Biocatalysis in organic media

The potentials of using organic reaction media in biotechnological conversions have already been demonstrated in several experimental studies. Examples of possible advantages are: possibility of higher substrate and/or product concentrations, favorable shift of reaction equilibria, reduced substrate and/or product inhibition, and facilitated product recovery. Especially water/organic solvent two-phase systems seem to possess several of these advantages. The solvent type will highly affect kinetics and stability of the (immobilized) biocatalyst, solubility and partitioning of reactants/products, and product recovery. Therefore the solvent choice can have a large influence on the economics of the two-liquid-phase biocatalytic process. Immobilization of the biocatalyst may be useful to provide protection against denaturating solvent effects. The polarity of the employed support material will also be decisive for the partitioning of substrates and products among the various phases.

A classification of biphasic systems, which is based on the possible types of theoretical concentration profiles and aqueous phase configurations, is discussed. Reversed micelles and aqueous two-liquid-phase systems can be considered as special cases. The design of two-liquid-phase bioreactors is dependent on the state of the biocatalyst, free or immobilized, and on the necessity for emulsification of one of the two liquid phases in the other. Many mass-transfer resistances, e.g. across the liquid/liquid interface, in the aqueous phase, across the liquid/solid interface, and in the biocatalyst phase, can limit the overall reaction rate. The epoxidation of alkenes in water/solvent two-phase systems is discussed to give an example of the scope of biotechnological processes that is obtained by using organic media. Finally, a design calculation of a packed-bed organic-liquid-phasel immobilized-biocatalyst reactor for the epoxidation of propene is given to illustrate some of the above aspects.     

Biocatalysis in Spain: A field of success and innovation

Abstract

This is the presentation of the Special Issue “Biocatalysis in Spain”, covering the work of several Spanish groups in the field of Biocatalysis and Biotransformations. Thus, both research articles and reviews allow one to draw an accurate view of the state-of-the-art development in Spain.     

Biocatalysis of lipoxygenase in selected organic solvent media

The biocatalysis of purified soybean lipoxygenase (LOX) (EC 1.13.11.12), using linoleic acid as a substrate model, was investigated in selected organic solvent media, including chloroform, dichloromethane, hexane, iso-octane, octane and toluene. The results indicated that there was a 2.6-fold increase in LOX activity in the monophasic iso-octane medium compared to that obtained in the aqueous medium. The results also showed that there was an increase of 2.2- and 1.8-fold in LOX activity in the monophasic reaction media of octane and hexane, respectively. However, an inhibitory effect on enzyme activity was observed when the monophasic reaction media of toluene, chloroform and dichloromethane were used. In addition, the results showed that the optimum concentration of octane and iso-octane in the biphasic medium containing the organic solvent and Tris–HCl buffer solution, was determined to be 3.5% and 4%, respectively, for LOX activity. Moreover, the biocatalysis of LOX in a ternary micellar system, containing either 3.5% octane or 4% iso-octane, Tris–HCl buffer solution and an emulsifier, resulted in an overall increase in enzyme activity. The K_m and V_max values, substrate specificity, optimum protein concentration, optimum reaction temperature as well as the enzymatically catalyzed end-products were investigated for LOX biocatalysis in both ternary micellar systems.     

非常规介质中细胞生化反应研究进展

对完整细胞在非常规介质中的生物催化反应进行了回顾 ,分别总结了产物为醇 ,甾体 ,有机酸 ,生物大分子及其它各类反应的研究进展。并从溶剂和细胞两种角度对主要的研究方法进行了阐述。     

Bacterial β‐Aminopeptidases: Structural Insights and Applications for Biocatalysis

