Activity of human P450 2D6 in biphasic solvent systems

Several limitations have restricted the use of P450 enzymes in synthesis, including the narrow substrate specificity of some P450 isoforms, the need for a redox partner and an expensive cofactor, incompatibility with organic solvents, and poor stability. We previously demonstrated that the natural redox partner and cofactor of the promiscuous P450s 3A4 and 2D6 can be efficiently substituted with some cheap hydrogen peroxide donors or organic peroxides. We report here that P450 2D6 maintains as much as 76% of its activity when used in buffer/organic emulsions. Product formation in biphasic solvent systems is comparable whether the natural redox partner and cofactor are used, or a surrogate. As reported for other enzymes, a correlation is observed between the logP and the suitability of a solvent for enzymatic activity. Moreover, the utility of our system was established by demonstrating the transformation of a novel hydrophobic substrate, not modified by P450 2D6 in the absence of organic solvent.     

Immobilization of enzymes with polyaziridines.

A novel method of enzyme immobilization using a low molecular weight prepolymer of tri-functional aziridines which can immobilize enzymes both by covalent attachment and entrapment within a gel matrix is described. The enzymes are immobilized on a solid support and exhibit an excellent retention of enzymatic activity. The immobilization procedure is essentially a single step process which can be easily performed at room temperature or 4 degrees C in either aqueous solution or in an inert organic solvent. The polyaziridines used in the immobilization are nontoxic, available in bulk at low cost and completely miscible with water and many organic solvents, thus providing one of the most satisfactory methods of immobilization available.     

嗜盐古菌几种常见胞外酶研究进展

嗜盐古菌是一类生活于极端高盐环境的化能异养型原核微生物,其所分泌的胞外酶(外泌酶)具有在高盐条件下仍能保持活性的特点,在制革工业、高盐有机废水处理和泡菜加工等腌制食品方面发挥重要用途.本文对嗜盐古菌的胞外蛋白酶、淀粉酶、酯酶等几种常见胞外酶的来源和基本酶学性质的最新研究进展进行综述,为更好地开发利用嗜盐古菌胞外酶资源提...     

Micellar enzymology

Experimental approaches to modelling the enzymatic function of biological membranes are discussed. Emphasis is given to pseudohomogeneous systems such as proteolipid complexes and enzymes in organic solvents; the latter are solubilized with phospholipids or synthetic surfactants. Methods for producing and studying such micellar systems are considered. The key research problems of micellar enzymology are formulated and its relation to enzyme membranology is discussed. Finally, the new potentialities are noted of applied enzymology (biotechnology) offered by application of a colloidal solution of water in organic solvents as a microheterogeneous medium for enzymatic reactions.     

Effect of poplar genotypes on mycorrhizal infection and secreted enzyme activities in mycorrhizal and non-mycorrhizal roots

The impact of ectomycorrhiza formation on the secretion of exoenzymes by the host plant and the symbiont is unknown. Thirty-eight F(1) individuals from an interspecific Populus deltoides (Bartr.)×Populus trichocarpa (Torr. & A. Gray) controlled cross were inoculated with the ectomycorrhizal fungus Laccaria bicolor. The colonization of poplar roots by L. bicolor dramatically modified their ability to secrete enzymes involved in organic matter breakdown or organic phosphorus mobilization, such as N-acetylglucosaminidase, β-glucuronidase, cellobiohydrolase, β-glucosidase, β-xylosidase, laccase, and acid phosphatase. The expression of genes coding for laccase, N-acetylglucosaminidase, and acid phosphatase was studied in mycorrhizal and non-mycorrhizal root tips. Depending on the genes, their expression was regulated upon symbiosis development. Moreover, it appears that poplar laccases or phosphatases contribute poorly to ectomycorrhiza metabolic activity. Enzymes secreted by poplar roots were added to or substituted by enzymes secreted by L. bicolor. The enzymatic activities expressed in mycorrhizal roots differed significantly between the two parents, while it did not differ in non-mycorrhizal roots. Significant differences were found between poplar genotypes for all enzymatic activities measured on ectomycorrhizas except for laccases activity. In contrast, no significant differences were found between poplar genotypes for enzymatic activities of non-mycorrhizal root tips except for acid phosphatase activity. The level of enzymes secreted by the ectomycorrhizal root tips is under the genetic control of the host. Moreover, poplar heterosis was expressed through the enzymatic activities of the fungal partner.     

