Application of whole cell rhodococcal biocatalysts in acrylic polymer manufacture

Rhodococci are ubiquitous in nature and their ability to metabolise a wide range of chemicals, many of which are toxic, has given rise to an increasing number of studies into their diverse use as biocatalysts. Indeed rhodococci have been shown to be especially good at degrading aromatic and aliphatic nitriles and amides and thus they are very useful for waste clean up where these toxic chemicals are present.The use of biocatalysts in the chemical industry has in the main been for the manufacture of high-value fine chemicals, such as pharmaceutical intermediates, though investigations into the use of nitrile hydratase, amidase and nitrilase to convert acrylonitrile into the higher value products acrylamide and acrylic acid have been carried out for a number of years. Acrylamide and acrylic acid are manufactured by chemical processes in vast tonnages annually and they are used to produce polymers for applications such as superabsorbents, dispersants and flocculants. Rhodococci are chosen for use as biocatalysts on an industrial scale for the production of acrylamide and acrylic acid due to their ease of growth to high biomass yields, high specific enzyme activities obtainable, their EFB class 1 status and robustness of the whole cells within chemical reaction systems.Several isolates belonging to the genus Rhodococcus have been shown in our studies to be among the best candidates for acrylic acid preparation from acrylonitrile due to their stability and tolerance to high concentrations of this reactive and disruptive substrate. A critical part of the selection procedure for the best candidates during the screening programme was high purity product with very low residual substrate concentrations, even in the presence of high product concentrations. Additionally the nitrile and amide substrate scavenging ability which enables rhodococci to survive very successfully in the environment leads to the formation of biocatalysts which are suitable for the removal of low concentrations of acrylonitrile and acrylamide in waste streams and for the removal of impurities in manufacturing processes.     

Engineered whole-cell biocatalyst-based detoxification and detection of neurotoxic organophosphate compounds

The development of efficient tools is required for the eco-friendly detoxification and effective detection of neurotoxic organophosphates (OPs). Although enzymes have received significant attention as biocatalysts because of their high specific activity, the uneconomic and labor-intensive processes of enzyme production and purification make their broad use in practical applications difficult. Because whole-cell systems offer several advantages compared with free enzymes, including high stability, a reduced purification requirement, and low preparation cost, they have been suggested as promising biocatalysts for the detoxification and detection of OPs. To develop efficient whole-cell biocatalysts with enhanced activity and a broad spectrum of substrate specificity, several factors have been considered, namely the selected strains, the chosen OP-hydrolyzing enzymes, where enzymes are localized in a cell, and which enhancer will assist the expression, function, and folding of the enzyme. In this article, we review the current investigative progress in the development of engineered whole-cell biocatalysts with excellent OP-hydrolyzing activity, a broad spectrum of substrate specificity, and outstanding stability for the detoxification and detection of OPs.     

Enzymatic and whole cell catalysis: Finding new strategies for old processes

The use of enzymes and whole bacterial cells has allowed the production of a plethora of compounds that have been used for centuries in foods and beverages. However, only recently we have been able to master techniques that allow the design and development of new biocatalysts with high stability and productivity. Rational redesign and directed evolution have lead to engineered enzymes with new characteristics whilst the understanding of adaptation mechanisms in bacterial cells has allowed their use under new operational conditions. Bacteria able to thrive under the most extreme conditions have also provided new and extraordinary catalytic processes. In this review, the new tools available for the improvement of biocatalysts are presented and discussed.     

Pictet–Spenglerases in alkaloid biosynthesis: Future applications in biocatalysis

Pictet–Spenglerases provide a key role in the biosynthesis of many biologically active alkaloids. There is increasing use of these biocatalysts as an alternative to traditional organic synthetic methods as they provide stereoselective and regioselective control under mild conditions. Products from these enzymes also contain privileged drug scaffolds (such as tetrahydroisoquinoline or β-carboline moieties), so there is interest in the characterization and use of these enzymes as versatile biocatalysts to synthesize analogs of the corresponding natural products for drug discovery. This review discusses all known Pictet–Spenglerase enzymes and their applications as biocatalysts.     

Recent advances in applied and mechanistic aspects of the enzymatic hydroxylation of steroids by whole-cell biocatalysts.

Recent advances in microbial steroid hydroxylation are covered, including new biocatalysts and substrate groups and new methodologies such as the use of low-water systems, immobilised biocatalysts, genetically constructed biocatalysts and enzyme mimics. Mechanistic factors that control the regiochemistry and stereochemistry of steroid hydroxylation are also discussed.     