β‐Aminopeptidases comprise a class of enzymes with functional and structural similarities. All members of the β‐aminopeptidases described to date were isolated from bacterial sources. Uniquely, they catalyze the hydrolysis of β³‐ and/or β²‐amino acid residues from amides and peptides that are otherwise considered proteolytically stable. Due to this unusual reactivity with β‐peptide substrates, β‐aminopeptidases have potential to be used as biocatalysts for β‐peptide synthesis and for the resolution of enantiomerically pure β‐amino acids from racemic substrate mixtures. β‐Aminopeptidases are formed from an inactive precursor by posttranslational autoproteolytic cleavage, exposing the catalytic nucleophile at the N‐terminus of the newly formed β‐polypeptide chain. Such an activation step is a characteristic trait of enzymes of the N‐terminal nucleophile (Ntn) hydrolase superfamily. However, classical Ntn hydrolases and β‐aminopeptidases differ by the fold of their catalytic cores and are hence likely to originate from distinct evolutionary ancestors. In this contribution, we review the existing literature on β‐aminopeptidases, including biochemical and functional studies, as well as structural investigations that recently allowed insights into the catalytic mechanisms of precursor processing and β‐peptide conversion.     

Natural products – learning chemistry from plants

Plant natural products (PNPs) are unique in that they represent a vast array of different structural features, ranging from relatively simple molecules to very complex ones. Given the fact that many plant secondary metabolites exhibit profound biological activity, they are frequently used as fragrances and flavors, medicines, as well as industrial chemicals. As the intricate structures of PNPs often cannot be mimicked by chemical synthesis, the original plant providers constitute the sole source for their industrial, large‐scale production. However, sufficient supply is not guaranteed for all molecules of interest, making the development of alternative production systems a priority. Modern techniques, such as genome mining and thorough biochemical analysis, have helped us gain preliminary understanding of the enzymatic formation of the valuable ingredients in planta. Herein, we review recent advances in the application of biocatalytical processes, facilitating generation of complex PNPs through utilization of plant‐derived specific enzymes and combinatorial biochemistry. We further evaluate the options of employing heterologous organisms harboring PNP biosynthetic pathways for the production of secondary metabolites of interest.     

Biocatalysis of tyrosinase using catechin as substrate in selected organic solvent media

The enzymatic activity of mushroom tyrosinase was investigated using catechin as substrate in selected organic solvent media. The results showed that optimal tyrosinase activity was obtained at pH 6.2, 6.6, 6.0 and 6.2 in the organic solvent media of heptane, toluene, dichloromethane, and dichloroethane, respectively, and at a temperature between 25°C and 27.5°C. In addition, the kinetic studies showed that the K_m values were 5.38, 1.03, 2.52 and 4.03 mM, for the tyrosinase-catechin biocatalysis in the reaction media of heptane, toluene, dichloromethane, and dichloroethane, respectively, while the corresponding V_max values were 1.22×10⁻³, 0.33×10⁻³, 1.47×10⁻³ and 1.20×10⁻³ δA per μg protein per second, respectively. The use of acetone as co-solvent for the tyrosinase-catechin biocatalysis showed that acetone concentrations ranging from 5% to 30% (v/v) in the heptane reaction medium produced a decrease of 4.3% to 96.7% in tyrosinase activity. The results also indicated that the presence of 12.5% acetone in the reaction medium of dichloromethane, and 22.0% in those of toluene and dichloroethane produced a maximal increase of 42.6%, 92.1% and 71.8%, respectively, in tyrosinase activity. However, the overall findings indicated that additional increases in acetone concentration resulted in an inhibition of tyrosinase activity.     

Biocatalysis in ionic liquids: the stereoconvergent hydrolysis of trans-β-methylstyrene oxide catalyzed by soluble epoxide hydrolase

Soluble epoxide hydrolase (sEH) was shown to catalyze hydrolysis of epoxides using the ionic liquids (ILs) [bmim][PF₆], [bmim][N(Tf)₂], and [bmim][BF₄] (where bmim=1-butyl-3-methylimidazolium, PF₆=hexafluorophosphate, N(Tf)₂=bis(trifluoromethylsulfonyl)imide, and BF₄=tetrafluoroborate) as reaction medium. Reaction rates were generally comparable with those observed in buffer solution, and when the cress enzyme was used the hydrolysis of trans-β-methylstyrene oxide gave, through a stereoconvergent process, the corresponding optically active (1S,2R)-erythro-1-phenylpropane-1,2-diol.     

