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Improved Variants (improved + variants)
Selected AbstractsA Diversified Library of Bacterial and Fungal Bifunctional Cytochrome P450 Enzymes for Drug Metabolite SynthesisADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 13 2009Roland Weis Abstract Innovative biohydroxylation catalysts for the preparation of drug metabolites were developed from scratch. A set of bacterial and fungal sequences of putative and already known bifunctional P450 enzymes was identified by protein sequence alignments, expressed in Escherichia coli and characterised. Notably, a fungal self-sufficient cytochrome P450 (CYP) from Aspergillus fumigatus turned out to be especially stable during catalyst preparation and application and also in presence of organic co-solvents. To enhance the catalytic activity and broaden the substrate specificity of those variants with high expression levels prominent single mutations were introduced. Selected improved variants were then used as lyophilised bacterial lysates for the synthesis of 4,-hydroxydiclofenac and 6-hydroxychlorzoxazone, the two metabolites of active pharmaceutical compounds diclofenac and chlorzoxazone representing the same metabolites as generated by human P450s. [source] Nature versus nurture in two highly enantioselective esterases from Bacillus cereus and Thermoanaerobacter tengcongensisMICROBIAL BIOTECHNOLOGY, Issue 1 2010Stephan Grosse Summary There is an increasing need for the use of biocatalysis to obtain enantiopure compounds as chiral building blocks for drug synthesis such as antibiotics. The principal findings of this study are: (i) the complete sequenced genomes of Bacillus cereus ATCC 14579 and Thermoanaerobacter tengcongensis MB4 contain a hitherto undescribed enantioselective and alkaliphilic esterase (BcEST and TtEST respectively) that is specific for the production of (R)-2-benzyloxy-propionic acid ethyl ester, a key intermediate in the synthesis of levofloxacin, a potent antibiotic; and (ii) directed evolution targeted for increased thermostability of BcEST produced two improved variants, but in either case the 3,5°C increase in the apparent melting temperature (Tm) of the mutants over the native BcEST that has a Tm of 50°C was outperformed by TtEST, a naturally occurring homologue with a Tm of 65°C. Protein modelling of BcEST mapped the S148C and K272R mutations at protein surface and the I88T and Q110L mutations at more buried locations. This work expands the repertoire of characterized members of the ,/,-fold hydrolase superfamily. Further, it shows that genome mining is an economical option for new biocatalyst discovery and we provide a rare example of a naturally occurring thermostable biocatalyst that outperforms experimentally evolved homologues that carry out the same hydrolysis. [source] Combinatorial engineering to enhance thermostability of amylosucrasePROTEIN SCIENCE, Issue 6 2008Stéphane Emond Abstract Amylosucrase is a transglucosidase that catalyzes amylose-like polymer synthesis from sucrose substrate. About 60,000 amylosucrase variants from two libraries generated by the MutaGen random mutagenesis method were submitted to an in vivo selection procedure leading to the isolation of more than 7000 active variants. These clones were then screened for increased thermostability using an automated screening process. This experiment yielded three improved variants (two double mutants and one single mutant) showing 3.5- to 10-fold increased half-lives at 50°C compared to the wild-type enzyme. Structural analysis revealed that the main differences between wild-type amylosucrase and the most improved variant (R20C/A451T) might reside in the reorganization of salt bridges involving the surface residue R20 and the introduction of a hydrogen-bonding interaction between T451 of the B, domain and D488 of flexible loop 8. This double mutant is the most thermostable amylosucrase known to date and the only one usable at 50°C. At this temperature, amylose synthesis by this variant using high sucrose concentration (600 mM) led to the production of amylose chains twice as long as those obtained by the wild-type enzyme at 30°C. [source] Microbial bio-production of a recombinant stimuli-responsive biosurfactantBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2009W. Kaar Abstract Biosurfactants have been the subject of recent interest as sustainable alternatives to petroleum-derived compounds in areas ranging from soil remediation to personal and health care. The production of naturally occurring biosurfactants depends on the presence of complex feed sources during microbial growth and requires multicomponent enzymes for synthesis within the cells. Conversely, designed peptide surfactants can be produced recombinantly in microbial systems, enabling the generation of improved variants by simple genetic manipulation. However, inefficient downstream processing is still an obstacle for the biological production of small peptides. We present the production of the peptide biosurfactant GAM1 in recombinant E. coli. Expression was performed in fusion to maltose binding protein using chemically defined minimal medium, followed by a single-step affinity capture and enzymatic cleavage using tobacco etch virus protease. Different approaches to the isolation of peptide after cleavage were investigated, with special emphasis on rapid and simple procedures. Solvent-, acid-, and heat-mediated precipitation of impurities were successfully applied as alternatives to post-cleavage chromatographic peptide purification, and gave peptide purities exceeding 90%. Acid precipitation was the method of choice, due to its simplicity and the high purification factor and recovery rate achieved here. The functionality of the bio-produced peptide was tested to ensure that the resulting peptide biosurfactant was both surface active and able to be triggered to switch between foam-stabilizing and foam-destabilizing states. Biotechnol. Bioeng. 2009;102: 176,187. © 2008 Wiley Periodicals, Inc. [source] | |||||||||||||