Understanding enzyme action on immobilised substrates

With increasing interest in automated synthesis and screening protocols, solid supported chemistry and biochemistry are attractive technologies. Studies with surface-immobilised substrates have been carried out to analyse enzyme accessibility, kinetics and thermodynamics. Several interesting new methods have been developed to monitor enzyme action on substrates attached to a solid phase such as polymer beads glass or gold surfaces. These include fluorescence measurements, MALDI-TOF mass spectrometry, and the use of quartz crystal microbalances to measure weight changes of immobilised molecules directly on the surface. Approaches that allow spatial resolution in single beads have also been reported. The ability of enzymes to reach the inside of beads is becoming better characterised and new supports have been developed that allow improved accessibility. The equilibrium position of reactions on the solid surface can be substantially shifted compared with reactions in solution, and this can be usefully exploited using hydrolases in reverse. Research is also starting to tackle the way in which kinetics are modified when the substrates are surface immobilised.     

Recent developments in the fungal transformation of steroids

Steroids constitute a vital part of the active ingredients in pharmaceuticals and intermediates used to produce medicines, and their application in chemical and agrochemical fields is also valued. The complex stereochemistry of steroids requires attention to regio- and stereoselectivity of the reaction during preparation, and therefore, biocatalytic methods are appropriate for their production. This work reviews the recent application of fungi for the transformation of different steroid substrates, new biotransformation techniques, recently characterized reactions, and practical aspects, covering the period from 1990 to 2014. The future prospects of fungal biotechnology and biotransformation in the biopharmaceutical industry are also considered.     

组合生物催化

自然界最有效的分子是由酶催化的反应所产生,并对这些产物进行自然选择,使其具有优化的生理活性,组合生物催化（Combinatorial Biocatalysis)利用酶反应的多样性,完成有机库（Organic Library)的反复合成,这些反复的反应,可以用分离的酶或全细胞,在天然或非天然的环境中、在溶液或固相中与底物进行反应。组合生物催化是组合方法的在药物发现和发展中产生和优化先导化合物（LeadCompound)的一个有力补充。     

Applications of oxidoreductases.

Oxidoreductases comprise the large class of enzymes that catalyze biological oxidation/reduction reactions. Because many chemical and biochemical transformations involve oxidation/reduction processes, developing practical biocatalytic applications of oxidoreductases has long been an important goal in biotechnology. During the past year, significant progress has been made in the development of oxidoreductase-based diagnostic tests and improved biosensors, in the design of innovative systems for regeneration of essential coenzymes, in the construction bioreactors for biodegradation of pollutants and for biomass processing, and in the development of oxidoreductase-based approaches for synthesis of polymers and oxyfunctionalized organic substrates.     

Estimations on the degradability of ores and bacterial leaching activity using short-time microcalorimetric tests

Abstract: This work is based on calorimetric measurements on batch cultures of ferrous iron-oxidizing thiobacilli and leaching cultures degrading solid substrates in the titration vessel under aerobic conditions. It has been demonstrated that heat-flow levels are a direct measure for oxidation rates of known reactions under non-limiting conditions. The total loss of heat-energy per unit of ferrous iron oxidized is identical under similar incubation conditions with different Thiobacillus ferrooxidans strains and different cell-densities in culture suspension. Under limiting conditions a reduced heat-loss occurred. This may have been due to either a more effective use of the substrate or to a shift to different redox reactions. Oxygen was proved to be a limiting factor in dense bacterial cultures with high substrate concentrations in the titration vessel. The experimental duration decreased significantly with the use of increasing cell densities. Therefore cell densities in degradation experiments with solid substrates were adjusted to between 5X10⁹ and 1x10¹⁰ cells/ml in short-term experiments. T. ferrooxidans is the most important species for short-term leaching experiments. Calorimetric measurements allow the best strains for a degradation of unknown substrates to be found. The calculated heat energy is a measure of the amount of converted substrate. High heat-flow only occurs if catabolic reactions take place and maintenance reactions and phenomena like adhesion of cells to surface do not cause significant loss of heat.     