Biocatalysis of silica gel-immobilized chlorophyllase in a ternary micellar system

A partially purified enzymic extract from Phaeodactylum tricornutum was immobilized on silica gel and the specific activity of chlorophyllase in its free and immobilized states were compared in a ternary micellar system. The storage stability of the free and immobilized chlorophyllase extracts, maintained at temperatures ranging from 4 to 35°C for a period of 0–20 h, was temperature-dependent. The results also showed that the specific activity of the free and immobilized chlorophyllase extracts was highest at 30°C for long-term incubation, using chlorophyll and pheophytin as substrates and that a three-fold increase in the specific activity of the immobilized chlorophyllase was observed in comparison to that obtained with the free counterpart. The findings indicated that when free and immobilized chlorophyllase extracts were recovered and reused with both substrates, the immobilized chlorophyllase extract could be recycled for longer periods of time, while the free enzyme extract showed no activity after the first cycle.     

From Enzyme Kinetics to Metabolic Network Modeling – Visualization Tool for Enhanced Kinetic Analysis of Biochemical Network Models

Model‐based analysis of enzyme kinetics allows the determination of optimal conditions for their use in biocatalysis. For biotransformations or fermentative approaches the modeling of metabolic pathways or complex metabolic networks is necessary to obtain model‐based predictions of steps which limit product formation within the network. To set up adequate kinetic models, relevant mechanistic information about enzyme properties is required and can be taken from in vitro studies with isolated enzymes or from in vivo investigations using stimulus‐response experiments which provide a lot of kinetic information about the metabolic network. But with increasing number of reaction steps and regulatory interdependencies in the network structure the amount of simulation data dramatically increases and the simulation results from the dynamic models become difficult to analyze and interpret. Demonstrated for an Escherichia coli model of the central carbon metabolism, methods for visualization and animation of simulation data were applied and extended to facilitate model analysis and biological interpretation. The dynamic metabolite pool and metabolic flux changes were visualized simultaneously by a software tool. In addition, a new quantification method for enzyme activation/inhibition was proposed, and this information was implemented in the metabolic visualization.     

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.     

Trends and developments in physical anthropology, 1990–91

The 1408 members of the American Association of Physical Anthropologists were surveyed by mail regarding professional background, training deficiencies, opinions regarding areas of future importance to the discipline, and teaching/research specializations. A total of 544 responses (39%) resulted; 71% were from professionals in the United States and 16% were from U.S. students. Survey results are compared to surveys done in 1971 and in 1978, and are broken into three cohorts: pre-1971 Ph.D.s, 1972–1978 Ph.D.s, and post-1979 Ph.D.s. Statistics and anatomy continue to be common training deficiencies across cohorts. Molecular/cell biology and writing are new training deficiencies that reflect contemporary concerns and trends in the discipline. Anatomy, genetics, ecology, and paleontology are still considered important to the future of physical anthropology; statistics, computer science, and the biomedical sciences are also thought to be of importance to its future. The most frequent teaching/research specializations are growth and development, evolutionary biology, and population studies. Genetics and primatology appear to be losing popularity; biomedical anthropology, statistics, and ecology appear to be gaining it. The survey results have implications for the future training of graduate students and for employment opportunities in physical anthropology.     

