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Nîmes, France

Bruhlmann F.,Firmenich | Fourage L.,Proteus | Fourage L.,Total S.A. | Ullmann C.,Proteus | And 4 more authors.
Journal of Biotechnology | Year: 2014

Directed evolution via iterative cycles of random and targeted mutagenesis was applied to the P450 domain of the subterminal fatty acid hydroxylase CYP102A1 of Bacillus megaterium to shift its regioselectivity towards the terminal position of palmitic acid. A powerful and versatile high throughput assay based on LC-MS allowed the simultaneous detection of primary and secondary oxidation products, which was instrumental for identifying variants with a strong preference for the terminal oxidation of palmitic acid. The best variants identified acquired up to 11 amino acid alterations. Substitutions at F87, I263, and A328, relatively close to the bound substrate based on available crystallographic information contributed significantly to the altered regioselectivity. However, non-obvious residues much more distant from the bound substrate showed surprising strong contributions to the increased selectivity for the terminal position of palmitic acid. © 2014 Elsevier B.V.

Bruhlmann F.,Firmenich | Bosijokovic B.,Firmenich | Ullmann C.,Proteus | Auffray P.,Proteus | And 3 more authors.
Journal of Biotechnology | Year: 2013

The performance of a 13-hydroperoxide lyase from guava, an enzyme of the CYP74 family, which is of interest for the industrial production of saturated and unsaturated C6-aldehydes and their derivatives, was improved by directed evolution. Four rounds of gene shuffling and random mutagenesis improved the functional expression in E. coli by offering a 15-fold higher product yield factor. The increased product yield factor relates to an improved total turnover number of the variant enzyme, which also showed higher solubility and increased heme content. Thermal stability was also dramatically improved even though there was no direct selection pressure applied for evolving this trait. A structure based sequence alignment with the recently solved allene oxide synthase of Arabidopsis thaliana showed that most amino acid alterations occurred on the surface of the protein, distant of the active site and often outside of secondary structures. These results demonstrate the power of directed evolution for improving a complex trait such as the total turnover number of a cytochrome P450, a critical parameter for process performance that is difficult to predict even with good structural information at hand. © 2012 Elsevier B.V.

Quemeneur M.,French National Institute for Agricultural Research | Quemeneur M.,Aix - Marseille University | Bittel M.,French National Institute for Agricultural Research | Trably E.,French National Institute for Agricultural Research | And 5 more authors.
International Journal of Hydrogen Energy | Year: 2012

Wheat straw is an abundant agricultural residue which can be used as raw material to produce hydrogen (H 2), a promising alternative energy carrier, at a low cost. Bioconversion of lignocellulosic biomass to produce H 2 usually involves three main operations: pretreatment, hydrolysis and fermentation. In this study, the efficiency of exogenous enzyme addition on fermentative H 2 production from wheat straw was evaluated using mixed-cultures in two experimental systems: a one-stage system (direct enzyme addition) and a two-stage system (enzymatic hydrolysis prior to dark fermentation). H 2 production from untreated wheat straw ranged from 5.18 to 10.52 mL-H 2 g-VS -1. Whatever the experimental enzyme addition procedure, a two-fold increase in H 2 production yields ranging from 11.06 to 19.63 mL-H 2 g-VS -1 was observed after enzymatic treatment of the wheat straw. The high variability in H 2 yields in the two step process was explained by the consumption of free sugars by indigenous wheat straw microorganisms during enzymatic hydrolysis. The direct addition of exogenous enzymes in the one-stage dark fermentation stage proved to be the best way of significantly improving H 2 production from lignocellulosic biomass. Finally, the optimal dose of enzyme mixture added to the wheat straw was evaluated between 1 and 5 mg-protein g-raw wheat straw -1. © 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.

Proteus, French Institute of Petroleum and French National Center for Scientific Research | Date: 2013-09-04

The invention relates to a polypeptide which has enhanced beta-glucosidase activity at a temperature of between about 30 C. and about 35 C.

The invention provides for methods of generating modified polynucleotide libraries by inserting and/or deleting at least three nucleotide residues in polynucleotide sequences. Theses methods may be used with other methods of gene modification such as gene shuffling. The invention further provides methods of directed molecular evolution using the modified polynucleotide libraries produced by these methods.

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