Thermophilic anaerobic digestion for sewage sludge stabilization

The microbially mediated biochemical reactions that occur during anaerobic digestion processes for methane production from soluble carbon energy substrates are well known, but in spite of this, the interactions within the multi-species cultures responsible for the overall process require more detailed elucidation. When the process feed comprises mixed, solid, carbon energy substrates, as in the case of waste sewage sludge stabilization, many aspects of both the process biochemistry and microbiology are unresolved. This mini-review seeks to identify some of these unresolved questions, particularly with respect to operation at thermophilic temperatures.     

The recent impact of solid-phase synthesis on medicinally relevant benzoannelated nitrogen heterocycles

Benzoannelated heterocycles such as benzodiazepines and indoles can be prepared efficiently through cyclization on solid supports, although no single approach is currently universal for the preparation of all benzoannelated N-heterocycle chemistries. In this review, a number of synthetic strategies for the generation of benzoannelated nitrogen heterocycles using resin-bound substrates have been described. Classical heterocycle forming reactions such as the Fischer indole, the Bischler-Napieralski tetrahydroisoquinoline, the Pictet-Spengler tetrahydro-beta-carboline, the Tsuge, the Nenitzescu and the Richter cinnoline reaction are presented. In addition, the Heck, Sonogashira, Wittig, Diels-Alder, and olefin metathesis reactions have been also used. Multicomponent reactions such as the Grieco three-component assembly have been exploited for the synthesis of heterocycles. Cyclative cleavage from the solid support is particularly suitable for the synthesis of heterocycles while particular emphasis has been focused on the synthesis of libraries and the use of combinatorial chemistry techniques. In addition, the most relevant pharmacological properties of benzoannelated nitrogen heterocycles are included.     

The use of optical spectroscopy in combinatorial chemistry.

Infrared and Raman spectroscopy allow direct spectral analysis of the solid-phase, thus avoiding the tedious cleavage of compounds from the solid support. With diagnostic bands in starting materials or products, infrared and Raman spectroscopy are efficient in monitoring each reaction step directly on the solid phase. Consequently, infrared and Raman spectroscopy have evolved as the premier analytical methodology for direct analysis on the solid support. While infrared transmission spectroscopy is a general analytical method for resin samples, internal reflection spectroscopy is especially suited for solid polymer substrates known as "pins" or "crowns." Single bead analysis is done best by infrared microspectroscopy, whereas photoacoustic spectroscopy allows totally nondestructive analysis of resin samples. With an automated accessory, diffuse reflection spectroscopy provides a method for high throughput on-bead monitoring of solid-phase reactions. Providing identification based on molecular structure, HPLC-FTIR is, therefore, complementary to LC-MS. Additionally, Raman spectroscopy as a complement to infrared spectroscopy can be applied to resin samples and-using a Raman microscope-to single beads. Fluorometry as an extremely sensitive spectroscopic detection method allows rapid quantification of organic reactions directly on the resin.     

The use of optical spectroscopy in combinatorial chemistry

Infrared and Raman spectroscopy allow direct spectral analysis of the solid‐phase, thus avoiding the tedious cleavage of compounds from the solid support. With diagnostic bands in starting materials or products, infrared and Raman spectroscopy are efficient in monitoring each reaction step directly on the solid phase. Consequently, infrared and Raman spectroscopy have evolved as the premier analytical methodology for direct analysis on the solid support. While infrared transmission spectroscopy is a general analytical method for resin samples, internal reflection spectroscopy is especially suited for solid polymer substrates known as “pins” or “crowns.” Single bead analysis is done best by infrared microspectroscopy, whereas photoacoustic spectroscopy allows totally nondestructive analysis of resin samples. With an automated accessory, diffuse reflection spectroscopy provides a method for high throughput on‐bead monitoring of solid‐phase reactions. Providing identification based on molecular structure, HPLC‐FTIR is, therefore, complementary to LC‐MS. Additionally, Raman spectroscopy as a complement to infrared spectroscopy can be applied to resin samples and—using a Raman microscope—to single beads. Fluorometry as an extremely sensitive spectroscopic detection method allows rapid quantification of organic reactions directly on the resin. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng (Comb Chem) 61:179–187, 1998/1999.     