Enzyme research and applications in biotechnological intensification of biogas production

Biogas technology provides an alternative source of energy to fossil fuels in many parts of the world. Using local resources such as agricultural crop remains, municipal solid wastes, market wastes and animal waste, energy (biogas), and manure are derived by anaerobic digestion. The hydrolysis process, where the complex insoluble organic materials are hydrolysed by extracellular enzymes, is a rate-limiting step for anaerobic digestion of high-solid organic solid wastes. Biomass pretreatment and hydrolysis are areas in need of drastic improvement for economic production of biogas from complex organic matter such as lignocellulosic material and sewage sludge. Despite development of pretreatment techniques, sugar release from complex biomass still remains an expensive and slow step, perhaps the most critical in the overall process. This paper gives an updated review of the biotechnological advances to improve biogas production by microbial enzymatic hydrolysis of different complex organic matter for converting them into fermentable structures. A number of authors have reported significant improvement in biogas production when crude and commercial enzymes are used in the pretreatment of complex organic matter. There have been studies on the improvement of biogas production from lignocellulolytic materials, one of the largest and renewable sources of energy on earth, after pretreatment with cellulases and cellulase-producing microorganisms. Lipids (characterised as oil, grease, fat, and free long chain fatty acids, LCFA) are a major organic compound in wastewater generated from the food processing industries and have been considered very difficult to convert into biogas. Improved methane yield has been reported in the literature when these lipid-rich wastewaters are pretreated with lipases and lipase-producing microorganisms. The enzymatic treatment of mixed sludge by added enzymes prior to anaerobic digestion has been shown to result in improved degradation of the sludge and an increase in methane production. Strategies for enzyme dosing to enhance anaerobic digestion of the different complex organic rich materials have been investigated. This review also highlights the various challenges and opportunities that exist to improve enzymatic hydrolysis of complex organic matter for biogas production. The arguments in favor of enzymes to pretreat complex biomass are compelling. The high cost of commercial enzyme production, however, still limits application of enzymatic hydrolysis in full-scale biogas production plants, although production of low-cost enzymes and genetic engineering are addressing this issue.     

Enzyme research and applications in biotechnological intensification of biogas production

Biogas technology provides an alternative source of energy to fossil fuels in many parts of the world. Using local resources such as agricultural crop remains, municipal solid wastes, market wastes and animal waste, energy (biogas), and manure are derived by anaerobic digestion. The hydrolysis process, where the complex insoluble organic materials are hydrolysed by extracellular enzymes, is a rate-limiting step for anaerobic digestion of high-solid organic solid wastes. Biomass pretreatment and hydrolysis are areas in need of drastic improvement for economic production of biogas from complex organic matter such as lignocellulosic material and sewage sludge. Despite development of pretreatment techniques, sugar release from complex biomass still remains an expensive and slow step, perhaps the most critical in the overall process. This paper gives an updated review of the biotechnological advances to improve biogas production by microbial enzymatic hydrolysis of different complex organic matter for converting them into fermentable structures. A number of authors have reported significant improvement in biogas production when crude and commercial enzymes are used in the pretreatment of complex organic matter. There have been studies on the improvement of biogas production from lignocellulolytic materials, one of the largest and renewable sources of energy on earth, after pretreatment with cellulases and cellulase-producing microorganisms. Lipids (characterised as oil, grease, fat, and free long chain fatty acids, LCFA) are a major organic compound in wastewater generated from the food processing industries and have been considered very difficult to convert into biogas. Improved methane yield has been reported in the literature when these lipid-rich wastewaters are pretreated with lipases and lipase-producing microorganisms. The enzymatic treatment of mixed sludge by added enzymes prior to anaerobic digestion has been shown to result in improved degradation of the sludge and an increase in methane production. Strategies for enzyme dosing to enhance anaerobic digestion of the different complex organic rich materials have been investigated. This review also highlights the various challenges and opportunities that exist to improve enzymatic hydrolysis of complex organic matter for biogas production. The arguments in favor of enzymes to pretreat complex biomass are compelling. The high cost of commercial enzyme production, however, still limits application of enzymatic hydrolysis in full-scale biogas production plants, although production of low-cost enzymes and genetic engineering are addressing this issue.     

