Production of vanillin from waste residue of rice bran oil by Aspergillus niger and Pycnoporus cinnabarinus

A new technology of transforming ferulic acid, which was from waste residue of rice bran oil, into vanillin was developed by a combination of fungal strains Aspergillus niger CGMCC0774 and Pycnoporus cinnabarinus CGMCC1115. Various concentrations of ferulic acid were compared, and the highest yield reached 2.2 g l(-1) of vanillic acid by A. niger CGMCC0774 in a 25 l fermenter when concentration of ferulic acid was 4 g l(-1). The filtrate of A. niger CGMCC0774 culture was concentrated and vanillic acid in the filtrate was bio-converted into vanillin by P. cinnabarinus CGMCC1115. The yield of vanillin reached 2.8 g l(-1) when 5 g l(-1) of glucose and 25 g of HZ802 resin were supplemented in the bioconversion medium. The 13C isotope analysis indicated that delta13C(PDB) of vanillin prepared was much different from chemically synthesized vanillin.     

黑曲霉CGMCC0774和朱红密孔菌CGMCC1115两步转化阿魏酸制备生物香兰素

在25 L发酵罐中黑曲霉Aspergillus niger CGMCC0774转化阿魏酸可生成香草酸2.24 g/L,摩尔转化率64.6%;朱红密孔菌Pycnoporus cinnabarinus CGMCC1115转化提取的香草酸可生成香草醛1.45 g/L,摩尔转化率为79.9%。将两步微生物转化有机串联,即用黑曲霉转化液加预先培养的朱红密孔菌Pycnoporus cinnabarinus CGMCC1115菌丝体继续转化,可产香草醛1.06 g/L,对原料阿魏酸的摩尔转化率34.0%。用米糠提取的天然阿魏酸做原料,两步串联微生物转化制备的生物香兰素经13C同位素的分析,符合生物香草素的等同要求。     

Estimation of kinetic parameters in a structured yeast model using regularisation.

In this work, a procedure for estimating kinetic parameters in biochemically structured models was developed. The approach is applicable when the structure of a kinetic model has been set up and the kinetic parameters should be estimated. The procedure consists of five steps. First, initial values were found in or calculated from literature. Hereafter using sensitivity analysis the most sensitive parameters were identified. In the third step physiological knowledge was combined with the parameter sensitivities to manually tune the most sensitive parameters. In step four, a global optimisation routine was applied for simultaneous estimation of the most sensitive parameters identified during the sensitivity analysis. Regularisation was included in the simultaneous estimation to reduce the effect of insensitive parameters. Finally, confidence intervals for the estimated parameters were calculated. This parameter estimation approach was demonstrated on a biochemically structured yeast model containing 11 reactions and 37 kinetic constants as a case study.     

Aspergillus niger I-1472 and Pycnoporus cinnabarinus MUCL39533, selected for the biotransformation of ferulic acid to vanillin, are also able to produce cell wall polysaccharide-degrading enzymes and feruloyl esterases

The filamentous fungal strains Aspergillus niger I-1472 and Pycnoporus cinnabarinus MUCL39533, previously selected for the bioconversion of ferulic acid to vanillic acid and vanillin respectively, were grown on sugar beet pulp. A large spectrum of polysaccharide-degrading enzymes was produced by A. niger and very few levels of feruloyl esterases were found. In contrast, P. cinnabarinus culture filtrate contained low amount of polysaccharide-degrading enzymes and no feruloyl esterases. In order to enhance feruloyl esterases in A. niger cultures, feruloylated oligosaccharide-rich fractions were prepared from sugar beet pulp or cereal bran and used as carbon sources. Number of polysaccharide-degrading enzymes were induced. Feruloyl esterases were much higher in maize bran-based medium than in sugar beet pulp-based medium, demonstrating the ability of carbon sources originating from maize to induce the synthesis of feruloyl esterases. Thus, A. niger I-1472 could be interesting to release ferulic acid from sugar beet pulp or maize bran.     

