Recent progress in biocatalyst discovery and optimization

Robertson, Dan E.; Steer, Brian A.

doi:10.1016/j.cbpa.2004.02.010

Cited by 77 publications

(33 citation statements)

References 55 publications

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“…Automation of the screening is particularly welcome as screening "hits" are typically low (less than two out of a total of 10,000 clones have industrially relevant enzymes) (Lorenz and Eck 2005). However, the screening hit rate can be improved by: (1) the use of different expression hosts, (2) the use of diverse strategies to enrich/select for community genomes with desired traits before metagenomic library construction, (3) the use of liquid enrichment cultures for the screening of multiple clones simultaneously, and (4) the development of novel high-throughput screening strategies compatible with fluorescence-activated cell sorting of the clones (throughput of 10 9 events per day) (Robertson and Steer 2004). In the sequence-driven analysis, clones of interest are identified based on the presence of conserved regions in phylogenetic marker genes (e.g., 16S rRNA genes) or in functional genes coding for key processes, such as oxygenases (Erwin et al 2005).…”

Section: Metagenomicsmentioning

confidence: 99%

Molecular Tools for Monitoring and Validating Bioremediation

et al. 2009

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Section: Metagenomicsmentioning

confidence: 99%

Molecular Tools for Monitoring and Validating Bioremediation

et al. 2009

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“…Applying these techniques and also rational protein design, the improvement of biocatalysts' activity, selectivity and stability are aimed (Bornscheuer and Pohl, 2001;Petrounia and Arnold, 2000;Polizzi et al, 2007;Robertson and Steer, 2004). To tap the full potential of these techniques, it is essential to combine them with adequate screening methods (Jaeger and Eggert, 2004;Reetz and Jaeger, 2002).…”

Section: Introductionmentioning

confidence: 99%

“Enzyme Test Bench,” a high‐throughput enzyme characterization technique including the long‐term stability

Rachinskiy

Schultze²,

Boy³

et al. 2009

Biotech & Bioengineering

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A new high throughput technique for enzyme characterization with specific attention to the long term stability, called "Enzyme Test Bench," is presented. The concept of the Enzyme Test Bench consists of short term enzyme tests in 96-well microtiter plates under partly extreme conditions to predict the enzyme long term stability under moderate conditions. The technique is based on the mathematical modeling of temperature dependent enzyme activation and deactivation. Adapting the temperature profiles in sequential experiments by optimal non-linear experimental design, the long term deactivation effects can be purposefully accelerated and detected within hours. During the experiment the enzyme activity is measured online to estimate the model parameters from the obtained data. Thus, the enzyme activity and long term stability can be calculated as a function of temperature. The engineered instrumentation provides for simultaneous automated assaying by fluorescent measurements, mixing and homogenous temperature control in the range of 10-85 +/- 0.5 degrees C. A universal fluorescent assay for online acquisition of ester hydrolysis reactions by pH-shift is developed and established. The developed instrumentation and assay are applied to characterize two esterases. The results of the characterization, carried out in microtiter plates applying short term experiments of hours, are in good agreement with the results of long term experiments at different temperatures in 1 L stirred tank reactors of a week. Thus, the new technique allows for both: the enzyme screening with regard to the long term stability and the choice of the optimal process temperature regarding such process parameters as turn over number, space time yield or optimal process duration. The comparison of the temperature dependent behavior of both characterized enzymes clearly demonstrates that the frequently applied estimation of long term stability at moderate temperatures by simple activity measurements after exposing the enzymes to elevated temperatures may lead to suboptimal enzyme selection. Thus, temperature dependent enzyme characterization is essential in primary screening to predict its long term behavior.

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“…Compared to chemical catalysts, enzymes have two key advantages. One is their high selectivity; the other is their great diversity in nature [10]. Whole-cell biocatalysis combines these benefits with simple, low-cost catalyst preparation and the possibility to develop efficient processes for cofactor regeneration and multistep conversions.…”

Section: Introductionmentioning

confidence: 99%

Enhanced uridine 5′-monophosphate production by whole cell of Saccharomyces cerevisiae through rational redistribution of metabolic flux

Liu

Chen

Liu

et al. 2011

Bioprocess Biosyst Eng

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A whole-cell biocatalytic process for uridine 5'-monophosphate (UMP) production from orotic acid by Saccharomyces cerevisiae was developed. To rationally redistribute the metabolic flux between glycolysis and pentose phosphate pathway, statistical methods were employed first to find out the critical factors in the process. NaH(2)PO(4), MgCl(2) and pH were found to be the important factors affecting UMP production significantly. The levels of these three factors required for the maximum production of UMP were determined: NaH(2)PO(4) 22.1 g/L; MgCl(2) 2.55 g/L; pH 8.15. An enhancement of UMP production from 6.12 to 8.13 g/L was achieved. A significant redistribution of metabolic fluxes was observed and the underlying mechanism was discussed.

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Recent progress in biocatalyst discovery and optimization

Cited by 77 publications

References 55 publications

Molecular Tools for Monitoring and Validating Bioremediation

Molecular Tools for Monitoring and Validating Bioremediation

“Enzyme Test Bench,” a high‐throughput enzyme characterization technique including the long‐term stability

Enhanced uridine 5′-monophosphate production by whole cell of Saccharomyces cerevisiae through rational redistribution of metabolic flux

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