α,β‐Unsaturated Aldehydes as Substrates for Asymmetric C?C Bond Forming Reactions with Thiamin Diphosphate (ThDP)‐Dependent Enzymes

The enzymes benzaldehyde lyase (BAL) from Pseudomonas fluorescens, benzoylformate decarboxylase (BFD) from Pseudomonas putida and pyruvate decarboxylase (PDC) from Saccharomyces cerevisiae provide different C C bond forming possibilities of α,β‐unsaturated aldehydes with aliphatic and aromatic aldehydes. Structure elucidation and determination of the absolute configuration of the products, which were obtained with high regio‐ and stereoselectivity were carried out. Selective 1,2‐reactivity with yields of 75% and >98% ee, for one single isomer ( A ) were obtained, by choosing the suitable enzyme in combination with the appropriate substrates. By varying enzymes or substrates the regioisomeric hydroxy ketones C , with up to >99% ee, can be obtained. The application of these new chiral building blocks in the synthesis of natural products or biological active substances is considerably facilitated by applying the different ThDP‐dependent enzymes as catalysts. Abbreviations: BAL, benzaldehyde lyase; BFD, benzoylformate decarboxylase; PDC, pyruvate decarboxylase; His, hexahistidine; 2‐HPP, 2‐hydroxy‐1‐phenylpropan‐1‐one; PAC, phenylacetylcarbinol; NTA, nitrilotriacetic acid; ThDP, thiamin diphosphate; wt, wild‐type.     

Surface‐Layer Lattices as Patterning Element for Multimeric Extremozymes

A promising new approach for the production of biocatalysts comprises the use of surface‐layer (S‐layer) lattices that present functional multimeric enzymes on their surface, thereby guaranteeing most accurate spatial distribution and orientation, as well as maximal effectiveness and stability of these enzymes. For proof of concept, a tetrameric and a trimeric extremozyme are chosen for the construction of S‐layer/extremozyme fusion proteins. By using a flexible peptide linker, either one monomer of the tetrameric xylose isomerase XylA from the thermophilic Thermoanaerobacterium strain JW/SL‐YS 489 or, in another approach, one monomer of the trimeric carbonic anhydrase from the methanogenic archaeon Methanosarcina thermophila are genetically linked to one monomer of the S‐layer protein SbpA of Lysinibacillus sphaericus CCM 2177. After isolation and purification, the self‐assembly properties of both S‐layer fusion proteins as well as the specific activity of the fused enzymes are confirmed, thus indicating that the S‐layer protein moiety does not influence the nature of the multimeric enzymes and vice versa. By recrystallization of the S‐layer/extremozyme fusion proteins on solid supports, the active enzyme multimers are exposed on the surface of the square S‐layer lattice with 13.1 nm spacing.     

Stable current outputs and phytate degradation by yeast‐based biofuel cell

In this paper, we report for the first time that Candida melibiosica 2491 yeast strain expresses enhanced phytase activity when used as a biocatalyst in biofuel cells. The polarization also results in an increase of the yeast biomass. Higher steady‐state electrical outputs, assigned to earlier production of an endogenous mediator, were achieved at continuous polarization under constant load. The obtained results prove that the C. melibiosica yeast‐based biofuel cell could be used for simultaneous electricity generation and phytate bioremediation. In addition, the higher phytase activity obtained by interruptive polarization suggests a new method for increasing the phytase yield from microorganisms. Copyright © 2014 John Wiley & Sons, Ltd.     

Immobilised biocatalysts—a critical review

This review examines the strengths and weaknesses of immobilisation when used as a technique for deploying enzymes and cells in systems .Suitable methods for preparing immobilised conjugatcs are described ; the potcntial benefits of immobilisation for industrial products. analysis and academic studies are discussed..     

Structure-Guided Modulation of the Catalytic Properties of [2Fe−2S]-Dependent Dehydratases

The FeS cluster-dependent dihydroxyacid dehydratases (DHADs) and sugar acid-specific dehydratases (DHTs) from the ilvD/EDD superfamily are key enzymes in the bioproduction of a wide variety of chemicals. We analyzed [2Fe−2S]-dependent dehydratases in silico and in vitro, deduced functionally relevant sequence, structure, and activity relationships within the ilvD/EDD superfamily, and we propose a new classification based on their evolutionary relationships and substrate profiles. In silico simulations and analyses identified several key positions for specificity, which were experimentally investigated with site-directed and saturation mutagenesis. We thus increased the promiscuity of DHAD from Fontimonas thermophila (FtDHAD), showing >10-fold improved activity toward D-gluconate, and shifted the substrate preference of DHT from Paralcaligenes ureilyticus (PuDHT) toward shorter sugar acids (recording a six-fold improved activity toward the non-natural substrate D-glycerate). The successful elucidation of the role of important active site residues of the ilvD/EDD superfamily will further guide developments of this important biocatalyst for industrial applications.     

Sulfur removal from coal through multiphase media containing biocatalysts

A novel bioprocess using micelle biocatalysts was developed to minimize several disadvantages of conventional microbial coal desulfurization processes. The multiphase biocatalysis process consists of an organic medium (mineral oil or a mixture with n-heptane), a surfactant and an aqueous phase containing Thiobacillus ferrooxidans organisms or their cell-free enzyme extracts. Water-in-oil emulsion and reverse micelle processes have been successful for removing sulfur from bituminous coal. The preliminary results indicated that, in the case of water-in-oil emulsion, the process that used the cell-free enzyme extracts of T. ferrooxidans showed higher sulfur reduction than that containing whole cells, and reverse micells were more effective than water-in-oil emulsion. With a high concentration of bacteria, more than 50% total sulfur removal was achieved through the reverse micelle system. These results indicate that multiphase biocatalysis may have a significant potential for developing biotechnical coal desulfurization processes.     

The chemical modification of enzymes to enhance solubilization in organic solvents for interaction with coal

Some enzymes can effectively function as catalysts in contact with organic solvents in a variety of ways; however, it is desirable to utilize the enzyme in a soluble form for interactions with a solid substrate such as coal. Dinitrofluorobenzene (DNFB) has been used as the reagent to add dinitrophenyl groups to enzymes, thus increasing hydrophobicity and solubility (up to 20 mg ml⁻¹) in less-polar organic solvents ranging from dioxane to benzene. Mixed reducing enzymes modified by DNFB and used in pyridine or benzene under hydrogen have been shown to enhance significantly the solubility in organic solvents of coals ranging from lignite to bituminous, with up to 35.3 wt% coal conversion. A fluidized-bed bioreactor appears to be the most effective contactor.     

Organic sulfur biodesulfurization in non-aqueous media

An alternative approach to water-based biodesulfurization systems was examined employing organic solvent-based reaction media. A gram-position microorganism, FE-9, was developed to function biocatalytically against organic sulfur-containing heterocycles in 100% dimethylformamide. Strain FE-9 converted dibenzothiophene to biphenyl and hydrogen sulfide under a hydrogen atmosphere, and to biphenyl, hydroxybiphenyl and sulfate under an air atmosphere. Thianthrene was converted to benzene and hydrogen sulfide under a hydrogen atmosphere, and to benzene and sulfate under an air atmosphere. The bioconversion of sulfur-containing heterocycles to aromatic hydrocarbons and hydrogen sulfide, and the oxidation of sulfide to sulfate under air, appears to represent two different biochemical events occurring in dimethylformamide. These studies indicate a strong potential for the development of biocatalysts that selectively abstract organosulfur from high-sulfur containing fossil fuels in an organic solvent-based reaction medium.