Metabolic potential of the organic‐solvent tolerant Pseudomonas putida DOT‐T1E deduced from its annotated genome

Pseudomonas putida DOT‐T1E is an organic solvent tolerant strain capable of degrading aromatic hydrocarbons. Here we report the DOT‐T1E genomic sequence (6 394 153 bp) and its metabolic atlas based on the classification of enzyme activities. The genome encodes for at least 1751 enzymatic reactions that account for the known pattern of C, N, P and S utilization by this strain. Based on the potential of this strain to thrive in the presence of organic solvents and the subclasses of enzymes encoded in the genome, its metabolic map can be drawn and a number of potential biotransformation reactions can be deduced. This information may prove useful for adapting desired reactions to create value‐added products. This bioengineering potential may be realized via direct transformation of substrates, or may require genetic engineering to block an existing pathway, or to re‐organize operons and genes, as well as possibly requiring the recruitment of enzymes from other sources to achieve the desired transformation.     

Lipases and (R)-oxynitrilases: useful tools in organic synthesis

The use of enzymes in organic solvents is currently of special relevance for the preparation of products of high added value. Lipases are the enzymes that have shown the greatest utility through enzymatic transesterification reactions. Over the last few years, we have shown the value of the enzymatic aminolysis and ammonolysis reactions for the preparation of amides and for the resolution of esters and amines. We have shown that the enzymatic alkoxycarbonylation is also of great utility in chemoselective reactions of natural products. Lyases, enzymes much less exploited in organic synthesis, are proving increasingly interesting, especially the use of (R)-oxynitrilases for the synthesis of optically active cyanohydrins.     

Salt-activation of nonhydrolase enzymes for use in organic solvents

Enzymatic reactions are important for the synthesis of chiral molecules. One factor limiting synthetic applications of enzymes is the poor aqueous solubility of numerous substrates. To overcome this limitation, enzymes can be used directly in organic solvents; however, in nonaqueous media enzymes usually exhibit only a fraction of their aqueous-level activity. Salt-activation, a technique previously demonstrated to substantially increase the transesterification activity of hydrolytic enzymes in organic solvents, was applied to horse liver alcohol dehydrogenase, soybean peroxidase, galactose oxidase, and xanthine oxidase, which are oxidoreductase and oxygenase enzymes. Assays of the lyophilized enzyme preparations demonstrated that the presence of salt protected enzymes from irreversible inactivation. In organic solvents, there were significant increases in activity for the salt-activated enzymes compared to nonsalt-activated controls for every enzyme tested. The increased enzymatic activity in organic solvents was shown to result from a combination of protection against inactivation during the freeze-drying process and other as-yet undetermined factors.     

Novel carbon-carbon bond formations for biocatalysis

Carbon-carbon bond formation is the key transformation in organic synthesis to set up the carbon backbone of organic molecules. However, only a limited number of enzymatic C-C bond forming reactions have been applied in biocatalytic organic synthesis. Recently, further name reactions have been accomplished for the first time employing enzymes on a preparative scale, for instance the Stetter and Pictet-Spengler reaction or oxidative C-C bond formation. Furthermore, novel enzymatic C-C bond forming reactions have been identified like benzylation of aromatics, intermolecular Diels-Alder or reductive coupling of carbon monoxide.     

Aspects Of Biocatalyst Stability In Organic Solvents

The stability of biocatalysis in systems containing organic solvents is reviewed. Among the examples presented are homogeneous mixtures of water and water-miscible organic solvents, aqueous/organic two-phase systems, solid biocatalysts suspended in organic solvents, enzymes in reverse micelles and modified enzymes soluble in water immiscible solvents. The stability of biocatalysts in organic solvents depends very much on the conditions. The hydrophobicity or the polarity of the solvent is clearly of great importance. More hydrophobic solvents (higher log P values) are less harmful to enzymes than less hydrophobic solvents. The water content of the system is a very important parameter. Some water is essential for enzymatic activity; however, the stability of enzymes decreases with increasing water content. Mechanisms of enzyme inactivation are discussed.     

