Immobilisation of laccase on Eupergit supports and its application for the removal of endocrine disrupting chemicals in a packed-bed reactor.

Laccase from Myceliophthora thermophila was covalently immobilised on Eupergit C and Eupergit C 250L yielding specific activities of up to 17 and 80 U/g, respectively. Due to its superior activity, Eupergit C 250L was chosen for further research. The somewhat lower catalytic efficiency (based on the ratio between the turnover number and the Michaelis constant, k(cat)/K(M)) of the immobilised enzyme in comparison with that of the free enzyme was balanced by its increased stability and broader operational window related to temperature and pH. The feasibility of the immobilised laccase was tested by using a packed bed reactor (PBR) operating in consecutive cycles for the removal of Acid Green 27 dye as model substrate. High degrees of elimination were achieved (88, 79, 69 and 57% in 4 consecutive cycles), while the levels of adsorption on the support varied from 18 to 6%, proving that dye removal took place mainly due to the action of the enzyme. Finally, a continuous PBR with the solid biocatalyst was applied for the treatment of a solution containing the following endocrine disrupting chemicals: estrone (E1), 17ß-estradiol (E2) and 17α-ethinylestradiol (EE2). At steady-state operation, E1 was degraded by 65% and E2 and EE2 were removed up to 80% and only limited adsorption of these compounds on the support, between 12 and 22%, was detected. In addition, a 79% decrease in estrogenic activity was detected in the effluent of the enzymatic reactor while only 14% was attained by inactivated laccase.

Photochemical regeneration of flavoenzymes - An Old Yellow Enzyme case-study.

Direct, NAD(P)H-independent regeneration of Old Yellow Enzymes represents an interesting approach for simplified reaction schemes for the stereoselective reduction of conjugated C=C-double bonds. Simply by illuminating the reaction mixtures with blue light in the presence of sacrificial electron donors enables to circumvent the costly and unstable nicotinamide cofactors and a corresponding regeneration system. In the present study, we characterise the parameters determining the efficiency of this approach and outline the current limitations. Particularly, the photolability of the flavin photocatalyst and the (flavin-containing) biocatalyst represent the major limitation en route to preparative application.

Non-enzymatic regeneration of nicotinamide and flavin cofactors for monooxygenase catalysis.

Biocatalytic oxygenation chemistry is a rapidly evolving field in which monooxygenases are the tools of choice. Monooxygenases catalyze many industrially important synthetic transformations; however, their use in preparative applications is hampered by their intrinsic requirement for reducing equivalents. As a result, non-enzymatic strategies--where the reducing equivalents are introduced directly into the catalytic cycle--are being developed to supersede the well-established enzymatic NAD(P)H regeneration systems currently in use. In this review we summarize and evaluate recent achievements in this area.

Synthetic cascades are enabled by combining biocatalysts with artificial metalloenzymes.

Enzymatic catalysis and homogeneous catalysis offer complementary means to address synthetic challenges, both in chemistry and in biology. Despite its attractiveness, the implementation of concurrent cascade reactions that combine an organometallic catalyst with an enzyme has proven challenging because of the mutual inactivation of both catalysts. To address this, we show that incorporation of a d(6)-piano stool complex within a host protein affords an artificial transfer hydrogenase (ATHase) that is fully compatible with and complementary to natural enzymes, thus enabling efficient concurrent tandem catalysis. To illustrate the generality of the approach, the ATHase was combined with various NADH-, FAD- and haem-dependent enzymes, resulting in orthogonal redox cascades. Up to three enzymes were integrated in the cascade and combined with the ATHase with a view to achieving (i) a double stereoselective amine deracemization, (ii) a horseradish peroxidase-coupled readout of the transfer hydrogenase activity towards its genetic optimization, (iii) the formation of L-pipecolic acid from L-lysine and (iv) regeneration of NADH to promote a monooxygenase-catalysed oxyfunctionalization reaction.

Ferritin-supported palladium nanoclusters: selective catalysts for aerobic oxidations in water.

Confinement of nanometallic Pd within the core of a hyperthermophilic ferritin cage (from Pyrococcus furiosus) is reported. The resulting nanostructured hybrid catalysts can be used for highly specific aerobic oxidation of alcohols in water.

Immobilization of laccase by encapsulation in a sol-gel matrix and its characterization and use for the removal of estrogens.

