Magnetic Combined Cross-Linked Enzyme Aggregates of Ketoreductase and Alcohol Dehydrogenase: An Efficient and Stable Biocatalyst for Asymmetric Synthesis of (R)-3-Quinuclidinol with Regeneration of Coenzymes In Situ

Chen, Yuhan; Jiang, Qi; Sun, Lili; Li, Qiang; Zhou, Liping; Chen, Qian; Li, Shanshan; Yu, Miao; Li, Wei

doi:10.3390/catal8080334

Cited by 17 publications

(10 citation statements)

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“…In addition, the use of extra polymers as a carrier leads to a ‘dilution of activity’ of the immobilized enzymes by introducing a huge portion of non‐catalytic ballast, which consequently results in lower space–time yields and lower productivities 20 . On the contrary, recent studies involving various enzymes immobilized as CLEAs have demonstrated activity recovery of more than 80% 21–23 . Owing to the low activity recovery and specific activity of resin‐bound immobilization, the carrier‐free method was further applied to immobilize muSDR.…”

Section: Discussionmentioning

confidence: 99%

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Efficient synthesis of an antiviral drug intermediate using overexpressed short‐chain dehydrogenase and cross‐linked enzyme aggregates stabilization

Yang

et al. 2020

J of Chemical Tech & Biotech

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BACKGROUND (2R,3S)‐N‐tert‐Butoxycarbonyl‐3‐amino‐1‐chloro‐2‐hydroxy‐4‐phenylbutane (1b) is key for the synthesis of the antiviral drug atazanavir. It can be obtained via the stereoselective bioreduction of (3S)‐3‐(N‐Boc‐amino)‐1‐chloro‐4‐phenyl‐butanone (1a) with short‐chain dehydrogenase/reductase (SDR) (EC 1.1.1.1). To develop a practical and cost‐effective biocatalytic approach with high stability and reusability, an SDR mutant (muSDR) from Novosphingobium aromaticivorans was overexpressed in Escherichia coli cell and immobilized by cross‐linked enzyme aggregates with enhanced stability and high efficiency. RESULTS muSDR gene was successfully cloned and overexpressed in Escherichia coli and reduced 1a to 1b with high stereoselectivity. Four resin‐bound muSDR and cross‐linked muSDR aggregates (muSDR‐CLEAs) were prepared and compared. With addition of Tween‐80, the muSDR‐CLEAs demonstrated much higher activity recovery than resin‐bound muSDR. Compared with free enzyme, muSDR‐CLEAs demonstrated enhanced thermal, pH, and storage stabilities and could obtain 1b with high substrate concentration, high de, and complete conversion. The reusability of CLEAs was also determined as no significant activity loss was observed after seven batches of reactions. The space time yield of the bioreduction using muSDR‐CLEAs was 226.6 g∙L−1∙day−1. CONCLUSION Overexpressed muSDR was efficiently immobilized, and it demonstrated superior stability and reusability. The recombinant muSDR and muSDR‐CLEAs are practical potential biocatalysts for industrial production of the atazanavir precursor. © 2020 Society of Chemical Industry (SCI)

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Section: Discussionmentioning

confidence: 99%

“…20 On the contrary, recent studies involving various enzymes immobilized as CLEAs have demonstrated activity recovery of more than 80%. [21][22][23] Owing to the low activity recovery and specific activity of resin-bound immobilization, the carrier-free method was further applied to immobilize muSDR.…”

Section: Discussionmentioning

confidence: 99%

Efficient synthesis of an antiviral drug intermediate using overexpressed short‐chain dehydrogenase and cross‐linked enzyme aggregates stabilization

Yang

et al. 2020

J of Chemical Tech & Biotech

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“…[73] It has been used, for example, in the co-immobilizationo faKRED and an ADH. [74] Nature also utilizes enzyme immobilization in the form of self-assembly of protein-based scaffolds, such as cellulosomes and microcompartment shell proteins,t op romote their stabilization and spatial organization,c reate optimum microenvironments forreactionsequences, and increase product yields. [75]…”

Section: Betterformulation and Recycling Of Enzymes Through Immobilizmentioning

confidence: 99%

Broadening the Scope of Biocatalysis in Sustainable Organic Synthesis

2019

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This Review is aimed at synthetic organic chemists who may be familiar with organometallic catalysis but have no experience with biocatalysis, and seeks to provide an answer to the perennial question: if it is so attractive, why wasn't it extensively used in the past? The development of biocatalysis in industrial organic synthesis is traced from the middle of the last century. Advances in molecular biology in the last two decades, in particular genome sequencing, gene synthesis and directed evolution of proteins, have enabled remarkable improvements in scope and substantially reduced biocatalyst development times and cost contributions. Additionally, improvements in biocatalyst recovery and reuse have been facilitated by developments in enzyme immobilization technologies. Biocatalysis has become eminently competitive with chemocatalysis and the biocatalytic production of important pharmaceutical intermediates, such as enantiopure alcohols and amines, has become mainstream organic synthesis. The synthetic space of biocatalysis has significantly expanded and is currently being extended even further to include new‐to‐nature biocatalytic reactions.