Immobilization of a recombinant strain producing glucose isomerase inside SiO<Subscript>2</Subscript>-xerogel and properties of prepared biocatalysts

An original method of immobilization of non-growing microorganism cells inside xerogel of silicium dioxide containing insoluble hydroxyl compounds of cobalt(II) has been developed. A recombinant strain producing glucose isomerase has been constructed on the basis of Escherichia coli with the use of a gene of Arthrobacter nicotianae. It was revealed that glucose isomerase activity and stability of biocatalysts prepared on the basis of the recombinant E. coli strain was 3–5 times greater compared with the biocatalysts prepared with the use of the donor strain A. nicotianae. Under conditions of continuous hydrolysis of 3 M fructose at 62–65°C in a fixed bed reactor, time of half-inactivation of the biocatalysts prepared from the recombinant strain and A. nicotianae was ∼60 and ∼25 days, respectively.     

Engineering ethanologenic Escherichia coli for levoglucosan utilization

Levoglucosan is a major product of biomass pyrolysis. While this pyrolyzed biomass, also known as bio-oil, contains sugars that are an attractive fermentation substrate, commonly-used biocatalysts, such as Escherichia coli, lack the ability to metabolize this anhydrosugar. It has previously been shown that recombinant expression of the levoglucosan kinase enzyme enables use of levoglucosan as carbon and energy source. Here, ethanologenic E. coli KO11 was engineered for levoglucosan utilization by recombinant expression of levoglucosan kinase from Lipomyces starkeyi. Our engineering strategy uses a codon-optimized gene that has been chromosomally integrated within the pyruvate to ethanol (PET) operon and does not require additional antibiotics or inducers. Not only does this engineered strain use levoglucosan as sole carbon source, but it also ferments levoglucosan to ethanol. This work demonstrates that existing biocatalysts can be easily modified for levoglucosan utilization.     

Biocatalysis for the synthesis of pharmaceuticals and pharmaceutical intermediates

Biocatalysis has been increasingly used for pharmaceutical synthesis in an effort to make manufacturing processes greener and more sustainable. Biocatalysts that possess excellent activity, specificity, thermostability and solvent-tolerance are highly sought after to meet the requirements of practical applications. Generating biocatalysts with these specific properties can be achieved by either discovery of novel biocatalysts or protein engineering. Meanwhile, chemoenzymatic routes have also been designed and developed for pharmaceutical synthesis on an industrial scale. This review discusses the recent discoveries, engineering, and applications of biocatalysts for the synthesis of pharmaceuticals and pharmaceutical intermediates. Key classes of biocatalysts include reductases, oxidases, hydrolases, lyases, isomerases, and transaminases.     

Immobilization of a recombinant strain producing glucose isomerase on SiO2-xerogel and properties of prepared biocatalysts

An original method of immobilization of nongrowing microorganism cells on xerogel of silicon dioxide containing insoluble hydroxyl compounds of cobalt(III) has been developed. A recombinant strain producing glucose isomerase has been constructed on the basis of Escherichia coli with the use of a gene of Arthrobacter nicotianae. It was revealed that glucose isomerase activity and stability of biocatalysts prepared on the basis of the recombinant E. coli strain was 3-5 times greater compared with the biocatalysts prepared with the use of the donor strain A. nicotianae. Under conditions of continuous hydrolysis of 3 M fructose at 62-65 degrees C in a fixed bed reactor, time of half-inactivation of the biocatalysts prepared from the recombinant strain and A. nicotianae was -60 and -25 days, respectively.     

Screening for novel biocatalysts

This review attempts to demonstrate the importance of goal-orientated screening for new biocatalysts. Examples of enzymes and microorganisms that have been developed and that have acquired commercial applications are described so as to illustrate the technological potential of biocatalysts. A survey of screening techniques and recently reported examples of screening from food, chemical, pharmaceutical and waste disposal applications etc. are also presented to demonstrate the feasibility of this approach for generating new biocatalysts. An appreciation of some of the difficulties involved, the achievements of Japanese researchers and some examples of the cornucopia of largely unrecognized and potentially valuable microbial activities are also given. An increased effort in screening would have the following benefits: an increased range of biocatalysts with different enzyme activities would be available and more biocatalysts with improved characteristics, suitable for use under industrial conditions, such as resistance to elevated temperatures, extremes of pH and organic solvents would be discovered. Secondly the manpower and other resources required to carry out screening programmes would be reduced, for instance by developing automated techniques. Thirdly, screening procedures would be made much more accessible to non-specialists. Fourthly, improved efforts and expertise in screening would supplement other emerging techniques such as protein engineering. The development of selective, non-random, goal-orientated screening techniques, methods of evaluating biocatalyst performance under operational conditions, and an approach that is more orientated towards commercially desirable goals are essential if these objectives are to be achieved. Screening of naturally occurring microorganisms still appears to be the best way to obtain new strains and/or enzymes for commercial applications. However, two major problems appear to exist. Firstly in identifying applications that are technically feasible and that have sufficient commercial potential to justify the research and development required to generate a new commercially viable biocatalyst and secondly the relatively small number of scientists outside Japan with skill and experience in screening for biocatalysts.     

