Enzymatic hydroxylation reactions

During the past 18 months, considerable progress has been made in the understanding of the key enzyme-substrate interactions that control the regioselectivity and stereoselectivity of the hydroxylation reaction performed by cytochrome-P450-dependent enzymes of mammalian origin. The manipulation of microbial hydroxylating enzymes, in both whole-cell and cell-free environments, has also been examined in the context of controlling the regioselectivity and stereoselectivity of the hydroxylation reaction. Several new applications for hydroxylating enzymes have been reported, and the construction of chimeric hydroxylating enzymes has been used both for mechanistic studies and for the production of enzymes with high hydroxylating activity for a defined substrate.     

Yeast whole-cell biocatalyst constructed by intracellular overproduction of Rhizopus oryzae lipase is applicable to biodiesel fuel production

Yeast whole-cell biocatalysts for lipase-catalyzed reactions were constructed by intracellularly overproducing Rhizopus oryzae lipase (ROL) in Saccharomvces cerevisiae MT8-1. The gene encoding lipase from R. orvzae IFO4697 was cloned, and intracellular overproduction systems of a recombinant ROL with a pro-sequence (rProROL) were constructed. When rProROL from R. oryzae IFO4697 was produced under the control of the 5'-upstream region of the isocitrate lyase gene of Candida tropicalis (UPR-ICL) at 30 degrees C for 98 h by two-stage cultivation using SDC medium (SD medium with 2% casamino acids) containing 2.0% and 0.5% glucose, intracellular lipase activity reached levels up to 474.5 IU/l. These whole-cell biocatalysts were permeabilized by air-drying and used for the synthesis of methyl esters (MEs), a potential biodiesel fuel, from plant oil and methanol in a solvent-free and water-containing system. The ME content in the reaction mixture was 71 wt% after a 165-h reaction at 37 degrres C with stepwise addition of methanol. These results indicate that an efficient whole-cell biocatalyst can be prepared by intracellular overproduction of lipase in yeast cells and their permeabilization.     

Maximizing the stability of metabolic engineering‐derived whole‐cell biocatalysts

A systematic and powerful knowledge‐based framework exists for improving the activity and stability of chemical catalysts and for empowering the commercialization of respective processes. In contrast, corresponding biotechnological processes are still scarce and characterized by case‐by‐case development strategies. A systematic understanding of parameters affecting biocatalyst efficiency, that is, biocatalyst activity and stability, is essential for a rational generation of improved biocatalysts. Today, systematic approaches only exist for increasing the activity of whole‐cell biocatalysts. They are still largely missing for whole‐cell biocatalyst stability. In this review, we structure factors affecting biocatalyst stability and summarize existing, yet not completely exploited strategies to overcome respective limitations. The factors and mechanisms related to biocatalyst destabilization are discussed and demonstrated inter alia based on two case studies. The factors are similar for processes with different objectives regarding target molecule or metabolic pathway complexity and process scale, but are in turn highly interdependent. This review provides a systematic for the stabilization of whole‐cell biocatalysts. In combination with our knowledge on strategies to improve biocatalyst activity, this paves the way for the rational design of superior recombinant whole‐cell biocatalysts, which can then be employed in economically and ecologically competitive and sustainable bioprocesses.     

Investigation on macrokinetics of heterogeneous process of monosaccharide isomerization using non-growing cells of a glucoisomerase producer <Emphasis Type="Italic">Arthrobacter nicotianae</Emphasis> immobilized inside SiO<Subscript>2</Subscript>-xerogel

Macrokinetic peculiarites of heterogeneous process of monosaccharide (glucose/fructose) isomerization using biocatalysts prepared by incorporation of non-growing cells of a glucose isomerase-producing strain Arthobacter nicotianae inside SiO₂-xerogel have been investigated. It was shown that the process proceeds in kinetic regime without diffusion limitation and biocatalyst activities at 60 and 75°C were 19 and 32 U/g, respectively. Time of equilibrium in the reaction of monosaccharide isomerization was a function of starting (“triggering”) glucose isomerase activity in a unit of reaction volume. When the activity exceeds 10 U/ml, equilibrium equimolar mixture of glucose and fructose was produced within a few hours. It was established that a continuous process carried out in a plug-flow packed-bed reactor is more efficient than a batch process accompanied with recycling, first of all, to significant improvement of operation stability of the designed biocatalysts. Under model conditions of industrial heterogeneous process of producing glucose-fructose syrups, the half-life time of inactivation of the biocatalysts was more than 500 h at (65 ± 5)°C.     

