β-(1→3)-linked tetrasaccharide. This result constitutes the first report of the modulation of the regioselectivity through site-directed mutagenesis for an endoglycosynthase. © 2007 Elsevier Ltd. All rights reserved.
Blanchard, S., Cottaz, S., Coutinho, P.M., Patkar, S., Vind, J., Boer, H., Koivula, A., Driguez, H., Armand, S.
"Mutation of fungal endoglucanases into glycosynthases and characterization of their acceptor substrate specificity"Journal of Molecular Catalysis B: Enzymatic, 44 (3-4), pp. 106-116. (2007)
Humicola insolens mutant Cel7B E197A is a powerful endo-glycosynthase displaying an acceptor substrate specificity restricted to β-d-glucosyl, β-d-xylosyl, β-d-mannosyl and β-d-glucosaminyl in +1 subsite. Our aim was to extend this substrate specificity to β-d-N-acetylglucosaminyl, in order to get access to a wider array of oligosaccharidic structures obtained through glycosynthase assisted synthesis. In a first approach a trisaccharide bearing a β-d-N-acetylglucosaminyl residue was docked at the +1 subsite of H. insolens Cel7B, indicating that the mutation of only one residue, His209, could lead to the expected wider acceptor specificity. Three H. insolens Cel7B glycosynthase mutants (H209A, H209G and H209A/A211T) were produced and expressed in Aspergillus oryzae. In parallel, sequence alignment investigations showed that several cellulases from family GH7 display an alanine residue instead of histidine at position 209. Amongst them, Trichoderma reesei Cel7B, an endoglucanase sharing the highest degree of sequence identity with Humicola Cel7B, was found to naturally accept a β-d-N-acetylglucosaminyl residue at +1 subsite. The T. reesei Cel7B mutant nucleophile E196A was produced and expressed in Saccharomyces cerevisiae, and its activity as glycosynthase, together with the H. insolens glycosynthase mutants, was evaluated toward various glycosidic acceptors. © 2006 Elsevier B.V. All rights reserved.
Eijsink, V.G.H., GÅseidnes, S., Borchert, T.V., Van Den Burg, B.
"Directed evolution of enzyme stability"
Biomolecular Engineering, 22 (1-3), pp. 21-30. (2005)
Modern enzyme development relies to an increasing extent on strategies based on diversity generation followed by screening for variants with optimised properties. In principle, these directed evolution strategies might be used for optimising any enzyme property, which can be screened for in an economically feasible way, even if the molecular basis of that property is not known. Stability is an interesting property of enzymes because (1) it is of great industrial importance, (2) it is relatively easy to screen for, and (3) the molecular basis of stability relates closely to contemporary issues in protein science such as the protein folding problem and protein folding diseases. Thus, engineering enzyme stability is of both commercial and scientific interest. Here, we review how directed evolution has contributed to the development of stable enzymes and to new insight into the principles of protein stability. Several recent examples are described. These examples show that directed evolution is an effective strategy to obtain stable enzymes, especially when used in combination with rational or semi-rational engineering strategies. With respect to the principles of protein stability, some important lessons to learn from recent efforts in directed evolution are (1) that there are many structural ways to stabilize a protein, which are not always easy to rationalize, (2) that proteins may very well be stabilized by optimizing their surfaces, and (3) that high thermal stability may be obtained without forfeiture of catalytic performance at low temperatures. © 2005 Elsevier B.V. All rights reserved.
Tindbaek, N., Svendsen, A., Oestergaard, P.R., Draborg, H.
"Engineering a substrate-specific cold-adapted subtilisin"
Protein Engineering, Design and Selection, 17 (2), pp. 149-156. (2004)
One region predicted to be highly flexible for a psychrophilic enzyme, TA39 subtilisin (S39), was transferred in silico to the mesophilic subtilisin, savinase (EC 220.127.116.11), from Bacillus lentus (clausii). The engineered hybrid and savinase were initially investigated by molecular dynamic simulations at 300 K to show binding region and global flexibility. The predicted S39 region consists of 12 residues, which due to homology between the subtilisins, results in a total change of eight residues. By site-directed modifications, the region was transferred to the binding region of savinase, thus a savinase-S39 hybrid, named H5, was constructed. The designed hybrid showed the same temperature optimum and pH profile as savinase, but H5 had higher specific activity on the synthetic substrate N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (AAPF) at all temperatures measured and, at the same time, H5 showed a decrease in thermostability. The H5 hybrid showed broader substrate specificity, measured at room temperature, due to an increase in catalytic efficiency on AAPF, AAPA and FAAF compared with savinase (N-succinyl-XXXX-pNA; XXXX = AAPF, AAPA and FAAF). The H5 hybrid showed increased activity at low temperature, increased binding region and global flexibility, as investigated by molecular dynamic simulations, and global destabilization from differential scanning calorimetry measurements. These psychrophilic characteristics indicated an increase in binding site flexibility, probably due to the modifications P129S, S130G, P131E, and thus we show that it is possible to increase low temperature activity and global flexibility by engineered flexibility in the binding region.