Yeast as an efficient biocatalyst for the production of lipid-derived flavours and fragrances

Responding to consumer' demand for natural products, biotechnology is constantly seeking new biocatalysts. In the field of hydrophobic substrate degradation, some yeast species known some years ago as non-conventional, have acquired their right to be considered as good biocatalysts. These Candida, Yarrowia, Sporobolomyces ... are now used for themselves or for their lipases in processes to produce flavours and fragrances. In this paper we present some examples of use of these biocatalysts to generate high-value compounds and discuss the new trends related to progress in the development of molecular tools or the mastering of the redox characteristics of the medium.     

Fungal biocatalysts in the biofiltration of VOC-polluted air

Gas-phase biofilters used for the treatment of waste gases were originally packed with compost or other natural filter beds containing indigenous microorganisms. Over the past decade much effort has been made to develop new carrier materials, more performant biocatalysts and new types of bioreactors. Elimination capacities reached nowadays are 5 to 10 times higher than those originally reported with conventional compost biofilters. With the recently developed inert filter beds, inoculation is a prerequisite for successful start-up and operation. Either non-defined mixed cultures or pure bacterial cultures have originally been used. The search for efficient fungal biocatalysts started only a few years ago, mainly for the biofiltration of waste gases containing hydrophobic compounds, such as styrene, alpha-pinene, benzene, or alkylbenzenes. In this review, recently isolated new fungal strains able to degrade alkylbenzenes and other related volatile organic pollutants are described, as well as their major characteristics and their use as biocatalysts in gas-phase biofilters for air pollution control. In biofiltration, the most extensively studied organism belongs to the genus Exophiala, although strains of Scedosporium, Paecilomyces, Cladosporium, Cladophialophora, and white-rot fungi are all potential candidates for use in biofilters. Encouraging results were obtained in most of the cases in which some of those organisms were present in gas-phase biofilters. They allow reaching high elimination capacities and are resistant to low pH values and to reduce moisture content.     

Chemical versus biochemical conversion: when and how to use biocatalysts

Developments in biotechnology have been extremely rapid the last two decades. This is particularly true for recombinant DNA technology, which has strongly stimulated the study of biocatalysis, and many examples of new applications of biocatalysts exist. Nevertheless, they have not yet been routinely applied in chemistry. In this study, the question is addressed as to when biocatalysts can be advantageously used in this field. If that is indeed the case, many decisions have to be made before they can be optimally utilized. In the present work, a decision tree is introduced which provides important questions to answer to come to such an optimal use. Examples are given to illustrate the decisionmaking. (c) 1996 John Wiley & Sons, Inc.     

An artificial self‐sufficient cytochrome P450 directly nitrates fluorinated tryptophan analogs with a different regio‐selectivity

Aromatic nitration is an immensely important industrial process to produce chemicals for a variety of applications, but it often suffers from multiple unsolved challenges. Enzymes as biocatalysts have been increasingly used for organic chemistry synthesis due to their high selectivity and environmental friendliness, but nitration has benefited minimally from the development of biocatalysis. In this work, we aimed to develop TxtE as practical biocatalysts for aromatic nitration. TxtE is a unique class I cytochrome P450 enzyme that nitrates the indole of l ‐tryptophan. To develop cost‐efficient nitration processes, we fused TxtE with the reductase domains of CYP102A1 (P450BM3) and of P450RhF to create class III self‐sufficient biocatalysts. The best engineered fusion protein was comparable with wild type TxtE in terms of nitration performance and other key biochemical properties. To demonstrate the application potential of the fusion enzyme, we nitrated 4‐F‐dl ‐tryptophan and 5‐F‐l ‐tryptophan in large scale enzymatic reactions. Tandem MS/MS and NMR analyses of isolated products revealed altered nitration sites. To our knowledge, these studies represent the first practice in developing biological nitration approaches and lay a solid basis to the use of TxtE‐based biocatalysts for the production of valuable nitroaromatics.     

Immobilization of biocatalysts with poly(vinyl alcohol) supports.

Two polymer materials, poly(vinyl alcohol) (PVA) superfine fibers and photocrosslinkable PVA bearing styrylpyridinium groups, have been developed to immobilize biocatalysts. The former has a large surface consisting of relatively large-size pores and the fibers can immobilize a large amount of biocatalyst on their surface by ionic interaction. The latter entraps many kinds of biocatalysts by cyclodimerization caused by visible light irradiation. The biocatalysts on/in these supports maintain high activity and thermal stability. These materials can easily be formed into various shapes suitable for various applications. A new bioreactor system was constructed for evaluating a variety of biocatalysts and supports.     