Exploring De Novo metabolic pathways from pyruvate to propionic acid

Industrial biotechnology provides an efficient, sustainable solution for chemical production. However, designing biochemical pathways based solely on known reactions does not exploit its full potential. Enzymes are known to accept non‐native substrates, which may allow novel, advantageous reactions. We have previously developed a computational program named Biological Network Integrated Computational Explorer (BNICE) to predict promiscuous enzyme activities and design synthetic pathways, using generalized reaction rules curated from biochemical reaction databases. Here, we use BNICE to design pathways synthesizing propionic acid from pyruvate. The currently known natural pathways produce undesirable by‐products lactic acid and succinic acid, reducing their economic viability. BNICE predicted seven pathways containing four reaction steps or less, five of which avoid these by‐products. Among the 16 biochemical reactions comprising these pathways, 44% were validated by literature references. More than 28% of these known reactions were not in the BNICE training dataset, showing that BNICE was able to predict novel enzyme substrates. Most of the pathways included the intermediate acrylic acid. As acrylic acid bioproduction has been well advanced, we focused on the critical step of reducing acrylic acid to propionic acid. We experimentally validated that Oye2p from Saccharomyces cerevisiae can catalyze this reaction at a slow turnover rate (10^?3 s^?1), which was unknown to occur with this enzyme, and is an important finding for further propionic acid metabolic engineering. These results validate BNICE as a pathway‐searching tool that can predict previously unknown promiscuous enzyme activities and show that computational methods can elucidate novel biochemical pathways for industrial applications. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:303–311, 2016     

Formation of a bilayer lipid membrane on rigid supports: an approach to BLM-based biosensors

Sensory transduction in living cells is thought to involve a change of electrical parameters at the receptor membrane following specific binding events at the membrane surface. Because of the complexity of the biomembrane structure and the environmental factors associated with it, experimental bilayer lipid membranes (BLMs) have been employed for elucidation of processes at the membrane level. This is because the BLM system can be easily probed by a host of powerful and sensitive electrochemical methods. Further, recent advances in microelectronics and biotechnology suggest that the development of a BLM-based electrochemical biosensor may be possible. This paper describes the use of bilayer lipid membranes on solid substrates for analysis of sensor development problems, with relevance to a possible novel type of biomolecular device. Some electrical parameters of the new structure were measured and compared to usual BLM results. The advantages of the self-assembled structure, together with the measuring system, are discussed in terms of stability and sensitivity.     

Course of pH during the formation of amoxicillin by a suspension-to-suspension reaction

Amoxicillin can be produced in an enzymatic suspension-to-suspension reaction in which the substrate(s) and product(s) are mainly present as solid particles, while the reaction takes place in the liquid phase. During these suspension-to-suspension reactions different subprocesses take place, such as dissolution/crystallization of substrates and products, enzymatic synthesis of the product(s), and undesired enzymatic hydrolysis of substrates and/or products. All these subprocesses are influenced by pH and also influence the pH because the reactants are weak electrolytes. This paper describes a quantitative model for predicting pH and concentrations of reactants during suspension-to-suspension reactions. The model is based on mass and charge balances, pH-dependent solubilities of the reactants, and enzyme kinetics. For the validation of this model, the kinetically controlled synthesis of amoxicillin from 6-aminopenicillanic acid and D-(p)hydroxyphenylglycine methyl ester was studied. The pH and the dissolved concentrations took a very different course at different initial substrate amounts. This was described quite reasonably by the model. Therefore, the model can be used as a tool to optimize suspension-to-suspension reactions of weak electrolytes.     

Biotechnological advantages of laboratory-scale solid-state fermentation with fungi

Despite the increasing number of publications dealing with solid-state (substrate) fermentation (SSF) it is very difficult to draw general conclusion from the data presented. This is due to the lack of proper standardisation that would allow objective comparison with other processes. Research work has so far focused on the general applicability of SSF for the production of enzymes, metabolites and spores, in that many different solid substrates (agricultural waste) have been combined with many different fungi and the productivity of each fermentation reported. On a gram bench-scale SSF appears to be superior to submerged fermentation technology (SmF) in several aspects. However, SSF up-scaling, necessary for use on an industrial scale, raises severe engineering problems due to the build-up of temperature, pH, O₂, substrate and moisture gradients. Hence, most published reviews also focus on progress towards industrial engineering. The role of the physiological and genetic properties of the microorganisms used during growth on solid substrates compared with aqueous solutions has so far been all but neglected, despite the fact that it may be the microbiology that makes SSF advantageous against the SmF biotechnology. This review will focus on research work allowing comparison of the specific biological particulars of enzyme, metabolite and/or spore production in SSF and in SmF. In these respects, SSF appears to possess several biotechnological advantages, though at present on a laboratory scale only, such as higher fermentation productivity, higher end-concentration of products, higher product stability, lower catabolic repression, cultivation of microorganisms specialized for water-insoluble substrates or mixed cultivation of various fungi, and last but not least, lower demand on sterility due to the low water activity used in SSF.     