Production of biodiesel fuel from soybean oil catalyzed by fungus whole-cell biocatalysts in ionic liquids

The methanolysis of soybean oil to produce a fatty acid methyl ester (ME, i.e., biodiesel fuel) was catalyzed by lipase-producing filamentous fungi immobilized on biomass support particles (BSPs) as a whole-cell biocatalyst in the presence of ionic liquids. We used four types of whole-cell biocatalysts: wild-type Rhizopus oryzae producing triacylglycerol lipase (w-ROL), recombinant Aspergillus oryzae expressing Fusarium heterosporum lipase (r-FHL), Candida antarctica lipase B (r-CALB), and mono- and diacylglycerol lipase from A. oryzae (r-mdlB). w-ROL gave the high yield of fatty acid methyl ester (ME) in ionic liquid [Emim][BF₄] or [Bmim][BF₄] biphasic systems following a 24 h reaction. While lipases are known to be severely deactivated by an excess amount of methanol (e.g. 1.5 Mequiv. of methanol against oil) in a conventional system, methanolysis successfully proceeded even with a methanol/oil ratio of 4 in the ionic liquid biphasic system, where the ionic liquids would work as a reservoir of methanol to suppress the enzyme deactivation. When only w-ROL was used as a biocatalyst for methanolysis, unreacted mono-glyceride remained due to the 1,3-positional specificity of R. oryzae lipase. High ME conversion was attained by the combined use of two types of whole-cell biocatalysts, w-ROL and r-mdlB. In a stability test, the activity of w-ROL was reduced to one-third of its original value after incubation in [Bmim][BF₄] for 72 h. The stability of w-ROL in [Bmim][BF₄] was greatly enhanced by cross-linking the biocatalyst with glutaraldehyde. The present study demonstrated that ionic liquids are promising candidates for use as the second solvent in biodiesel fuel production by whole-cell biocatalysts.     

Preparation of immobilized whole cell biocatalyst and biodiesel production using a packed-bed bioreactor

Rhizopus oryzae NBRC 4697 was selected from among promising candidates as a biocatalyst for biodiesel production. This microorganism was immobilized on to polyurethane foam coated with activated carbon for reuse, and, for biodiesel production. Vacuum drying of the immobilized cells was found to be more efficient than natural or freeze-drying processes. Although the immobilized cells were severely inhibited by a molar ratio of methanol to soybean oil in excess of 2.0, stepwise methanol addition (3 aliquots at 24-h feeding intervals) significantly prevented methanol inhibition. A packed-bed bioreactor (PBB) containing the immobilized whole cell biocatalyst was then operated under circulating batch mode. Stepwise methanol feeding was used to mitigate methanol inhibition of the immobilized cells in the PBB. An increase in the feeding rate (circulating rate) of the reaction mixture barely affected biodiesel production, while an increase in the packing volume of the immobilized cells enhanced biodiesel production noticeably. Finally, repeated circulating batch operation of the PBB was carried out for five consecutive rounds without a noticeable decrease in the performance of the PBB for the three rounds.     