Laccase from Myceliophthora thermophila was immobilized by encapsulation in a sol-gel matrix based on methyltrimethoxysilane and tetramethoxysilane. The amount of laccase used for the preparation of the hydrogel was in the range 2.2-22 mg of protein/mL sol and the corresponding enzymatic activities were in the range 5.5-17.0 U/g biocatalyst. The kinetic parameters of the encapsulated laccase showed that the immobilized enzyme presented lower affinity for the substrate 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS). However, the stability of laccase was significantly enhanced after immobilization; thus, both pH and thermal stability improved about 10-30% and tolerance to different inactivating agents (NaN(3) , ZnCl(2) , CoCl(2) , CaCl(2) , methanol, and acetone) was 20-40% higher. The reusability of the immobilized laccase was demonstrated in the oxidation of ABTS for several consecutive cycles, preserving 80% of the initial laccase activity after 10 cycles. The feasibility of the immobilized biocatalyst was tested for the continuous elimination of Acid Green 27 dye as a model compound in a packed-bed reactor (PBR). Removals of 70, 58, 57, and 55% were achieved after four consecutive cycles with limited adsorption on the support: only 10-15%. Finally, both batch stirred tank reactor (BSTR) operated in several cycles and PBR, containing the solid biocatalyst were applied for the treatment of a solution containing the endocrine disrupting chemicals (EDCs): estrone (E1), 17ß-estradiol (E2), and 17α-ethinylestradiol (EE2). Eliminations of EDCs in the BSTR were higher than 85% and the reusability of the biocatalyst for the degradation of those estrogens was demonstrated. In the continuous operation of the PBR, E1 was degraded by 55% and E2 and EE2 were removed up to 75 and 60%, at steady-state conditions. In addition, a 63% decrease in estrogenic activity was detected.

Engineering of Candida antarctica lipase B for hydrolysis of bulky carboxylic acid esters.

Candida antarctica lipase B (CALB) is a widely used biocatalyst with high activity and specificity for a wide range of primary and secondary alcohols. However, the range of converted carboxylic acids is more narrow and mainly limited to unbranched fatty acids. To further broaden the biotechnological applications of CALB it is of interest to expand the range of converted carboxylic acid and extend it to carboxylic acids that are branched or substituted in close proximity of the carboxyl group. An in silico library of 2400 CALB variants was built and screened in silico by substrate-imprinted docking, a four step docking procedure. First, reaction intermediates of putative substrates are covalently docked into enzyme active sites. Second, the geometry of the resulting enzyme-substrate complex is optimized. Third, the substrate is removed from the complex and then docked again into the optimized structure. Fourth, the resulting substrate poses are rated by geometric filter criteria as productive or non-productive poses. Eleven enzyme variants resulting from the in silico screening were expressed in Escherichia coli BL21 and measured in the hydrolysis of two branched fatty acid esters, isononanoic acid ethyl ester and 2-ethyl hexanoic acid ethyl esters. Five variants showed an initial increase in activity. The variant with the highest wet mass activity (T138S) was purified and further characterized. It showed a 5-fold increase in hydrolysis of isononanoic acid ethyl ester, but not toward sterically more demanding 2-ethyl hexanoic acid ethyl ester.

United States population estimates and components of change: 1970 to 1987.

PIP: This report analyzes year-to-year change in the US population from 1970 to 1987, including natural increase and net civilian immigration. Data are drawn from Current Population Reports. 1) The January 1, 1988 total population including Armed Forces overseas was over 245 million. This reflects a .9% increase over January 1, 1987, and an increase of 18 million since the April 1, 1980 census. 2) In the beginning of 1988, Whites numbered 206,979 million, Blacks 30,083, and Blacks and other races 38,130. 3) The crude birth rate dipped from 15.9 in 1980 to 15.6 in 1987; there is no evidence of a consistent trend since 1980. 4) The 3.8 million births in 1987 reflect a continuation of the gradual increase births that has been occurring since the mid-1970s, an increase attributed to Baby Boomers. 5) There was an 8.7/1000 death rate in 1987. This rate has fluctuated in the 8.5-8.7/1000 range since 1977 after declining from 9.4/1000 in 1972. The 2.1 million deaths in 1987 continue the gradual increase that has occurred since the end of the 1940s. This increase is attributed to the growth in population and to the population's continued aging. 6) Net immigrants/1000 population dropped slightly to 2.5 in 1987, down from 2.7 in 1986 and 3.7 in 1980. 7) Rates of growth for both Blacks and Whites have declined substantially since 1960; Blacks declined by about 1/3 (from 2.2%) and Whites by more than 1/2 (from 1.5%). The population of other races increased by 4.5% in 1987. The Black population grew by 1.5% in 1987, compared with a growth of .7% for the White population.^ieng