Use of apple seed meal as a new source of beta-glucosidase for enzymatic glucosylation of 4-substituted benzyl alcohols and tyrosol in monophasic aqueous-dioxane medium

A facile method for enzymatic glycosylation of 4-substituted benzyl alcohols and tyrosol with glucose in a monophasic aqueous-dioxane medium was reported, using a crude meal of apple seed as a new catalyst. The corresponding beta-d-glucosides were synthesized in moderate yields (13.1-23.1%), among which the salidroside was obtained in 15.8% yield.     

Preparation of L-aspartic acid by means of immobilized Alcaligenes metalcaligenes cells

Two types of biocatalysts based on immobilized cells of Alcaligenes metalcaligenes exhibiting aspartate ammonia-lyase activity (EC 4.3.1.1) were developed for the enzymic preparation of L-aspartic acid from ammonium fumarate. The first type of the biocatalyst consists in individual covalently crosslinked and permeabilized cells(I), while the second type is represented by cell aggregates (II). For the above preparation, biocatalyst I can be used only discontinuously in a mixed reactor. After termination of the reaction between individual cycles of its use, the biocatalyst is returned to the reactor in the form of a highly concentrated cell suspension or paste. Biocatalyst II can be used discontinuously or continuously in a fixed-bed column of the catalyst. The effects of pH, substrate concentration and temperature on the reaction velocity and effectivity of enzymic conversion was investigated. Optimal parameters of the reaction are as follows: pH 8.5, initial substrate concentration, 1.35 mol/L, temperature for discontinuous process, 37 degrees C, and temperature for continuous process, 25 degrees C. Under these conditions the enzymic conversion of substrate to product is quantitative. Under optimal toring conditions, the specific activity of both catalysts does not change within a period of one year. The operational half-life of the biocatalyst II during continuous use in a fixed-bed column of the catalyst under standard reaction conditions depends on the quality of the substrate. The discontinuous preparation of L-asparatic acid with the aid of biocatalyst I and continuous preparation of this product with the aid of biocatalyst II have been verified under pilot-plant conditions.     

Performance of an immobilized fuculose-1-phosphate aldolase for stereoselective synthesis

A derivative of fuculose-1-phosphate aldolase, immobilized with high loading on glyoxal–agarose gels, has been characterized and evaluated as a biocatalyst for an aldol addition reaction. The reaction of the solid biocatalyst was diffusion-controlled for conversion of its natural substrate. Nevertheless, when catalyzing the synthesis of a biologically active aminopolyol, the lower reaction rate with non-natural substrates led to a process controlled by the intrinsic enzyme kinetics. The resulting biocatalyst has high synthetic specific activity and has been successfully used in batch synthesis reactions with high conversion. In addition, the immobilized aldolase has been employed in fed-batch synthesis, increasing the selectivity of the reaction and obtaining high conversion (88%).     

Microbial oxidation of naphthalene to cis-1,2-naphthalene dihydrodiol using naphthalene dioxygenase in biphasic media

The selective oxidation of aryl substrates to chiral cis-1,2-dihydrodiols is an industrially important reaction for the production of intermediates that can be used to produce fine chemicals, pharmaceuticals, and many other bioactive natural products. More specifically, the oxidation of naphthalene to produce optically pure (+)-cis-(1R,2S)-1,2-napthalene dihydrodiol (NDHD) to be used as a chiral synthon for specialty chemicals has gained much interest. Escherichia coli JM109(DE3) pDTG141 expresses naphthalene dioxygenase which catalyzes this reaction. Poor substrate solubility and substrate toxicity are barriers to using the power of these enzymes in large-scale aqueous whole cell systems. A biphasic reaction system was chosen to overcome these barriers. The optimal biphasic conditions for E. coli JM109(DE3) pDTG141 were determined to be 20% dodecane as the organic solvent containing 40 g/L naphthalene. The productivity of the biotransformation using resting cells was 1.75 g-diol/g-cdw/h for the first 6 h with 20% organic phase, which was increased from 0.59 g-diol/g-cdw/h for growing cells with 40% organic phase. The biocatalytic activity was retained for at least 12 h. The biocatalyst could be recycled for at least four runs in both suspended and immobilized form. The stability of the 12 h recycle was improved by immobilization in calcium alginate beads. The process has been improved both environmentally and economically by reducing the amount of solvent used and by recycling the biocatalyst.     