"Protein engineering of cellulases"
Biochimica et Biophysica Acta-Protein Structure and Molecular Enzymology, 1543(2), 239-252 (2000)
Cellulases are enzymes which hydrolyse the beta -1, 4- glucosidic linkages of cellulose. They fall into 13 of the 82 glycoside hydrolase families identified by sequence analysis, but they are traditionally divided into two classes termed 'endoglucanases' (EC 18.104.22.168) and 'cellobiohydrolases' (22.214.171.124). Both types of cellulases degrade soluble cellodextrins and amorphous cellulose but, with a few notable exceptions, it is only the cellobiohydrolases which degrade crystalline cellulose efficiently. Site-directed mutagenesis has been central to the characterisation of cellulases, ranging from the identification and characterisation of putative catalytic and binding residues, the trapping of enzyme-substrate complexes by crystallography through to the construction of new and improved biocatalysts including 'glycosynthases'. Whilst studies on soluble substrates and substrate analogues have provided a wealth of information, understanding the mechanism of degradation of the natural substrate, crystalline cellulose, remains a great challenge.
F. Xu; A.E. Palmer; D.S. Yaver; R.M. Berka; G.A. Gambetta; S.H. Brown; E.I. Solomon.
"Targeted mutations in a Trametes villosa laccase: Axial perturbations of the T1 copper."
J. Biol. Chem., 274, 12372-12375 (1999)
Trametes villosa laccase was mutated on a tetrapeptide segment near the type 1 site. The mutations F463M and F463L were at the position corresponding to the type 1 copper axial methionine (M517) ligand in Zucchini ascorbate oxidase. The mutations E460S and A461E were near the T1 copper site. The mutated Trametes laccases were expressed in an Aspergillus oryzae host and characterized. The E460S mutation failed to produce a transformant with meaningful expression. The F463L and A461E mutations did not significantly alter the molecular and enzymological properties of the laccase. In contrast, the F463M mutation resulted in a type 1 copper site with an EPR signal intermediate between that of the wild type laccase and plastocyanin, an altered UV-visible spectrum, and a decreased redox potential (by 0.1 V). In oxidizing phenolic substrate, the mutation led to a more basic optimal pH as well as an increase in kcat and Km. These effects are attributed to a significant perturbation of the T1 copper center caused by the coordination of the axial methionine (M463) ligand.
F. Xu; R.M. Berka; J.A. Wahleithner; B.A. Nelson; J.R. Shuster; S.H. Brown; A.E. Palmer; E.I. Solomon.
"Site-directed mutations in fungal laccase: Effect on redox potential, activity and pH profile."
Biochem. J., 334, 63-70 (1998)
A Myceliophthora thermophila laccase and a Rhizoctonia solani laccase were mutated on a pentapeptide segment believed to be near the type-1 Cu site. The mutation L513F in Myceliophthora laccase and the mutation L470F in Rhizoctonia laccase took place at a position corresponding to the type-1 Cu axial methionine (M517) ligand in Zucchini ascorbate oxidase. The triple mutations V509L,S510E,G511A in Myceliophthora laccase and L466V,E467S,A468G in Rhizoctonia laccase involved a sequence segment whose homologue in ascorbate oxidase is flanked by the M517 and a type-1 Cu-ligating histidine (H512). The single mutation did not yield significant changes in the enzymic properties (including any significant increase in the redox potential of the type-1 Cu). In contrast, the triple mutation resulted in several significant changes. In comparison with the wild type, the Rhizoctonia and Myceliophthora laccase triple mutants had a phenol-oxidase activity whose pH optimum shifted 1 unit lower and higher, respectively. Although the redox potentials were not significantly altered, the Km, kcat and fluoride inhibition of the laccases were greatly changed by the mutations. The observed effects are interpreted as possible mutation-induced structural perturbations on the molecular recognition between the reducing substrate and laccase and on the electron transfer from the substrate to the type-1 Cu centre.