Stoichiometry of soil enzyme activity at global scale

Extracellular enzymes are the proximate agents of organic matter decomposition and measures of these activities can be used as indicators of microbial nutrient demand. We conducted a global-scale meta-analysis of the seven-most widely measured soil enzyme activities, using data from 40 ecosystems. The activities of beta-1,4-glucosidase, cellobiohydrolase, beta-1,4-N-acetylglucosaminidase and phosphatase g(-1) soil increased with organic matter concentration; leucine aminopeptidase, phenol oxidase and peroxidase activities showed no relationship. All activities were significantly related to soil pH. Specific activities, i.e. activity g(-1) soil organic matter, also varied in relation to soil pH for all enzymes. Relationships with mean annual temperature (MAT) and precipitation (MAP) were generally weak. For hydrolases, ratios of specific C, N and P acquisition activities converged on 1 : 1 : 1 but across ecosystems, the ratio of C : P acquisition was inversely related to MAP and MAT while the ratio of C : N acquisition increased with MAP. Oxidative activities were more variable than hydrolytic activities and increased with soil pH. Our analyses indicate that the enzymatic potential for hydrolyzing the labile components of soil organic matter is tied to substrate availability, soil pH and the stoichiometry of microbial nutrient demand. The enzymatic potential for oxidizing the recalcitrant fractions of soil organic material, which is a proximate control on soil organic matter accumulation, is most strongly related to soil pH. These trends provide insight into the biogeochemical processes that create global patterns in ecological stoichiometry and organic matter storage.     

多孔纳米材料固定化酶研究进展

酶是一种天然生物催化剂,有催化效率高、底物选择性强和绿色环保等优点,但酶结构不稳定且重复利用率低,制约了其产业化应用。随着技术的发展,酶的固定化可以提高酶的活性和稳定性,为生物酶的工程化应用带来了新的机遇。多孔纳米材料具有比表面积大、孔隙率高、机械和化学性能稳定等特点和优异的成本效益,是理想的固定化酶载体。本文综述了近些年来金属有机框架、共价有机框架和多孔微球等纳米材料固定化酶的研究进展和应用,重点介绍了载体固定酶的方式,并总结了每种载体的特点,最后讨论了多孔纳米材料固定化酶面临的挑战和发展趋势。     

The principles of enzymes stabilization: VI. Catalysis by water-soluble enzymes entrapped into reversed micelles of surfactants in organic solvents

• 1.1.|The possibility of stabilizing water-soluble enzymes againsts the inactivating action of organic solvents by means of surfactants has been studied. Several enzymes (α-chymotrypsin (EC 3.4.21.1), trypsin (EC 3.4.21.4), pyrophosphatase (EC 3.6.1.1), peroxidase (EC 1.11.1.7), lactate dehydrogenase (EC 1.1.1.27) and pyruvate kinase (EC 2.7.1.40)) were used to demonstrate that enzymes can be entrapped into reversed micelles formed by surfactants (Aerosol OT, cetyltrimethylammonium bromide, Brij 56) in an organic solvent (benzene, chloroform, octane, cyclohexane). The enzymes solubilized in this way retain their catalytic activity and substrate specificity.
• 2.2.|A kinetic theory has been put forward that describes enzymatic reactions occurring in a micelle-solvent pseudobiphasic system. In terms of this theory, an explanation is given for the experimental dependence of the Michaelis-Menten equation parameters on the concentrations of the components of a medium (water, organic solvent, surfactant) and also on the combination of the signs of the charges in the substrate molecules and on interphase (++, +−, −−).
• 3.3.|The results obtained by us may prove important for applications of enzymes in organic synthesis and for studying the state and role of water in the structure of biomembranes and active centres of enzymes.

     

Catalytic mechanisms, basic roles, and biotechnological and environmental significance of halogenating enzymes

The understanding of enzymatic incorporation of halogen atoms into organic molecules has increased during the last few years. Two novel types of halogenating enzymes, flavindependent halogenases and α-ketoglutarate-dependent halogenases, are now known to play a significant role in enzyme-catalyzed halogenation. The recent advances on the halogenating enzymes RebH, SyrB2, and CytC3 have suggested some new mechanisms for enzymatic halogenations. This review concentrates on the occurrence, catalytic mechanisms, and biotechnological applications of the halogenating enzymes that are currently known.     

Enzymology in monophasic organic media.