Novel screening methods--the key to cloning commercially successful biocatalysts

Providing sufficient biocatalyst to support the demands of multi tonne product supply can be problematical. Here we describe how screening for and cloning a gamma-lactamase overcame biocatalyst supply issues, and greatly improved the actual biocatalytic process. The isolation of an expressing gamma-lactamase clone from a gene library necessitated a combination of classical molecular biology techniques together with innovative screening methods to identify a functional clone. Once isolated the enzyme was characterised with regard to its process performance and proved to be active at 500 g L(-1) substrate. Further development of the recombinant fermentation and downstream processing has resulted in the ability to produce sufficient biocatalyst from one 5001 fermentation to resolve 5 metric tonnes of (+/-)-lactam, whilst simplifying the process chemistry greatly.     

An integrated process: ester synthesis in an enzymatic membrane reactor and water sorption

In the case of such reactions as ester synthesis, water is produced during the reaction. Because these reactions are carried out in hydrophobic solvents an additional (water) phase in the system must not be allowed, i.e. the concentration of water saturation in the organic solvent should not be exceeded. In such a case, the reaction kinetics and product equilibrium concentration undergo undesirable changes because of the partition coefficient of the components and hampered process of product separation. Hence, removal of the water produced in the reaction determines whether the process is successful or not. For this purpose, the integrated process with water sorption in the column with molecular sieves was applied. Integration of the process of synthesis and dehydration of a reaction phase, in which a biocatalyst is suspended and not dissolved as in water solutions, requires holding up of the catalyst in the reactor before directing the stream of reaction mixture to dehydration process. This hold-up and a possibility of multiple use of the catalyst may be accomplished by using a separating barrier, e.g. an ultrafiltration membrane or by permanent fixing of the catalyst to the matrix, e.g. a polymeric membrane. The efficiency and activity of a biocatalyst (lipase CAL-B) immobilized on a polymer membrane by sorption and chemical binding, were determined. A subject of study was the synthesis of geranyl acetate, one of the most known aromatic compound. A hydrophobic (polypropylene) matrix was shown to be a much better carrier in the reactions performed in an organic solvent than a hydrophilic (polyamide) membrane being tested. The reaction kinetics of geranyl acetate synthesis with the use of geraniol and acetic acid as substrates, was described by the equation defining the "Ping-Pong Bi Bi" mechanism that was related additionally to the inhibition of a substrate (acetic acid). The following constants of kinetic equation were obtained k(3)(')=0.344 mol g(-1)h(-1), K(mA)=0.257 mol l(-1), K(mG)=1.629 and K(iA)=0.288 for the native enzyme and v(max,Gel)=111.579 mol l(-1)h(-1), K(mA)=0.255 mol l(-1), K(mG)=1.91 mol l(-1), K(iA)=0.238 mol l(-1) for the one immobilized by sorption on a polypropylene membrane. Half-life time of the native enzyme activity was 204 h and stability of the immobilized preparation was 70 h. With respect to the reaction kinetics and stability of the native enzyme and immobilized preparation, from both types of membrane bioreactor more attractive appears to be the one in which the membrane is used not as a catalyst layer but only as a barrier that immobilizes the native enzyme within the bioreactor volume. When an integrated process proceeds, the method to collect water in the sorption column during the process, appeared to work very well. The reaction proceeded with a very high efficiency (after 120 h alpha=98.2% for native enzyme and 83.2% for immobilized enzyme) and due to low water concentration in the system ( approximately 0.000% v/v) the second phase was not created.     