Kinetic deuterium isotope effects for 7-alkoxycoumarin O-dealkylation reactions catalyzed by human cytochromes P450 and in liver microsomes. Rate-limiting C-H bond breaking in cytochrome P450 1A2 substrate oxidation

7-Ethoxy (OEt) coumarin has been used as a model substrate in many cytochrome P450 (P450) studies, including the use of kinetic isotope effects to probe facets of P450 kinetics. P450s 1A2 and 2E1 are known to be the major catalysts of 7-OEt coumarin O-deethylation in human liver microsomes. Human P450 1A2 also catalyzed 3-hydroxylation of 7-methoxy (OMe) coumarin at appreciable rates but P450 2E1 did not. Intramolecular kinetic isotope effects were used as estimates of the intrinsic kinetic deuterium isotope effects for both 7-OMe and 7-OEt coumarin dealkylation reactions. The apparent intrinsic isotope effect for P450 1A2 (9.4 for O-demethylation, 6.1 for O-deethylation) showed little attenuation in other competitive and noncompetitive experiments. With P450 2E1, the intrinsic isotope effect (9.6 for O-demethylation, 6.1 for O-deethylation) was attenuated in the noncompetitive intermolecular experiments. High noncompetitive intermolecular kinetic isotope effects were seen for 7-OEt coumarin O-deethylation in a baculovirus-based microsomal system and five samples of human liver microsomes (7.3-8.1 for O-deethylation), consistent with the view that P450 1A2 is the most efficient P450 catalyzing this reaction in human liver microsomes and indicating that the C-H bond-breaking step makes a major contribution to the rate of this P450 (1A2) reaction. Thus, the rate-limiting step appears to be the chemistry of the breaking of this bond by the activated iron-oxygen complex, as opposed to steps involved in the generation of the reactive complex. The conclusion about the rate-limiting step applies to all of the systems studied with this model P450 1A2 reaction including human liver microsomes, the most physiologically relevant.     

On a general model structure for macroscopic biological reaction rates

Macroscopic modelling of bioprocesses requires the determination of a biological reaction scheme and a kinetic model. The a priori selection of an appropriate kinetic model structure is usually made difficult by the lack of detailed bioprocess knowledge and the profusion of apparently similar biological kinetic laws. Moreover, parameter identification is made arduous and time-consuming by the strong non-linearities involved in kinetic laws. In most cases, these kinetic structures are non-linearizable and no first parameter estimation can be deduced easily. In order to avoid such identification problems, Bogaerts et al. [Bogaerts, Ph., Castillo, J., Hanus, R., 1999. A general mathematical modelling technique for bioprocesses in engineering applications. Syst. Anal. Model. Simul. 35, 87-113] have developed a general linearizable kinetic structure which allows the representation of activation and/or inhibition effects of each component in the culture. This paper further generalizes this structure in order to improve the way saturation effects are taken into account, and in turn, improve the biological interpretation of the model parameters. The main advantage of the proposed structure lies in an associated systematic estimation procedure. The usefulness of the proposed model is tested with simulated as well as with experimental data.     

Overproduction of laccase by a monokaryotic strain of Pycnoporus cinnabarinus using ethanol as inducer