Advances in recovery of novel biocatalysts from metagenomes

Metagenomics has accelerated the process of discovery of novel biocatalysts by enabling scientists to tap directly into the entire diversity of enzymes held within natural microbial populations. Their characterization has revealed a great deal of valuable information about enzymatic activity in terms of factors which influence their stability and activity under a wide range of conditions. Many of the biocatalysts have particular properties making them suitable for biotechnological applications. A diverse array of strategies has been developed to optimize each step of the process of generating and screening metagenomic libraries for novel biocatalysts. This review covers the diversity of metagenome-derived enzymes characterized to date, and the strategies currently being developed to optimize discovery of novel metagenomic biocatalysts.     

Physiological and Biochemical Characteristics of Immobilized Champagne Yeasts and Their Participation in Champagnizing Processes: A Review

Methods for immobilizing champagne yeasts, physiological and biochemical characteristics of the immobilized cells, and problems of their utilization in the production of quality champagne wines are reviewed. Studies aimed at the development of efficient biocatalysts for champagnizing wines using bottle fermentation (methode champenoise) and tank processing (bulk, or Charmat process) based on the use of immobilized yeast cells are described. Data on the industrial use of such biocatalysts in countries manufacturing champagne wines are presented. Problems and prospects of further research in this field are discussed.     

生物催化剂发现与改造的研究进展

生物催化是指将酶或生物有机体用于有用的化学转化的过程,在人们对传统化学催化的环境影响抱有忧虑的情况下,生物催化提供了一种有吸引力的选择。在过去的几十年里,对生物催化剂的研究每出现一次大的进步,生物催化的发展就会出现一次高潮。因此,生物催化剂的发现与改造已成为当今研究的热点。宏基因组文库技术的出现克服了许多微生物不可培养的障碍,人们能够从自然资源中获得丰富的潜在的生物催化剂。而基于理性设计的分子改造技术的发展,可以使得人们对潜在的生物催化剂进行快速而有效的改造以满足工业化生产的需求。随着生物催化剂发现与改造的手段不断进步,更多的优良生物催化剂得到了广泛的应用,生物催化在工业生产中也得到了更深入的应用。结合作者的研究工作,总结了生物催化剂发现与改良的一些研究进展,以为获得更多优良的、能够实现工业应用的生物催化剂奠定理论基础。     

Physiological and biochemical characteristics of immobilized champagne yeasts and their participation in sparkling processes

Methods for immobilizing champagne yeasts, physiological and biochemical characteristics of the immobilized cells, and problems of their utilization in the production of quality champagne wines are reviewed. Studies aimed at the development of efficient biocatalysts for champaignizing wines using bottle fermentation (method champenoise) and tank processing (bulk, or Charmat process), based on the use of immobilized yeast cells, are described. Data on the industrial use of such biocatalysts in countries manufacturing champagne wines are presented. Problems and prospects of further research in this field are discussed.     

Selective immobilization of Bacillus subtilis lipase A from cell culture supernatant: Improving catalytic performance and thermal resistance

Bacillus subtilis lipase A (BSLA) has been extensively studied through protein engineering; however, its immobilization and behavior as an insoluble biocatalyst have not been extensively explored. In this work, for the first time, a direct immobilization of recombinant BSLA from microbial culture supernatant was reported, using chemically modified porous with different electrostatic, hydrophobic, hydrophilic, and hydrophilic−hydrophobic enzyme-support interactions. The resulting biocatalysts were evaluated based on their immobilization kinetics, activity expression (pH 7.4), thermal stability (50 °C), solvent resistance and substrate preference. Biocatalysts obtained using glyoxyl silica support resulted in the selective immobilization of BSLA, resulting in an activity recovery of 50 % and an outstanding aqueous stabilization factor of 436, and 9.5 in isopropyl alcohol, compared to the free enzyme. This selective immobilization methodology of BSLA allows to efficiently generate immobilized biocatalysts, thus avoiding laborious purification steps from cell culture supernatant, which is usually a limiting step when large amounts of enzyme variants or candidates are assessed as immobilized biocatalysts. Direct enzyme immobilization from cell supernatant provides an interesting tool which can be used to facilitate the development and assessment of immobilized biocatalysts from engineered enzyme variants and mutant libraries, especially in harsh conditions, such as high temperatures or non-aqueous solvents, or against non-water-soluble substrates. Furthermore, selective immobilization approaches from cell culture supernatant or clarified lysates could help bridging the gap between protein engineering and enzyme immobilization, allowing for the implementation of immobilization steps in high throughput enzyme screening platforms for their potential use in directed evolution campaigns.