Polyhydroxylated steroidal constituents from the fresh rhizomes of Tupistra yunnanensis

Six new polyhydroxylated steroidal saponins, tupistrosides A-F (1-6), together with nine known steroids, were isolated from the fresh rhizomes of Tupistra yunnanensis. Their structures were elucidated to be (25S)-1beta,4beta,5beta-trihydroxy-spirostane-3beta-yl O-alpha-l-arabinopyranoside (1), 1beta,24beta-dihydroxy-spirost-5,25(27)-dien-3alpha-yl O-beta-d-glucopyranoside (2), (22S,25S)-1alpha,2beta,3alpha,5alpha-tetrahydroxy-furo-spirostane-26-yl O-beta-d-glucopyranoside (3), 1beta,3alpha,22 xi-trihydroxy-furost-5,25(27)-dien-26-yl O-beta-d-glucopyranoside (4), 26-O-beta-d-glucopyranosyl-1beta,22-dihydroxy-furost-5-en-3alpha-yl O-beta-d-glucopyranoside (5) and 22-methoxy-1beta,2beta,3beta,4beta,5beta,7alpha-hexahydroxy-furost-25(27)-en-6-one-26-yl O-beta-d-glucopyranoside (6), respectively, by means of spectroscopic analysis and the results of acid hydrolysis.     

Production of 6-aminopenicillanic acid by immobilized Pleurotus ostreatus

Summary 6-Aminopenicillanic acid from penicillin V is produced by Pleurotus ostreatus immobilized by entrapment in a chitosan matrix. In these carriers the cell concentration increases after network formation by irreversible shrinking of the biocatalyst. Specific activity of the biocatalyst for the hydrolysis reaction is 1,31 mol.min^-1. (g wet weight of catalyst)^-1 corresponding to a relative activity of 38%. Catalytic half-life of immobilized Pl. ostreatus is 25 days compared to 2.5 days for free suspended cells.This paper is part of the Dissertation of Michael Kluge, Technical University Braunschweig 1981.     

Preparative scale Baeyer-Villiger biooxidation at high concentration using recombinant Escherichia coli and in situ substrate feeding and product removal process

An efficient biocatalytic process based on the use of adsorbent resin (in situ substrate feeding and product removal) makes experiments at high substrate concentration possible by overcoming limitations due to substrate and product inhibition. This process was successfully applied to the preparative scale Baeyer-Villiger biooxidation of (-)-(1S,5R)-bicyclo[3.2.0]hept-2-en-6-one (25 g). Whole cells of recombinant E. coli (1 liter) overexpressing cyclohexanone monooxygenase were used as a biocatalyst and the substrate was preloaded onto the adsorbent resin. The corresponding lactone was obtained in 75-80% yield. Time for cell growth and biotransformation is about 24 h each and oxygen supply can be improved by using a tailor-made bubble column.     

D-malate production by permeabilized Pseudomonas pseudoalcaligenes; optimization of conversion and biocatalyst productivity

For the development of a continuous process for the production of solid D-malate from a Ca-maleate suspension by permeabilized Pseudomonas pseudoalcaligenes, it is important to understand the effect of appropriate process parameters on the stability and activity of the biocatalyst. Previously, we quantified the effect of product (D-malate2 -) concentration on both the first-order biocatalyst inactivation rate and on the biocatalytic conversion rate. The effects of the remaining process parameters (ionic strength, and substrate and Ca2 + concentration) on biocatalyst activity are reported here. At (common) ionic strengths below 2 M, biocatalyst activity was unaffected. At high substrate concentrations, inhibition occurred. Ca2+ concentration did not affect biocatalyst activity. The kinetic parameters (both for conversion and inactivation) were determined as a function of temperature by fitting the complete kinetic model, featuring substrate inhibition, competitive product inhibition and first-order irreversible biocatalyst inactivation, at different temperatures simultaneously through three extended data sets of substrate concentration versus time. Temperature affected both the conversion and inactivation parameters. The final model was used to calculate the substrate and biocatalyst costs per mmol of product in a continuous system with biocatalyst replenishment and biocatalyst recycling. Despite the effect of temperature on each kinetic parameter separately, the overall effect of temperature on the costs was found to be negligible (between 293 and 308 K). Within pertinent ranges, the sum of the substrate and biocatalyst costs per mmol of product was calculated to decrease with the influent substrate concentration and the residence time. The sum of the costs showed a minimum as a function of the influent biocatalyst concentration.     