Synthesis of substituted catechols using nitroarene dioxygenases

The nitroarene dioxygenases are in the class of Rieske iron-containing oxygenases that incorporate atmospheric oxygen into substrates via electrophilic attack on the substrate. In their native role, the nitroarene dioxygenases start degradative pathways by hydroxylating nitro-substituted, and adjacent unsubstituted carbons of nitroaromatic compounds. The reaction yields the corresponding nitro-cis-cyclohexadienediol, which is unstable and spontaneously re-aromatizes to form a catechol and nitrite. In bacterial metabolism, the specificity of the hydroxylation determines subsequent steps in degradation pathways. Experiments were done to find whether the specificity could be exploited to direct the hydroxylation of multiply substituted aromatic substrates and thereby produce novel catechols. Recombinant strains carrying genes for nitroarene dioxygenases were used for transformation of various substituted nitroaromatic compounds. The reactions were analyzed using HPLC to track substrate consumption and product formation, then GC–MS and NMR to identify the reaction products. A number of substituted catechols were obtained using the recombinant biocatalysts. The nitro-substituted carbon was the primary site for dioxygenase hydroxylation. When substrates included nitro and halogen substituents, the halogen-substituted positions were also targeted, but less frequently than the nitro-substituted site. The production of catechols was limited in batch fermentations, likely due to toxicity of the quinones that result from air oxidation of catechols. The nitroarene dioxygenases will serve as catalysts for direct synthesis of highly substituted catechols, however, the reaction conditions must be engineered to overcome product toxicity and allow sustained accumulation of catecholic products.     

A recombinant Escherichia coli whole cell biocatalyst harboring a cytochrome P450cam monooxygenase system coupled with enzymatic cofactor regeneration

A cytochrome P450cam monooxygenase (P450cam) system from the soil bacterium Pseudomonas putida requires electron transfer among three different proteins and a cofactor, nicotinamide adenine dinucleotide (NADH), for oxygenation of its natural substrate, camphor. Herein, we report a facile way to significantly enhance the catalytic efficiency of the P450cam system by the coupling of its native electron transfer system with enzymatic NADH regeneration catalyzed by glycerol dehydrogenase (GLD) in Escherichia coli whole cell biocatalysts. Recombinant E. coli harboring the P450cam system, but lacking GLD, exhibited little activity for camphor hydroxylation. In contrast, coexpression of GLD with the proteinaceous electron transfer components of P450cam resulted in about tenfold improvement in the substrate conversion, implying that the whole cell biocatalyst utilized molecular oxygen, endogenous NADH, and glycerol in the cell for catalysis. The addition of glycerol to the reaction media further promoted camphor hydroxylation, suggesting that exogenous glycerol is also available for GLD in the host cell and actively participates in the catalytic cycle. These results clearly show the utility of GLD towards functional reconstruction of the native P450cam system. The present approach may also be useful for E. coli whole cell biocatalysts with the other NADH-dependent oxygenases and oxidoreductases.     

Accelerated biocatalyst stability testing for process optimization

The deactivation of protein biocatalysts even at relatively low temperatures is one of the principal drawbacks to their use. To aid in the development of novel biocatalysts, we have derived an equation for both time- and temperature-dependent activity of the biocatalyst based on known concepts such as transition state theory and the Lumry-Eyring model. We then derived an analytical solution for the total turnover number (ttn), under isothermal operation, as a function of the catalytic constant kcat, the unfolding equilibrium constant K, and the intrinsic first-order deactivation rate constant(s) k(d,i). Employing an immobilized glucose isomerase biocatalyst in a CSTR and utilizing a linear temperature ramp beyond the Tm of the enzyme, we demonstrate an accelerated method for extracting the thermodynamic and kinetic constants describing the biocatalyst system. In addition, we demonstrate that the predicted biocatalyst behavior at different temperatures and reaction times is consistent with the experimental observations.     

Directed evolution of enzymes for applied biocatalysis

Directed evolution has rapidly emerged as a powerful new strategy for improving the characteristics of enzymes in a targeted manner. By coupling various protocols for generating large variant libraries of genes, together with high-throughput screens that select for specific properties of an enzyme, such as thermostability, catalytic activity and substrate specificity, it is now possible to optimize biocatalysts for specific applications. However, further work is required to broaden the range of screens that can be used, particularly in terms of reaction type, such as hydroxylation and carbon-carbon bond formation, and functional characteristics, such as enantioselectivity and regioselectivity, so that directed evolution can be used in a routine manner for biocatalyst development.     

