Screening Of Biocatalysts Research Articles

Screening of biocatalysts for transformation of sucrose to fructooligosaccharides

• FTase production by molds, yeasts and bacteria is demonstrated. • FTase from A. flavus generated upto 63% (w/w) of FOS from sucrose. • Major fructooligosaccharides produced were GF 2 (kestose), GF 3 (nystose) and GF 4 (fructofuranosyl-nystose). Twenty microorganisms comprising of sixteen molds, two yeasts and two bacteria were evaluated for their ability to produce fructosyltransferase (FTase) and generate fructooligosaccharides (FOS) from sucrose. FTase production by these microorganisms was studied over a period of 120 h on medium containing 20% (w/v) sucrose as the sole carbon source. High FTase levels (35–31 U/ml) were observed in culture filtrates of Aspergillus flavus , Aspergillus niger , Aspergillus terreus and Penicillium islandicum . Higher concentrations of FOS were generated from 50% (w/v) sucrose using culture filtrates of A. flavus NFCCI 2364 (63.40%, w/w), A. niger SI 19 (54.94%, w/w), A. flavus NFCCI 2785 (44.61%, w/w), P. islandicum MTCC 4926 (43.56%, w/w), A. terreus NFCCI 2347 (24.17%, w/w) and Fusarium solani NFCCI 2315 (15.25%, w/w). Kestose, nystose and 1-fructofuranosyl nystose were the predominant oligosaccharides as revealed by HPLC analysis.

On the feasibility of in situ steroid biotransformation and product recovery in microchannels

Microchannel reactor technologies are gaining widespread use in a large range of areas, which comprise biotechnology and chemistry. The small volumes involved and the favorable mass and heat transfer inherent to these devices make them particularly useful for the screening of biocatalysts and rapid characterization of bioconversion systems. In the present work, the enzymatic oxidation of cholesterol to 4-cholesten-3-one performed within microchannels by cholesterol oxidase, was studied in a two-phase system, comprising an organic phase as substrate and product pool and an aqueous phase with dissolved enzyme. A mathematical model based on mass balances for cholesterol, 4-cholesten-3-one and dissolved oxygen concentrations, comprising double-substrate Michaelis–Menten kinetics and the velocity profile of two immiscible fluids, was developed in order to describe and predict the process of cholesterol oxidation. The numerical procedure of solving the non-linear 3D model was based on an implicit finite-difference method improved by non-equidistant differences. In a Y-shape microreactor geometry, roughly up to 70% conversion of cholesterol was achieved at residence times below 1 min. The suitable adjustment of the ratio of the fluid flow rates was performed by taking into account the viscosity of the fluids involved. This allowed for phase separation to be reestablished at the Y-shaped exit from the microreactor and thereby enabled in situ product separation from the aqueous phase containing the enzyme.

Miniaturization in Biocatalysis

The use of biocatalysts for the production of both consumer goods and building blocks for chemical synthesis is consistently gaining relevance. A significant contribution for recent advances towards further implementation of enzymes and whole cells is related to the developments in miniature reactor technology and insights into flow behavior. Due to the high level of parallelization and reduced requirements of chemicals, intensive screening of biocatalysts and process variables has become more feasible and reproducibility of the bioconversion processes has been substantially improved. The present work aims to provide an overview of the applications of miniaturized reactors in bioconversion processes, considering multi-well plates and microfluidic devices, update information on the engineering characterization of the hardware used, and present perspective developments in this area of research.

Quantitative matrix-assisted laser desorption/ionization mass spectrometry for the determination of enzyme activities

Quantitative matrix-assisted laser desorption/ionization (MALDI) time-of-flight (ToF) mass spectrometry (MS) was applied for the determination of concentrations of low-molecular-weight (<400 Da) substrates and products of enzyme-catalyzed reactions. Isotope-labeled and fluorinated internal standards were used for the quantification. Automated quantitative MALDI-ToF MS analysis of quenched samples allowed the direct and simultaneous observation of time-dependent decrease of substrate concentration and increase of product concentration without any need for prepurification or desalting steps. The results showed good agreement with established but more elaborate analytical methods. MALDI-ToF MS thus is an interesting alternative tool for the determination of enzyme activities. Due to automated and miniaturized measurement it is especially suitable for the screening of biocatalysts.

Screening of commercial enzymes for the enantioselective hydrolysis of R,S-naproxen ester

This study focused on the identification of suitable lipase or esterase activity for enantiomeric resolution of ( R,S)-naproxen. For an economically viable reaction the enantiomeric ratio ( E) should preferably be >100, while maximising the conversion will reduce the mass of material that requires racemisation and recycling. Hence the aim was to find an enzyme that yields ( S)-naproxen with an enantiomeric excess of more than 98%, a substrate conversion in excess of 40% of the racemate, and an E of >100. ( R,S)-Naproxen ethyl ester (NEE) (50 mg) was used as substrate for enzyme hydrolysis reactions at 37 °C for 4 h. Biocatalyst screening was performed in buffered aqueous solvent on a 1 ml scale. The reactions were stopped with 2 ml MeCN, filtered through cotton wool and analysed by HPLC to determine the percentage m/m and R/ S ratio. Eight commercially available enzymes were selected for optimisation of enantioselectivity through statistically designed experiments where the reaction conditions were varied. ChiroCLEC-CR from Altus and ESL001-01 from Diversa provided acceptable enantiomeric excess, but only ChiroCLEC-CR met the specification set for the enantiomeric ratio ( E).

Methods for biocatalyst screening.

Biocatalysts are now widely accepted as useful alternative tools to classic organic synthetic techniques for the regio- and enantioselective synthesis under mild reaction conditions in many fields of chemistry. The development of techniques for the rational or evolutionary design of novel or modified enzymes has increased the need for fast and reliable methods for the identification of the most powerful catalysts. We present a short overview on screening techniques in this area. Beside classical methods such as spectrophotometry and fluorimetry, a number of new approaches like methods based on the measurement of pH changes or IR-thermography have been recently developed. Additionally the use of electrospray and matrix-assisted laser desorption/ionization mass spectrometry has gained increasing influence in this field of biotechnology.

Construction of a Diverse Fab Expression Library from Autoimmunized Mice Based on an Improved Preparation of Cloning Arms from Bacteriophage Vectors: A New Library with Potential for Screening of Biocatalysts

This paper describes an improved procedure for the preparation of cloning arms from vectors Lc1 and Hc2 for the construction of cDNA libraries in phage δ for expression in E. coli of the combinatorial Fab fragments of the immunoglobulin repertoire from autoimmunized mice. It was found that annealing, dialysis and dephorphorylation after the restriction enzyme digestion of vectors were critical for the high efficient preparation of cloning arms. The construction of Fab cDNA libraries involves the use of primer-directed PCR amplification products of total RNA from autoimmunized mice. Fab production was confirmed by the immunoassay and sequence analysis. The partial sequenced genes for Fab fragments were different from each other in the FR4, CDR3 and FR3 regions, indicating a diverse nature of the library. This library may be used to screen for catalytic Fab's.