Improvement of the enzymatic synthesis of ethyl valerate by esterification reaction in a solvent system

The present study reports the improved enzymatic synthesis of ethyl valerate (green apple flavor) by esterification reaction of ethanol and valeric acid in heptane medium. Lipase from Thermomyces lanuginosus (TLL) was immobilized by physical adsorption on polyhydroxybutyrate (PHB) particles and used as a potential biocatalyst. The effect of certain parameters that influence the ester synthesis was evaluated by factorial design. The experimental conditions that maximized the synthesis of ethyl valerate were 30.5°C, 18% m/v of biocatalyst (TLL–PHB), absence of molecular sieves, agitation of 234?rpm, and 1,000?mM of each reactant (ethanol and valeric acid). Under these conditions, conversion percentage ≈92% after 105?min of reaction was observed. Soluble TLL was also used as biocatalyst and the highest conversion was of 82% after 120?min of reaction. Esterification reaction performed in a solvent-free system exhibited conversion of 13% after 45?min of reaction catalyzed by immobilized lipase, while the soluble lipase did not exhibit catalytic activity. The synthesis of the ester was confirmed by Fourier transform infrared spectroscopy and gas chromatography–mass spectrometry analyses. After six consecutive cycles of ethyl valerate synthesis, the prepared biocatalyst retained ≈86% of its original activity.     

Testing for diffusion limitations in salt-activated enzyme catalysts operating in organic solvents

The dramatic activation of serine proteases in nonaqueous media resulting from lyophilization in the presence of KCl is shown to be unrelated to relaxation of potential substrate diffusional limitations. Specifically, lyophilizing subtilisin Carlsberg in the presence of KCl and phosphate buffer in different proportions, ranging from 99% (w/w) enzyme to 1% (w/w) enzyme in the final lyophilized solids, resulted in biocatalyst preparations that were not influenced by substrate diffusion. This result was made evident through use of a classical analysis whereby initial catalytic rates, normalized per weight of total enzyme in the catalyst material, were measured as a function of active enzyme for biocatalyst preparations containing different ratios of active to inactive enzyme. The active enzyme content of a given biocatalyst preparation was controlled by mixing native subtilisin with subtilisin preinactivated with PMSF, a serine protease inhibitor, and lyophilizing the enzyme mixture in the presence of different fractions of KCl and phosphate buffer. Plots of initial reaction rates as a function of percent active subtilisin in the biocatalyst were linear for all biocatalyst preparations. Thus, enzyme activation (reported elsewhere to be as high as 3750-fold in hexane for the transesterification of N-Ac-L-Phe-OEt with n-PrOH) is a manifestation of intrinsic enzyme activation and not relaxation of diffusional limitations resulting from diluted enzyme preparations. Similar activation is reported herein for thermolysin, a nonserine protease, thereby demonstrating that enzyme activation due to lyophilization in the presence of KCl may be a general phenomenon for proteolytic enzymes.     

Immobilized cells of recombinant Escherichia coli strain for continuous production of L-aspartic acid

For L-aspartic acid biosynthesis, high production cells of Escherichia coli mutant B-715 and P1 were immobilized in chitosan gel using a technique developed in our laboratory. The immobilization process reduced initial activity of the intact cells, however, the biocatalyst produced was very stabile for long-term use in multi-repeated batch or continuous processes. Temperature influence on the conversion of ammonium fumarate to L-aspartic acid was investigated. In long-term experiments, over 603 hours, the temperature 40 degrees C was found to be the best for both biocatalyst stability and high conversion rate. The optimum substrate concentration was 1.0 M. Continuous production of L-aspartic acid was investigated in three types of column bioreactors characterized by different volumes as well as different high to biocatalyst bed volume rations (Hz/Vz). The highest conversion rate, 99.8%, and the productivity 6 g/g/h (mass of L-aspartic acid per dry mass of cells in biocatalyst per time unit) was achieved in the bioreactor with the highest value Hz/Vz = 3.1, and liquid hour space velocity value of 5.2, defined as the volume of feeding substrate passed per volume of catalyst in bioreactor per one hour.     