Biocatalysis in ionic liquids for lignin valorization: Opportunities and recent developments

Lignin holds tremendous potential as a renewable feedstock for upgrading to a number of high-value chemicals and products that are derived from the petroleum industry at present. Since lignin makes up a significant fraction of lignocellulosic biomass, co-utilization of lignin in addition to cellulose and hemicelluloses is vital to the economic viability of cellulosic biorefineries. The recalcitrant nature of lignin, originated from the molecule's compositional and structural heterogeneity, however, poses great challenges toward effective and selective lignin depolymerization and valorization. Ionic liquid (IL) is a powerful solvent that has demonstrated high efficiency in fractionating lignocellulosic biomass into sugar streams and a lignin stream of reduced molecular weight. Compared to thermochemical methods, biological lignin deconstruction takes place at mild temperature and pressure while product selectivity can be potentially improved via the specificity of biocatalysts (lignin degrading enzymes, LDEs). This review focuses on a lignin valorization strategy by harnessing the biomass fractionating capabilities of ILs and the substrate and product selectivity of LDEs. Recent advances in elucidating enzyme-IL interactions as well as strategies for improving enzyme activity in IL are discussed, with specific emphases on biocompatible ILs, thermostable and IL-tolerant enzymes, enzyme immobilization, and surface charge engineering. Also reviewed is the protein engineering toolsets (directed evolution and rational design) to improve the biocatalysts' activity, stability and product selectivity in IL systems. The alliance between IL and LDEs offers a great opportunity for developing a biocatalytic route for lignin valorization.     

Life in Sulfidic Environments: Historical Perspective and Current Research Trends

SYNOPSIS. The discovery of the deep-sea hydrothermal vents andtheir associated fauna in 1977 was a watershed for the recentinterest in the biology of sulfidic environments. From the initialquestions concerning how organisms survived at the high sulfideconcentrations around the vents, research quickly focused onthe previously unrecognized sulfuroxidizing chemoautotrophicinvertebrate/bacterial symbioses whose nature was discoveredin 1980. There followed a successful and frenzied search forother reducing habitats harboring non-vent chemoautotrophicsymbioses. The interest in the biology of species living insulfidic habitats has since expanded to include the non-symbioticspecies, species with sulfuroxidizing symbionts and specieswith methanotrophic symbionts, all of which must be adaptedto tolerate sulfide and many of which are adapted to utilizesulfide in various ways.     

Trends in Overweight and Obesity in Portugal: The National Health Surveys 1995–6 and 1998–9

Objective: To assess overweight and obesity trends in the Portuguese population. Research Methods and Procedures: National Health Surveys, conducted in 1995–6 (17, 989 men; 20, 249 women) and 1998–9 (17, 923 men; 20, 302 women), were used. Results: In men, the prevalence of overweight and obesity increased from 39.9% (95% CI: 39.2 to 40.6) and 10.3% (95% CI: 9.9 to 10.7), respectively, in 1995–6, to 42.5% (95% CI: 41.8 to 43.2) and 11.5% (95% CI: 11.0 to 12.0), respectively, in 1998–9. In women, prevalence of obesity increased from 12.7% (95% CI: 12.2 to 13.2) in 1995–6 to 14.2% (95% CI: 13.7 to 14.6) in 1998–9, whereas the prevalence of overweight remained stable: 32.2% (95% CI: 31.6 to 32.9) in 1995–6 and 32.3% (95% CI: 31.6 to 32.9) in 1998–9. In men, prevalence of overweight and obesity were higher among former smokers and educational group of 6 to 12 years, and prevalence of obesity was higher in the Lisbon region. In women, prevalence of overweight and obesity were higher among never smokers, and prevalence of obesity was higher among educational group of ?6 years. Prevalence of obesity was higher in the Alentejo region, and overweight was higher in the Center region. Finally, prevalence of obesity increased in both sexes for all strata studied (age, smoking status, educational level, and geographic region), whereas prevalence of overweight increased only in men. Discussion: In Portugal, overweight and obesity levels are related to sociodemographic factors; the increase in obesity levels stresses the need for preventive measures.     

Application of lipase catalysis in organic solvents for selective protection–deprotection of bioactive compounds

Lipases are useful catalysts to realise in non-conventional medium esterifications and alcoholysis of polyhydroxylated compounds with high level of regioselectivity. This paper describes some examples of regioprotection–deprotection of flavonoids and conduritols, realised in our laboratory, using Pseudomonas cepacia, Mucor miehei and Candida cylindracea (C. rugosa) lipases.     