Studies on the accumulation of heavy metal elements in biological systems

Summary Cells of a Daucus carota suspension culture were entrapped in a matrix of calcium alginate. The immobilised cells, incubated in a buffer mixture of sucrose, nitrate, KCl, CaCl₂, 2-(N-morpholino)-ethane sulphonic acid at pH 5.5, hydroxylated digitoxigenin. When compared under the same incubation conditions, freely suspended cells biotransformed digitoxigenin at a faster rate. Periplogenin formation was maximal at pH 5.3 and temperatures of 26°–34°C. The hydroxylase activity of the entrapped cells adapted to the presence of 20 mM CaCl₂ over a 12 day incubation. The diffusion barrier established on entrapment of the cells could not be overcome by addition of detergents or methanol. Controlled addition of chloroform (at 1/4 and 1/2 saturation) did stimulate hydroxylation of digitoxigenin without adversely affecting cell viability. The rate of hydroxylation of digitoxigenin was linear over an immobilised cell concentration of 0–7 mg dry weight and a digitoxigenin concentration of 0–20 mg/L. Five consecutive batch bioconversions at a rate greater than 60% could be achieved before the biocatalyst was inactivated. The results are discussed in relation to improving the hydroxylation reaction by immobilised D. carota and other reactions performed by immobilised plant cells.     

Biodiesel production using Aspergillus niger as a whole‐cell biocatalyst in a packed‐bed reactor

Enzymatic lipase transesterification of palm oil to biodiesel in a packed‐bed reactor (PBR) using a novel strain of the fungus Aspergillus niger, immobilized within polyurethane biomass support particles (BSPs), was investigated. A three‐step addition of methanol was used to reduce lipase inhibition by immiscible methanol. The influence of water content and PBR flow rate was investigated. FAME yield was enhanced with an increase of PBR flow rate in the range of 0.15–30 L h^?1, where inefficient mixing of the reaction mixture at lower flow rates resulted in low conversion rates i.e. 69% after 72‐h reaction. Adding the third mole equivalent of methanol resulted in lipase inhibition due to methanol migration into the accumulated glycerol layer. Glutaraldehyde (GA) solution (0.5 vol.%) was used to stabilize lipase activity, which led to a high FAME yield (>90%) in the PBR after 72‐h of reaction time at a flow rate of 15 L h^?1, and a water content of 15%. Moreover, a high conversion rate (>85%) was maintained after four palm oil batch conversion cycles in the PBR. In contrast, lipase activity of non‐GA‐treated cells decreased with each PBR batch cycle, where only 70% FAME was produced after the forth PBR cycle. Transesterification of palm oil in a PBR system using BSPs‐immobilized A. niger as a whole‐cell biocatalyst is a viable process for enzymatic biodiesel production.     

Metabolism of biphenyl by Aspergillus toxicarius: induction of hydroxylating activity and accumulation of water-soluble conjugates.

Biphenyl metabolism in Aspergillus toxicarius occurs by successive hydroxylations in the 4- and 4'-positions, followed by conjugation with sulfate to produce 4-hydroxybiphenyl-O-sulfonic acid and 4,4'-dihydroxybiphenyl-O-sulfonic acid. The hydroxylation reactions normally occur only after a prolonged lag period after which the appearance of the monohydroxylated compound precedes the dihydroxylated compound. The accumulation of the monohydroxy compound is transient; therefore, it is an intermediate in the hydroxylating pathway. The onset of hydroxylating activity can be greatly accelerated when the culture is primed with the intermediate or product of the reaction (4-hydroxybiphenyl or 4,4'-dihydroxybiphenyl) at the time of biphenyl addition; a concentration of 0.05 mg 4,4'-dihydroxybiphenyl per ml produces optimal induction. Water-soluble conjugates of 4-hydroxybiphenyl and 4,4'-dihydroxybiphenyl were found in cultures of A. toxicarius grown in the presence of biphenyl plus inducer. The conjugate was shown to be the sulfate ester; no glucuronide or other conjugate species was found in any phase of the transformation. As with hydroxylating activity, the sulfotransferase activity appeared to be induced by the products of biphenyl metabolism.     

