High-Throughput Screening of Thermostable Esterases for Industrial Bioconversions

Lagarde, Delphine; Nguyen, Hong-Khanh; Ravot, Gilles; Wahler, Denis; Reymond, Jean‐Louis; Hills, G. Loftus; Veit, T.; Lefèvre, Fabrice

doi:10.1021/op020019h

Cited by 49 publications

(21 citation statements)

References 14 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, if the reaction can be performed at higher temperature at which the substrates or products become liquid, there is no need to add (and remove at the end) organic solvents. Therefore, the reaction can be performed at lower cost (by reducing the product recovery process) in an environmental friendly process (by reducing harsh chemical conditions) (Lagarde et al, 2002).…”

Section: Protéus's Toolboxmentioning

confidence: 99%

Enzymes for industrial applications

Robic¹,

Ullmann²,

Auffray³

et al. 2017

OCL

View full text Add to dashboard Cite

-Protéus is a biotechnology company specializing in the discovery, engineering and production of enzymes for industrial applications, as well as in the development of innovative bioprocesses involving these enzymes. Protéus is a subsidiary of the PCAS Group, actor in fine chemicals and specialty products and producer of high-value complex molecules. Enzymes allow considering unique functionalizations that are difficult to achieve by conventional chemical means. Examples involving the screening of our ready to use toolbox of lipases, the engineering of the well-known lipase CalB and the specific modifications of lipids will be presented. Keywords: lipids / lipases / protein engineering / bioprocesses / biocatalysisRésumé -Développement d'enzymes pour des applications industrielles. Protéus est une société de biotechnologie spécialisée dans la découverte, l'ingénierie et la production d'enzymes pour des applications industrielles, ainsi que dans le développement de bioprocédés innovants mettant en oeuvre ces enzymes. Protéus est une filiale du groupe PCAS actif dans le développement, la fabrication et la commercialisation de produits de chimie fine et de spécialités. Les enzymes permettent de produire de nouvelles molécules dans un contexte de chimie verte (économie d'atomes, conditions douces de mise en oeuvre, amélioration de la sélectivité, réduction de la toxicité) et d'envisager des fonctionnalisations uniques qui sont difficiles à obtenir par des moyens chimiques classiques. Des exemples de réalisation seront présentés avec notamment le criblage de notre toolbox enzymatique, l'ingénierie de la lipase issue de Candida antarctica et la fonctionnalisation de liaisons CH non activées.

show abstract

Section: Protéus's Toolboxmentioning

confidence: 99%

Enzymes for industrial applications

Robic¹,

Ullmann²,

Auffray³

et al. 2017

OCL

View full text Add to dashboard Cite

show abstract

“…After centrifugation (4 min at 4000 g) and resuspension in 50 mL of 200 mM PIPES buffer at pH 7.0, cells were incubated for 1 hour at 90 8C. After this incubation, residual activities were determined by incubating the cells at 60 8C with a synthetic C10 ester CLIPS-O TM as substrate (as described by Lagarde et al [14] ), and reading the corresponding absorbance at 414 nm. These values were compared to the value obtained with cells expressing the WT-CALB tested in the same conditions.…”

Section: Screening Of Enzyme Variants (Gfp and Lipase B)mentioning

confidence: 99%

“…After centrifugation (4 min at 4000 g), E. coli MC1061(DE3) clones expressing the CALB improved lipase variants were resuspended in 50 mL of 200 mM PIPES buffer at pH 7.0 and incubated at different times: 5, 10, 15 and 30 min at 90 8C. Residual activities were determined at 60 8C using the synthetic C 10 ester CLIPS-O TM as substrate (as described by Lagarde et al [14] ), and reading the corresponding absorbance at 414 nm.…”

Section: Characterization Of Calb Variantsmentioning

confidence: 99%

Rational Strategies for Directed Evolution of Biocatalysts –Application to Candida antarctica lipase B (CALB)

Chodorge¹,

Fourage²,

Ullmann³

et al. 2005

Adv Synth Catal

View full text Add to dashboard Cite

Provided that the industrial constraints have been properly defined, the directed evolution technologies available today enable one to design tailormade enzymes and biocatalytic routes for chemical processes. Family shuffling has proved to be a successful strategy using traditional recombination protocols. However, when starting from a single gene, the first step is to create the appropriate population of parental genes to ensure an efficient recombination during gene shuffling. Our recent work focused on the determination of rational directed evolution strategies that can be applied for the creation of an improved biocatalyst within the requested industrial timelines. For this purpose, we have developed a rational approach that first explores the "protein plasticity" (ability of the protein to accept mutations with a limited loss of activity) of the enzyme, which enables us to estimate (i) the "optimal mutation load" (number of mutations introduced per gene that gives the highest frequency of improved variants) and (ii) the "ad minima size sample" (minimal number of clones to be screened) that can be used to rapidly improve this enzyme. We have then applied this approach to create in a few weeks variants of the well known lipase B with a seven fold improvement in thermostability.