Multiplexed Capillary Electrophoresis as Analytical Tool for Fast Optimization of Multi‐Enzyme Cascade Reactions – Synthesis of Nucleotide Sugars

Nucleotide sugars are considered as bottleneck and expensive substrates for enzymatic glycan synthesis using Leloir‐glycosyltransferases. Synthesis from cheap substrates such as monosaccharides is accomplished by multi‐enzyme cascade reactions. Optimization of product yields in such enzyme modules is dependent on the interplay of multiple parameters of the individual enzymes and governed by a considerable time effort when convential analytic methods like capillary electrophoresis (CE) or HPLC are applied. We here demonstrate for the first time multiplexed CE (MP‐CE) as fast analytical tool for the optimization of nucleotide sugar synthesis with multi‐enzyme cascade reactions. We introduce a universal separation method for nucleotides and nucleotide sugars enabling us to analyze the composition of six different enzyme modules in a high‐throughput format. Optimization of parameters (T, pH, inhibitors, kinetics, cofactors and enzyme amount) employing MP‐CE analysis is demonstrated for enzyme modules for the synthesis of UDP‐α‐D‐glucuronic acid (UDP‐GlcA) and UDP‐α‐D‐galactose (UDP‐Gal). In this way we achieve high space‐time‐yields: 1.8 g/L?h for UDP‐GlcA and 17 g/L?h for UDP‐Gal. The presented MP‐CE methodology has the impact to be used as general analytical tool for fast optimization of multi‐enzyme cascade reactions.     

Conservation of Drill Populations in Bakossiland,Cameroon: Historical Trends and Current Status

Very little information is available on the current status of drill populations in Cameroon. We report on drill group sizes and status in Bakossiland, a mountainous area spanning 2000 km² in the Littoral and South West Provinces of southwestern Cameroon. Between 1970 and 2002 direct visual counts of drill groups (n = 105) yielded group size estimates ranging from 5 to 400 (mean ± S.E.M.; 93.1 ± 8.4). We encountered solitary adult male drills on 8 occasions. Groups were at all elevations (150–2000 m) in 5 habitat types: lowland, premontane, submontane and montane forests and montane savannah at 2000 m). Group sizes did not vary with respect to elevation, habitat type or season (wet and dry mo). However, over the past decade drills have been virtually hunted out of the Mwenzekong Mountains (Banyang Mbo Wildlife Sanctuary), and they are greatly reduced in the southern Bakossi forests of Mwendolengo, Edib Hills and Mungo River. The species became extinct in the Loum Forest Reserve in the late 1970s, and until recently was thought to have become extinct on Mount Mwanenguba. Since 1994 on Mount Kupe, the drill population has begun to recover, largely due to protection afforded by the Bakossi traditional chiefs. Traditional powers and values are still influential in the region. A new national park—Bakossi Mountains National Park— and associated Protected Areas are currently under gazettment. We discuss the effectiveness of conservation strategies in relation to the survival of drills in the area.     

Trends and Disparities in Provider Diagnosis of Overweight Analysis of NHANES 1999–2004

Rates of overweight and obesity are disproportionately high within minority populations. This study examined the trends in provider diagnosis of overweight from 1999 to 2004 and examined whether there were differences in provider diagnosis based on race/ethnicity. We examined data from 4,071 adults with BMI ≥30 who participated in the National Health and Nutrition Examination Surveys (NHANES) (1999–2004). Provider diagnosis was determined by self‐report. From 1999 to 2004, the provider diagnosis of overweight decreased from 71 to 64% (P = 0.003). After controlling for potential confounders, non‐Hispanic blacks and Mexican Americans were less likely to report a provider diagnosis of overweight compared to non‐Hispanic whites. Odds ratio (OR) (95% confidence interval (CI)) for non‐Hispanic blacks was 0.6 (95% CI, 0.4–0.8) and for Mexican Americans was 0.7 (95% CI, 0.4–1.0) compared to non‐Hispanic whites. Reasons for this disparity warrant further investigation.