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“…Magnetic CLEAs of other enzymes have been described. For example, m-combi-CLEAs have been prepared from ketoreductases (KREDs) and glucose dehydrogenase for cofactor regeneration with glucose and used in the synthesis of chiral alcohols [149,206,207]. Cui and coworkers [170] prepared m-CLEAs of phenyl alanine ammonia lyase (PAL EC 4.3.1.24) and showed that storage and operational stabilities were superior to those of the simple CLEA.…”

Section: Other Potential Applicationsmentioning

confidence: 99%

CLEAs, Combi-CLEAs and ‘Smart’ Magnetic CLEAs: Biocatalysis in a Bio-Based Economy

2019

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Biocatalysis has emerged in the last decade as a pre-eminent technology for enabling the envisaged transition to a more sustainable bio-based economy. For industrial viability it is essential that enzymes can be readily recovered and recycled by immobilization as solid, recyclable catalysts. One method to achieve this is via carrier-free immobilization as cross-linked enzyme aggregates (CLEAs). This methodology proved to be very effective with a broad selection of enzymes, in particular carbohydrate-converting enzymes. Methods for optimizing CLEA preparations by, for example, adding proteic feeders to promote cross-linking, and strategies for making the pores accessible for macromolecular substrates are critically reviewed and compared. Co-immobilization of two or more enzymes in combi-CLEAs enables the cost-effective use of multiple enzymes in biocatalytic cascade processes and the use of "smart" magnetic CLEAs to separate the immobilized enzyme from other solids has raised the CLEA technology to a new level of industrial and environmental relevance. Magnetic-CLEAs of polysaccharide-converting enzymes, for example, are eminently suitable for use in the conversion of first and second generation biomass.Catalysts 2019, 9, 261 2 of 31 and deprotection steps. This affords synthetic routes that, compared with conventional organic syntheses, are more step economic, more energy efficient, generate less waste and provide products in exquisite stereochemical purities that are difficult to compete with.Consequently, in the last two decades biocatalysis has emerged as an important technology for meeting the growing demand for green and sustainable processing [2][3][4]. This embraces the whole gamut of chemicals manufacture, from the enantioselective synthesis of chiral drugs [5][6][7][8][9][10][11][12] to the conversion of renewable biomass to liquid fuels and commodity chemicals [13][14][15]. This raises the perennial question: if biocatalysis is so superior why has it not been extensively used in the past? The answer is simple: thanks to advances in molecular biology and biotechnology, biocatalysis has undergone a spectacular leap forward in the last two decades:-Many more enzymes have been identified through (meta)genome mining, that is the in silico analysis of publicly accessible genome sequence data bases that have been generated as a result of next generation genome sequencing [16]. -Advances in gene synthesis have enabled the synthesis of identified genes, ready for cloning into a host production organism, in a few weeks at relatively low cost. This has significantly reduced the cost of development and subsequent production of enzymes at industrial scale. -Advances in protein engineering, using directed evolution techniques [17,18], have enabled optimization of enzyme performance under the challenging conditions encountered in industrial-scale processes, namely, high (stereo)selectivities, activities and space-time yields with non-natural substrates at high substrate concentrations, in the presence of organic solve...

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Magnetic Combined Cross-Linked Enzyme Aggregates of Ketoreductase and Alcohol Dehydrogenase: An Efficient and Stable Biocatalyst for Asymmetric Synthesis of (R)-3-Quinuclidinol with Regeneration of Coenzymes In Situ

Cited by 17 publications

References 58 publications

Efficient synthesis of an antiviral drug intermediate using overexpressed short‐chain dehydrogenase and cross‐linked enzyme aggregates stabilization

Efficient synthesis of an antiviral drug intermediate using overexpressed short‐chain dehydrogenase and cross‐linked enzyme aggregates stabilization

Broadening the Scope of Biocatalysis in Sustainable Organic Synthesis

CLEAs, Combi-CLEAs and ‘Smart’ Magnetic CLEAs: Biocatalysis in a Bio-Based Economy

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