Over the past year, an important area of research has been directed towards the fundamental aspects of enzymes and new applications of enzymology in monophasic organic media. Much of this research has focused on the factors that influence enzymatic catalysis in monophasic organic solvents, including the importance of enzyme-associated water, and the effect of organic solvents on enzyme structure and thermodynamic features. From an applications perspective, new advances in the use of enzymes in organic and polymer syntheses and optical resolutions have been made.     

Recent advances of enzymatic reactions in ionic liquids

The tremendous potential of room temperature ionic liquids as an alternative to environmentally harmful ordinary organic solvents is well recognized. Ionic liquids, having no measurable vapor pressure, are an interesting class of tunable and designer solvents, and they have been used extensively in a wide range of applications including enzymatic biotransformation. In fact, ionic liquids can be designed with different cation and anion combinations, which allow the possibility of tailoring reaction solvents with specific desired properties, and these unconventional solvent properties of ionic liquids provide the opportunity to carry out many important biocatalytic reactions that are impossible in traditional solvents. As compared to those observed in conventional organic solvents, the use of enzymes in ionic liquids has presented many advantages such as high conversion rates, high enantioselectivity, better enzyme stability, as well as better recoverability and recyclability. To date, a wide range of pronounced approaches have been taken to further improve the performance of enzymes in ionic liquids. This review presents the recent technological developments in which the advantages of ionic liquids as a medium for enzymes have been gradually realized.     

Recent advances in exploiting ionic liquids for biomolecules: Solubility,stability and applications

The technological utility of biomolecules (e.g. proteins, enzymes and DNA) can be significantly enhanced by combining them with ionic liquids (ILs) – potentially attractive ”green“ and ”designer“ solvents – rather than using in conventional organic solvents or water. In recent years, ILs have been used as solvents, cosolvents, and reagents for biocatalysis, biotransformation, protein preservation and stabilization, DNA solubilization and stabilization, and other biomolecule‐based applications. Using ILs can dramatically enhance the structural and chemical stability of proteins, DNA, and enzymes. This article reviews the recent technological developments of ILs in protein‐, enzyme‐, and DNA‐based applications. We discuss the different routes to increase biomolecule stability and activity in ILs, and the design of biomolecule‐friendly ILs that can dissolve biomolecules with minimum alteration to their structure. This information will be helpful to design IL‐based processes in biotechnology and the biological sciences that can serve as novel and selective processes for enzymatic reactions, protein and DNA stability, and other biomolecule‐based applications.     

Extracellular enzyme kinetics scale with resource availability

Microbial community metabolism relies on external digestion, mediated by extracellular enzymes that break down complex organic matter into molecules small enough for cells to assimilate. We analyzed the kinetics of 40 extracellular enzymes that mediate the degradation and assimilation of carbon, nitrogen and phosphorus by diverse aquatic and terrestrial microbial communities (1160 cases). Regression analyses were conducted by habitat (aquatic and terrestrial), enzyme class (hydrolases and oxidoreductases) and assay methodology (low affinity and high affinity substrates) to relate potential reaction rates to substrate availability. Across enzyme classes and habitats, the scaling relationships between apparent V_max and apparent K_m followed similar power laws with exponents of 0.44 to 0.67. These exponents, called elasticities, were not statistically distinct from a central value of 0.50, which occurs when the K_m of an enzyme equals substrate concentration, a condition optimal for maintenance of steady state. We also conducted an ecosystem scale analysis of ten extracellular hydrolase activities in relation to soil and sediment organic carbon (2,000–5,000 cases/enzyme) that yielded elasticities near 1.0 (0.9 ± 0.2, n = 36). At the metabolomic scale, the elasticity of extracellular enzymatic reactions is the proportionality constant that connects the C:N:P stoichiometries of organic matter and ecoenzymatic activities. At the ecosystem scale, the elasticity of extracellular enzymatic reactions shows that organic matter ultimately limits effective enzyme binding sites. Our findings suggest that one mechanism by which microbial communities maintain homeostasis is regulating extracellular enzyme expression to optimize the short-term responsiveness of substrate acquisition. The analyses also show that, like elemental stoichiometry, the fundamental attributes of enzymatic reactions can be extrapolated from biochemical to community and ecosystem scales.