show abstract

“…For example, organic solvents are often needed to solubilize substrates and products. If the reaction can be performed at higher temperature, there is no need to add (and remove at the end) organic solvents, therefore the reaction can be performed at lower cost in an environmental friendly process [10]. From this point of view, the exploration for new microbial enzyme sources is vital for the advancement of new thermostable and organic solvent resistant enzymes and their applications [11].…”

Section: Introductionmentioning

confidence: 99%

Purification and characterization of a novel thermostable esterase from Thermus sp. NCCB 100425T

Akatin

2015

Turk J Bioch

View full text Add to dashboard Cite

Purification and characterization of a novel thermostable esterase from Thermus sp. NCCB 100425 T [Thermus sp. NCCB 100425 T ' den yeni bir ısıya dayanıklı esterazın saflaştırılması ve karakterizasyonu] ABSTRACT Objective: The purpose of the present study was to purify and characterize an esterase from a thermophilic bacterium Thermus sp. NCCB 100425 T and to check its suitability for industrial applications. Methods: Thermus sp. NCCB 100425 T esterase was purified by using ammonium sulphate precipitation and hydrophobic interaction chromatography and then characterized biochemically. Results: The purity of the enzyme was observed as a single band on native-and SDS-PAGE. In the presence of p-nitrophenyl acetate (pNPA) as a substrate, the optimum pH and temperature of the enzyme were found to be 7.5 and 60°C, respectively. K m and V max values are calculated as 18.32 mM and 96.15 U/mg protein, respectively, with pNPA. pH stability was investigated in the range of pH 4.0-9.0 at 4°C and 60°C. After 7 days incubation, activity of pure enzyme was retained 85-90% for all pH at 4°C and 59±5.2% for pH 8.0 at 60°C. It was determined that approximately 80% of enzyme activity was retained between 30-60°C after 7 days incubation. In the presence of 10% ethanol and DMSO, the enzyme activity was retained 96±2.7% and 78±2.5%, respectively. Additionally, it was detected that some metal ions affect the enzyme activity at different ratios. T esterazı amonyum sülfat çöktürmesi ve hidrofobik etkileşim kromatografisi kullanılarak saflaştırıldı ve biyokimyasal özellikleri incelendi. Bulgular: Doğal-ve SDS-PAGE'de tek bant şeklinde görülen saf enzimin, p-nitrofenil asetat (pNPA) varlığında optimum pH ve sıcaklık değerleri sırasıyla 7,5 ve 60°C olarak belirlendi. Yine pNPA bir substrat olarak kullanıldığında K m ve V max değerlerinin 18,32 mM ve 96,15 U/ mg protein olduğu tespit edildi. pH kararlılığı pH 4,0-9,0 aralığında 4°C ve 60°C'de incelendi. 7 günlük bir inkübasyondan sonra saf enzimin aktivitesinin 4°C'de bütün pH'larda yaklaşık %85-90 oranında korunduğu, bu oranın 60°C'de pH 8,0'de ise %59±5,2 olduğu görüldü. 30-60°C'ler arasında 7 gün inkübe edilen enzim, orijinal aktivitesini %80 oranında korudu. %10 nihai konsantrasyondaki etanol ve DMSO varlığında enzim aktivitesi sırasıyla %96±2,7 ve %78±2,5 oranlarında korundu. Ayrıca, bazı metal iyonlarının enzim aktivitesini farklı oranlarda etkilediği tespit edildi. Sonuç: Bütün bu sonuçlar değerlendirildiğinde Thermus sp. NCCB 100425 T esterazının özel-likle yüksek sıcaklık-ve pH-kararlılığı nedeniyle endüstriyel ve/veya klinik uygulamalar için avantajlara sahip olabileceği açıkça görülmektedir. Thus, this study can be considered as a step to obtain an industrially suitable esterase having excellent features. ) and solvents were purchased from Merck A.G. (Darmstadt, Germany). All chemicals were analytical and microbiological grade. Conclusion Materials and Methods Chemicals Bacterial strain and crude enzyme extractionThermus sp. NCCB 100425 T bacterial strain isolated from a geotherm...

show abstract

High-Throughput Screening of Thermostable Esterases for Industrial Bioconversions

Cited by 49 publications

References 14 publications

Enzymes for industrial applications

Enzymes for industrial applications

Rational Strategies for Directed Evolution of Biocatalysts –Application to Candida antarctica lipase B (CALB)

Purification and characterization of a novel thermostable esterase from Thermus sp. NCCB 100425T

Contact Info

Product

Resources

About