Stable and efficient immobilization technique of aldolase under consecutive microwave irradiation at low temperature

Tiwari

et al. 2013

IJMS

387

195

Enzymes found in nature have been exploited in industry due to their inherent catalytic properties in complex chemical processes under mild experimental and environmental conditions. The desired industrial goal is often difficult to achieve using the native form of the enzyme. Recent developments in protein engineering have revolutionized the development of commercially available enzymes into better industrial catalysts. Protein engineering aims at modifying the sequence of a protein, and hence its structure, to create enzymes with improved functional properties such as stability, specific activity, inhibition by reaction products, and selectivity towards non-natural substrates. Soluble enzymes are often immobilized onto solid insoluble supports to be reused in continuous processes and to facilitate the economical recovery of the enzyme after the reaction without any significant loss to its biochemical properties. Immobilization confers considerable stability towards temperature variations and organic solvents. Multipoint and multisubunit covalent attachments of enzymes on appropriately functionalized supports via linkers provide rigidity to the immobilized enzyme structure, ultimately resulting in improved enzyme stability. Protein engineering and immobilization techniques are sequential and compatible approaches for the improvement of enzyme properties. The present review highlights and summarizes various studies that have aimed to improve the biochemical properties of industrially significant enzymes.

Section: Immobilization To Upgrade Industrial Enzymesmentioning

confidence: 99%

“…Microwave irradiation technology has been reported by several researchers to overcome the diffusion limitation. For instance, penicillin acylase from Bacillus megaterium was covalently immobilized on mesocellular silica foams [155]. …”

Section: Immobilization To Upgrade Industrial Enzymesmentioning

confidence: 99%

From Protein Engineering to Immobilization: Promising Strategies for the Upgrade of Industrial Enzymes

Tiwari

et al. 2013

IJMS

387

195

J of Chemical Tech & Biotech

“…Thus, large quantities of the enzyme are necessary to obtain industrially useful product yields . This issue can be circumvented by improving its resistance and catalytic activity using a molecular evolution approach, or by applying a cell‐free lysate or a whole‐cell biocatalyst instead of an isolated enzyme or by immobilization on appropriate supports …”

Section: Introductionmentioning

confidence: 99%

2‐Deoxyribose‐5‐phosphate aldolase from Thermotoga maritima in the synthesis of a statin side‐chain precursor: characterization, modeling and optimization

Švarc

Blažević

Vasić-Rački

et al. 2019

BACKGROUND Producing statin side‐chains by sequential aldol condensation catalyzed by 2‐deoxyribose‐5‐phosphate aldolase (DERA) (EC 4.1.2.4) is the best‐known and most promising synthetic route. The advantage of this process is the formation of two stereocenters in a single step starting from inexpensive achiral components, acetaldehyde and chloroacetaldehyde. The limitation for the industrial‐scale application is enzyme inactivation caused by substrates as well by the intermediate produced by single aldol addition. RESULTS The DERA enzyme from Thermotoga maritima (DERATm) was investigated extensively in this work. The influence of aldehydes on DERATm stability was studied in detail. Mathematical models in different reactor configurations were developed based on experimentally determined kinetic parameters of all reactions included in the synthesis of statin side‐chain. Models also included enzyme inactivation by all compounds with negative impact on its stability. Mathematical model‐based optimization enabled optimization of process conditions and reactor configuration. The fed‐batch reactor proved to be the most suitable choice achieving product concentration of 78 g L−1, productivity of 56 g L day−1 and yield of 95% under an optimal condition. CONCLUSION The validated mathematical model and applied methodology can be exploited for further process improvement and for process design of similar systems. © 2019 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

“…The problem of DERA inactivation in the presence of aldehydes has been addressed before either by employing random (epPCR, DNA shuffling) and/or targeted mutagenesis [61], [63] or, alternatively, by screening of environmental DNA libraries [44] and immobilization procedures [64]–[66]. The immobilization approach aimed at improving stability of DERA was attempted also in our laboratory (unpublished data), however this approach was found to be economically unfavorable.…”

Section: Introductionmentioning

confidence: 99%

A Highly Productive, Whole-Cell DERA Chemoenzymatic Process for Production of Key Lactonized Side-Chain Intermediates in Statin Synthesis

Ošlaj¹,

Jérôme²,

Orkić³

et al. 2013

PLoS ONE

Employing DERA (2-deoxyribose-5-phosphate aldolase), we developed the first whole-cell biotransformation process for production of chiral lactol intermediates useful for synthesis of optically pure super-statins such as rosuvastatin and pitavastatin. Herein, we report the development of a fed-batch, high-density fermentation with Escherichia coli BL21 (DE3) overexpressing the native E. coli deoC gene. High activity of this biomass allows direct utilization of the fermentation broth as a whole-cell DERA biocatalyst. We further show a highly productive bioconversion processes with this biocatalyst for conversion of 2-substituted acetaldehydes to the corresponding lactols. The process is evaluated in detail for conversion of acetyloxy-acetaldehyde with the first insight into the dynamics of reaction intermediates, side products and enzyme activity, allowing optimization of the feeding strategy of the aldehyde substrates for improved productivities, yields and purities. The resulting process for production of ((2S,4R)-4,6-dihydroxytetrahydro-2H-pyran-2-yl)methyl acetate (acetyloxymethylene-lactol) has a volumetric productivity exceeding 40 g L−1 h−1 (up to 50 g L−1 h−1) with >80% yield and >80% chromatographic purity with titers reaching 100 g L−1. Stereochemical selectivity of DERA allows excellent enantiomeric purities (ee >99.9%), which were demonstrated on downstream advanced intermediates. The presented process is highly cost effective and environmentally friendly. To our knowledge, this is the first asymmetric aldol condensation process achieved with whole-cell DERA catalysis and it simplifies and extends previously developed DERA-catalyzed approaches based on the isolated enzyme. Finally, applicability of the presented process is demonstrated by efficient preparation of a key lactol precursor, which fits directly into the lactone pathway to optically pure super-statins.