Heterogeneity of molecular forms of phenol oxidase from grape leaves

The substrate specificity and some kinetic properties of the monomeric (Mr = 26 000--35 000) and dimeric (Mr = 55 000--70 000) forms of phenol oxidase from vine leaves were studied. These forms possess different hydroxylating and o-diphenol oxidase activities. A kinetic analysis demonstrated that the monomeric form of the enzyme possesses a higher affinity for monophenols and can more effectively accomplish the hydroxylation reaction as compared to the dimeric one. During vine vegetation the ratio of molecular forms of phenol oxidase is altered manifesting itself in quantitative and qualitative changes of enzymatic activity. During plant maturation the dimeric fraction is predominant. The maturation process is associated with a sharp rise of the o-phenol oxidase activity, a disappearance of the hydroxylating activity and a substantial deceleration of phenol compounds production.     

Production of enantiomerically pure (S)-phenyl(pyridin-2-yl)methanol with Lactobacillus paracasei BD101

Abstract

Asymmetric reduction studies of heteroaryl ketones, including phenyl(pyridin-2-yl)methanone in enantioselective form with biocatalysts are very few, and chiral heteroaryl alcohols have been synthesized generally in the small scale. In this study, seven bacterial strains have been used to produce the (S)-phenyl(pyridin-2-yl)methanol in high enantiomeric excess and yield. Among the tested strains, Lactobacillus paracasei BD101, was found to be the best biocatalyst for the reducing phenyl(pyridin-2-yl)methanone to the (S)-phenyl(pyridin-2-yl)methanol at gram scale. The asymmetric bioreduction conditions were systematically optimized using L. paracasei BD101, which demonstrated excellent enantioselectivity and high level of conversion for the bioreduction reaction. (S)-phenyl(pyridin-2-yl)methanol, which is an analgesic, was produced enantiomerically pure form in the first time on gram scale using a biocatalyst. In total, 5.857?g of (S)-phenyl(pyridin-2-yl)methanol in enantiomerically pure form (>99% enantiomeric excess) was obtained in 52?h with 93% yield using whole cells of L. paracasei BD101. Enantiomerically pure (S)-phenyl (pyridin-2-yl)methanol, which is an analgesic, was first produced in the gram scale using a biocatalyst with excellent ee (>99%) and yield (93%).     

Design of an automated bioreactor system to screen immobilised whole cell catalysts

An automated CSTR system has been developed to study the stability of immobilised whole cell biocatalysts for ammonium acrylate production. Two potentially useful amidase-active rhodococci have been identified: One possesses an amidase of much higher activity than the other but of much lower stability. The activity and stability of each biocatalyst over a range of temperatures has been determined and used to compare the productivity of the biocatalysts in a bioconversion.     

Immobilization of steapsin lipase on macroporous immobead-350 for biodiesel production in solvent free system

Commercially available steapsin lipase was immobilized on macroporous polymer beads (IB-350) and further investigated for biodiesel production under solvent free conditions. The fatty acid methyl ester (biodiesel) synthesis was carried out by the methanolysis of fresh and used cooking sunflower oil. The enzymatic reaction for biodiesel synthesis was optimized with various reaction parameters and the obtained reaction conditions were 1: 6 molar ratio (oil: methanol), 50 mg biocatalyst and 20% water content at 45°C for 48 h under solvent free conditions. It was observed that 94% of biodiesel was produced under the optimized reaction conditions. The four step addition of methanol at the interval of 12 h was found to be more effective. Moreover the biocatalyst was effectively reused for four consecutive recycles and was appreciably stable for 90 days. The results obtained highlight potential of immobilized steapsin lipase for biodiesel production.     