Autodisplay of functional CYP106A2 in Escherichia coli

Cytochrome P450 enzymes catalyse a wide variety of reactions, including the hydroxylation and epoxidation of CC bonds, and dealkylation reactions. There is high interest in these reactions for biotechnology and pharmaceutical processes. Many P450s require membrane surroundings and have substrates that do not cross biological membranes. To circumvent these obstacles, CYP106A2 from Bacillus megaterium was expressed on the outer membrane of Escherichia coli cells by Autodisplay. Exposure on the surface was confirmed by a protease accessibility test and flow cytometry after immunolabelling. HPLC assays showed that 0.5 ml of cells displaying the enzyme (OD??? = 6) converted 9.13 μmol of deoxycorticosterone to 15β-OH-deoxycorticosterone within 1h. Imipramine and abietic acid were also accepted as substrates. The number of active enzyme molecules per cell was calculated to be 20,000. Surprisingly, surface-exposed CYP106A2 was active in E. coli BL21 without the external addition of the heme group. However, when CYP106A2 was expressed on the surface of an E. coli strain lacking the TolC channel protein (JW5503), enzymatic activity was almost completely abolished. The activity of CYP106A2 on the surface of E. coli JW5503 could be restored by the external addition of the heme group. This suggests, as has been reported before, that E. coli uses a TolC-dependent mechanism to export heme into the growth media, where it can be scavenged by a surface-displayed apoenzyme. Our results indicate that Autodisplay enables the functional surface display of P450 enzymes and provides a new platform to access their synthetic potential.     

Characterization of membrane-bound dehydrogenases of Gluconobacter oxydans 621H using a new system for their functional expression

Acetic acid bacteria are used in biotechnology due to their ability to incompletely oxidize a great variety of carbohydrates, alcohols, and related compounds in a regio- and stereo-selective manner. These reactions are catalyzed by membrane-bound dehydrogenases (mDHs), often with a broad substrate spectrum. In this study, the promoters of six mDHs of Gluconobacter oxydans 621H were characterized. The constitutive promoter of the alcohol dehydrogenase and the glucose-repressed promoter of the inositol dehydrogenase were used to construct a shuttle vector system for the fully functional expression of mDHs in the multi-deletion strain G. oxydans BP.9 that lacks its mDHs. This system was used to express each mDH of G. oxydans 621H, in order to individually characterize the substrates, they oxidize. From 55 tested compounds, the alcohol dehydrogenase oxidized 30 substrates and the polyol dehydrogenase 25. The substrate spectrum of alcohol dehydrogenase overlapped largely with the aldehyde dehydrogenase and partially with polyol dehydrogenase. Thus, we were able to resolve the overlapping substrate spectra of the main mDHs of G. oxydans 621H. The described approach could also be used for the expression and detailed characterization of substrates used by mDHs from other acetic acid bacteria or a metagenome.

     

A computer model for the growth and differentiation of a fungal colony on solid substrate

Mold growth and differentiation are closely related to the formation of secondary products. In solid-substrate fermentations this interrelationship is often more completely realized than in submerged cultures. Solid substrate reactions are used commercially in a limited manner in the western world, but are relatively common in Asia. Basic studies in solid-substrate fermentation should yield results applicable to all types of commercial mold fermentations for the production of a secondary product. This paper presents a relatively simple model for the growth of a mold colony on a solid surface with a defined medium utilizing glucose. Unlike submerged cultures the model must account for both cellular differentiation and the spatial heterogeneity in the system. Model parameters were estimated independently using literature values. The results of the simulation studies suggest that mass transfer limitations are at least partially responsible for the proliferation of differentiated structures on solid substrates as compared to liquid cultures. Since the concentration profile depends on the depth of the substratum, conditions that enhance conidia production can be achieved by controlling the depth of the solid medium.     