Combined immobilized lipases for effective biodiesel production from spent coffee grounds

This work describes the enzymatic transesterification of the oil extracted from SCGs for synthesis of biodiesel as a promising alternative to diesel fuels based on petroleum. Biocatalysts from various sources were tested for biodiesel synthesis using coffee oil among which CaCO₃- immobilized Staphylococcus aureus and Bacillus stearothermophilus showed the highest conversion yields (61 ± 2.64% and 64.3 ± 1.53%, respectively) in 4 h. In further optimizing reaction parameters, methanol to oil molar ratio, biocatalyst quantity, water content, as well as incubation time and temperature markedly improved oil-to-biodiesel conversion up to 99.33 ± 0.57 % in a solvent free reaction after 12 h at 55 °C. A mixture of inexpensive CaCO₃-immobilized bacterial lipases at a 1:1 ratio was the best environment-friendly catalyst for biofuel synthesis as well as the ideal trade-off between conversion and cost. Obtained coffee biodiesel remained stable beyond 40 days at ambient storage conditions and its chemical characteristics were comparable to those of other known biodiesels according to the European requirements (EN14214). Collectively, SCGs, after oil extraction, could be an ideal substrate for the production of an environment-friendly biodiesel by using appropriate mixture of CaCO₃-immobilized lipases.     

Dormancy Release,Germination, and Electrolyte Leakage from Apple Embryos during Stratification in the Presence of Sucrose

The dynamics of dormancy release during the stratification of apple (Malus domestica Borkh.) seeds was quantitatively described by three characteristics of seeds germination: the percentage of seeds that germinated by the tenth day, mean germination time, and the sum of seeds germinated in each of ten days (Timson's parameter), which allowed the assessment of the viability, the rate of dormancy release, and seed heterogeneity. We showed that apple seeds were characterized by a combined (physical and physiological) type of dormancy, with the seed coat and the embryo envelope being involved in the maintenance of physical dormancy. The addition of sucrose to the stratification medium accelerated the release of seed dormancy and improved all characteristics that determine seed germinability. Electrolyte leakage from embryos hardly changed during stratification, which agrees with the fact that all seeds remained viable throughout the entire period of dormancy. We assume that the release of seed dormancy is not a single-stage process.     

Enhanced Asymmetric Reduction of Ethyl 3‐Oxobutyrate by Baker's Yeast via Substrate Feeding and Enzyme Inhibition

The moderate enantioselectivity of wild form baker's yeast can be considerably increased either by using continuous feeding to maintain a low substrate concentration throughout the reaction, or by the selective inhibition of competing enzymatic pathways. The reduction of ethyl 3‐oxobutyrate to ethyl (S)‐3‐hydroxybutyrate was used as a model reaction. With the substrate feeding method, the enantioselectivity could be increased from 75 % to as high as 98 %. The increased selectivity originates from the much higher substrate binding constant of the (R)‐specific enzymes, so that these enzymes remain essentially inactive if a low concentration of ethyl 3‐oxobutyrate is maintained in the bioreactor. Alternatively, the enantioselectivity of baker's yeast can be improved by selectively blocking competing enzymatic pathways. It was found that vinyl acetate is a selective inhibitor for the (R)‐specific enzymes. Ethyl (S)‐3‐hydroxybutyrate with an enantiomeric excess of 98 % was obtained by pre‐incubation of baker's yeast in 100 mM of vinyl acetate solution for 1 h. These results suggest that by selecting appropriate process conditions, natural baker's yeast can be a competitive biocatalyst for the large‐scale production of chiral secondary alcohols.     

Integrated organic–aqueous biocatalysis and product recovery for quinaldine hydroxylation catalyzed by living recombinant Pseudomonas putida