Reconstitution of the In Vitro Activity of the Cyclosporine-Specific P450 Hydroxylase from Sebekia benihana and Development of a Heterologous Whole-Cell Biotransformation System

The cytochrome P450 enzyme CYP-sb21 from Sebekia benihana is capable of catalyzing the site-specific hydroxylation of the immunosuppressant cyclosporine (CsA), leading to the single product γ-hydroxy-N-methyl-l-Leu4-CsA (CsA-4-OH). Unlike authentic CsA, this hydroxylated CsA shows significantly reduced immunosuppressive activity while it retains a side effect of CsA, the hair growth stimulation effect. Although CYP-sb21 was previously identified to be responsible for CsA-specific hydroxylation in vivo, the in vitro activity of CYP-sb21 has yet to be established for a deeper understanding of this P450 enzyme and further reaction optimization. In this study, we reconstituted the in vitro activity of CYP-sb21 by using surrogate redox partner proteins of bacterial and cyanobacterial origins. The highest CsA site-specific hydroxylation activity by CYP-sb21 was observed when it was partnered with the cyanobacterial redox system composed of seFdx and seFdR from Synechococcus elongatus PCC 7942. The best bioconversion yields were obtained in the presence of 10% methanol as a cosolvent and an NADPH regeneration system. A heterologous whole-cell biocatalyst using Escherichia coli was also constructed, and the permeability problem was solved by using N-cetyl-N,N,N-trimethylammonium bromide (CTAB). This work provides a useful example for reconstituting a hybrid P450 system and developing it into a promising biocatalyst for industrial application.     

On ortho-diphenol oxidase activity of dopamine-β-monooxygenase

Dopamine-β-monooxygenase obtained from chromaffin granules revealed, besides hydroxylating, the oxidase activity towards catechol and catecholamines. The oxidase reaction results in the formation of hydrogen peroxide and corresponding o-quinones which, in the case of catecholamines, further spontaneously transform to adrenochrome or related compounds. The oxidase activity, like hydroxylation, is inhibited by copper chelators and inorganic anions with hign affinities to copper atoms. Thus, dopamine-β-monooxygenase may be considered also as a “non-blue” copper-containing oxidase. For the oxidase activity of dopamine-β-monooxygenase, in contrast to its hydroxylating function, the additional reducing cofactor, such as ascorbate, is not necessary.     

The involvement of cytochrome P-450 in hepatic microsomal steroid hydroxylation reactions supported by sodium periodate, sodium chlorite, and organic hydroperoxides.

The mechanism of steroid hydroxylation in rat liver microsomes has been investigated by employing NaIO4, NaClO2, and various organic hydroperoxides as hydroxylating agents and comparing the reaction rates and steroid products formed with those of the NADPH-dependent reaction. Androstenedione, testosterone, progesterone, and 17beta-estradiol were found to act as good substrates. NaIO4 was by far the most effective hydroxylating agent followed by cumene hydroperoxide, NADPH, NaClO2, pregnenolone 17alpha-hydroperoxide, tert-butyl hydroperoxide, and linoleic acid hydroperoxide. Androstenedione was chosen as the model substrate for inducer and inhibitor studies. The steroid was converted to its respective 6beta-, 7alpha, 15-, and 16alpha-hydroxy derivatives when incubated with microsomal fractions fortified with hydroxylating agent. Evidence for cytochrome P-450 involvement in androstenedione hydroxylation included a marked inhibition by substrates and modifiers of cytochrome P-450 and by reagents which convert cytochrome P-450 to cytochrome P-420. The ratios of the steroid products varied according to the type of hydroxylating agent used and were also modified by in vivo phenobarbital pretreatment. It was suggested that multiple forms of cytochrome P-450 exhibiting different affinities for hydroxylating agent are responsible for these different ratios. Horse-radish peroxidase, catalase, and metmyoglobin could not catalyze androstenedione hydroxylation. Addition of NaIO4, NaClO2, cumene hydroperoxide and other organic hydroperoxides to microsomal suspensions resulted in the appearance of a transient spectral change in the difference spectrum characterized by a peak at about 440 nm and a trough at 420 nm. The efficiency of these oxidizing agents in promoting steroid hydroxylation in microsomes appeared to be related to their effectiveness in eliciting the spectral complex. Electron donors, substrates, and modifiers of cytochrome P-450 greatly diminished the magnitude of the spectral change. It is proposed that NaIO4, NaClO2, and organic hydroperoxides promote steroid hydroxylation by forming a transient ferryl ion (compound I) of cytochrome P-450 which may be the common intermediate hydroxylating species involved in hydroxylations catalyzed by cytochrome P-450.