Gas phase transesterification reactions catalyzed by lipolytic enzymes

Porcine pancreatic lipase and Fusarium solani cutinase were used to catalyze transesterification reactions between methyl propionate, ethyl propionate, and a series of primary alcohols at high temperatures in a continuous packed-bed gas-solid reactor, in which the solid phase is composed of the enzyme and the substrates and products are in a gaseous form. In this type of system, enzyme activity was found to depend essentially on the water activity (A(w)) of the enzyme preparation.     

A Comparison of Hybridization Efficiency between Flat Glass and Channel Glass Solid Supports

Two different solid supports, channel glass and flat glass, were compared for their affect on the sensitivity and efficiency of DNA hybridization reactions. Both solid supports were tested using a set of arrayed, synthetic oligonucleotides that are designed to detect short insertion/deletion polymorphisms (SIDPs). A total of 13 different human SIDPs were chosen for analysis. Capture probes, designed for this test set, were covalently immobilized on substrates. Hybridization efficiency was assessed using fluorescently labeled stacking probes which were preannealed to the target and then hybridized to the support-bound oligonucleotide array; the hybridization pattern was detected by fluorescence imaging. It was found that structural features of nucleic acid capture probes tethered to a solid support and the molecular basis of their interaction with targets in solution have direct implications on the hybridization process. Our results demonstrate that channel glass has a number of practical advantages over flat glass including higher sensitivity and a faster hybridization rate.     

Lignin and ligninase]

Ligninases (lignin peroxidases) are heme-containing peroxidases excreted by some white-rot fungi as components of their lignolytic multienzyme complexes. These peroxidases functioning at rather acidic media catalyze oxidative cleavage of both synthetic non-phenolic lignin models and many other oxidation-proof compounds (chloroorganic pesticides, carcinogenic hydrocarbons, etc.). Data on the ligninase structure and functions not only shed light of the lignin biodegradation but also open new perspectives in peroxidation chemistry and biotechnology. Many aspects of ligninase catalytic mechanism can be understood in comparative studies of congruent chemical reactions, e.g., peroxidisulfate-supported oxidation, as well as of ligninase-like activity of some plant and animal peroxidases which is also manifested at low pH. Ligninases are not only more powerful oxidative agents than other peroxidases, but also, in contrast to latters, appear to be able to control the contributions of C-C and C-O bond splitting in primary radical-cations of substrates. The contribution of the oxidative-hydrolytic dealkylation of radical cations can be considered as one of classification criteria for lignolytic enzymes.     

The control of lipase-catalysed transesterification and esterification reaction rates. Effects of substrate polarity, water activity and water molecules on enzyme activity

The reaction rate of two lipase-catalysed reactions, esterification and transesterification, were studied in a liquid/solid two-phase system in order to investigate the effect of water partition between the enzyme preparation and the liquid phase composed of only the reactants, i.e. without the conventional solvents. Lipase from Candida cylindracea was used for these studies. The enzyme was inactive in dehydrated systems. In the case of monoester synthesis, the reaction rate increased with increasing water activity. The reaction rates of the non-specific C. cylindracea lipase-catalysed reactions were very sensitive to the nature of the substrates in this unusual system. For instance, the transesterification reaction rate of ethyl propionate was 48 times higher with nonanol than heptanol in the case of dehydrated substrates, but only 2.2 times higher in the case of water-saturated substrates. The results presented here demonstrate the absolute necessity to consider the polarity of every substrate, because of its ability to modify the water partition between the solid phase (enzyme preparation) and the liquid phase (substrate and product), which results in drastic changes in enzyme activity. Contrary to esterification, which is known to be activated by the water produced, the rate of transesterification remained constant at the beginning of the reaction. However, when transesterification and esterification were carried out in the same liquid phase, the transesterification reaction rate was controlled by the water produced by the concomitant esterification. Activation effects of the water molecules produced during the enzymatic reaction were of exactly the same order of magnitude for both reactions.