In an earlier study, biocatalytic carbon oxyfunctionalization with water serving as oxygen donor, e.g., the bioconversion of quinaldine to 4-hydroxyquinaldine, was successfully achieved using resting cells of recombinant Pseudomonas putida, containing the molybdenum-enzyme quinaldine 4-oxidase, in a two-liquid phase (2LP) system (ütkür et al. J Ind Microbiol Biotechnol 38:1067-1077, 2011). In the study reported here, key parameters determining process performance were investigated and an efficient and easy method for product recovery was established. The performance of the whole-cell biocatalyst was shown not to be limited by the availability of the inducer benzoate (also serving as growth substrate) during the growth of recombinant P. putida cells. Furthermore, catalyst performance during 2LP biotransformations was not limited by the availability of glucose, the energy source to maintain metabolic activity in resting cells, and molecular oxygen, a possible final electron acceptor during quinaldine oxidation. The product and the organic solvent (1-dodecanol) were identified as the most critical factors affecting biocatalyst performance, to a large extent on the enzyme level (inhibition), whereas substrate effects were negligible. However, none of the 13 alternative solvents tested surpassed 1-dodecanol in terms of toxicity, substrate/product solubility, and partitioning. The use of supercritical carbon dioxide for phase separation and an easy and efficient liquid-liquid extraction step enabled 4-hydroxyquinaldine to be isolated at a purity of >99.9% with recoveries of 57 and 84%, respectively. This study constitutes the first proof of concept on an integrated process for the oxyfunctionalization of toxic substrates with a water-incorporating hydroxylase.     

Calcium alginate bead immobilization of cells containing tyrosine ammonia lyase activity for use in the production of p-hydroxycinnamic acid

An Escherichia coli catalyst with tyrosine ammonia lyase activity (TAL) has been stabilized for repeated use in batch conversions of high tyrosine solids to p-hydroxycinnamic acid (pHCA). The TAL biocatalyst was stabilized by controlling the reaction pH to 9.8 +/- 0.1 and immobilizing the cells within a calcium alginate matrix that was cross-linked with glutaraldehyde and polyethyleneimine (GA/PEI). We found a GA range where the bead-encapsulated TAL was not inactivated, and the resulting cross-linking provided the beads with the mechanical stability necessary for repeated use in consecutive batch reactions with catalyst recycle. The GA/PEI calcium alginate TAL catalyst was used in 41 1-L batch reactions where 50 g L(-1) tyrosine was converted to 39 +/- 4 g L(-1) pHCA in each batch. The practical usefulness and ease of this process was demonstrated by scaling up the TAL bead immobilization and using the immobilized TAL catalyst in four 125-L bioconversion reactions to produce over 12 kg of purified pHCA.     

Effect of paclobutrazol on water stress-induced abscisic Acid in apple seedling leaves

Abscisic acid (ABA) was quantitated by enzyme-linked immunosorbent assay (ELISA) in water-stressed leaves from control apple seedlings, and also from apple seedlings treated for 28 days with paclobutrazol ([2RS, 3RS]-1-[4-chlorophenyl]-4,4-dimethyl-2-[1,2,4-triazol-1-yl] pentan-3-ol). The ELISA quantitative estimates were also validated by gas chromatography-electron capture detector and lettuce seed germination inhibition bioassay. Paclobutrazol treatment reduced endogenous ABA levels by about one-third, and prevented the marked accumulation of water-stress-induced ABA that occurred in untreated seedlings. The presence of ABA in the apple leaf extracts was confirmed by gas chromatography-mass spectrometry.     

Formation of benzoquinol moiety in cornoside by salidroside mono-oxygenase,a cytochrome P450 enzyme,from <Emphasis Type="Italic">Abeliophyllum distichum </Emphasis>cell suspension cultures

A microsomal fraction prepared from Abeliophyllum distichumNakai (Oleaceae) cell suspension cultures oxidized salidroside, a glucoside of 4-hydroxyphenylethyl alcohol, to cornoside possessing a unique benzoquinol ring. The enzyme named salidroside mono-oxygenase required NADPH as the only cofactor, and molecular oxygen. The reaction was strongly inhibited by CO as well as several cytochrome P450 inhibitors, such as cytochrome c and miconazole, indicating the involvement of a cytochrome P450 enzyme. Salidroside mono-oxygenase accepted salidroside as the only substrate, but did not oxidize 4-hydroxyphenylethyl alcohol, the salidroside aglucone, and 4-hydroxybenzoic acid. The optimum pH of the reaction was 7.5, and apparent K(m) values for salidroside and NADPH were 44 micro M and 33 micro M, respectively. The benzoquinol ring formation mechanism is discussed in comparison to the mechanism for ipso substitution of 4-hydroxybenzoate by active oxygen species followed by elimination leading to hydroquinone.