AIMS: Laccase production by the monokaryotic strain Pycnoporus cinnabarinus ss3 was studied using ethanol as inducer in the culture medium. METHODS AND RESULTS: The effect of ethanol was tested at 10, 20, 30, 35 and 45 g l-1 and compared with that of ferulic acid, known until now as the most efficient inducer for laccase expression by P. cinnabarinus ss3. In the presence of 35 g l-1 ethanol, laccase activity (266 600 U l-1) and productivity (19 000 U l-1 day-1) were nine and fivefold higher compared with ferulic acid-induced cultures, and 155- and 65-fold higher compared with non-induced cultures, respectively. In vivo, ethanol added to the culture medium of P. cinnabarinus ss3 favoured a continuous and high expression of laccase gene. Under these conditions, P. cinnabarinus ss3 produced preferentially the isoenzyme LAC I. Ethanol added in vitro to the purified P. cinnabarinus ss3 laccase typically inhibited the enzymatic activity. CONCLUSIONS: In spite of an initial inhibitory effect on mycelial growth, ethanol was shown to be a very strong inducer for laccase expression by P. cinnabarinus ss3 allowing an average yield of 1-1.5 g l-1 laccase. SIGNIFICANCE AND IMPACT OF THE STUDY: This study identified P. cinnabarinus ss3 as an outstanding producer of laccase in the presence of ethanol as inducer. Ethanol is an inexpensive agricultural by-product and the process is simple to scale-up for industrial production.     

Assessment of formal and low structured kinetic modeling of polyhydroxyalkanoate synthesis from complex substrates

A formal kinetic mathematical model for poly-(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxybutyrate) [P(3HB-co-3HV-co-4HB)] terpolyester synthesis from glucose and galactose derived from whey permeate supplemented with gamma-butyrolactone by the archaeon Haloferax mediterranei was created. Further, a low structured mathematical model for poly-3-hydroxybutyrate synthesis from whey permeate by Pseudomonas hydrogenovora was developed. In both cases, biosyntheses for obtaining the experimental data used for compiling the models were performed via fed-batch cultivations. The model developed for H. mediterranei consists of 10 differential and 11 algebraic equations, including 27 kinetic constants. The model compiled for P. hydrogenovora encompasses 10 differential and 3 algebraic equations, including 36 kinetic constants. Both models were solved by Runge-Kuta variable step numerical integration with Monte Carlo parameter optimization procedure. Difficulties arising from the modeling of redirection of metabolic fluxes from biomass growth toward polyhydroxyalkanoate synthesis and byproducts are discussed.     

Modeling of uncertainties in biochemical reactions

Mathematical modeling is an indispensable tool for research and development in biotechnology and bioengineering. The formulation of kinetic models of biochemical networks depends on knowledge of the kinetic properties of the enzymes of the individual reactions. However, kinetic data acquired from experimental observations bring along uncertainties due to various experimental conditions and measurement methods. In this contribution, we propose a novel way to model the uncertainty in the enzyme kinetics and to predict quantitatively the responses of metabolic reactions to the changes in enzyme activities under uncertainty. The proposed methodology accounts explicitly for mechanistic properties of enzymes and physico‐chemical and thermodynamic constraints, and is based on formalism from systems theory and metabolic control analysis. We achieve this by observing that kinetic responses of metabolic reactions depend: (i) on the distribution of the enzymes among their free form and all reactive states; (ii) on the equilibrium displacements of the overall reaction and that of the individual enzymatic steps; and (iii) on the net fluxes through the enzyme. Relying on this observation, we develop a novel, efficient Monte Carlo sampling procedure to generate all states within a metabolic reaction that satisfy imposed constrains. Thus, we derive the statistics of the expected responses of the metabolic reactions to changes in enzyme levels and activities, in the levels of metabolites, and in the values of the kinetic parameters. We present aspects of the proposed framework through an example of the fundamental three‐step reversible enzymatic reaction mechanism. We demonstrate that the equilibrium displacements of the individual enzymatic steps have an important influence on kinetic responses of the enzyme. Furthermore, we derive the conditions that must be satisfied by a reversible three‐step enzymatic reaction operating far away from the equilibrium in order to respond to changes in metabolite levels according to the irreversible Michelis–Menten kinetics. The efficient sampling procedure allows easy, scalable, implementation of this methodology to modeling of large‐scale biochemical networks. Biotechnol. Bioeng. 2011;108: 413–423. © 2010 Wiley Periodicals, Inc.     

Two parallel pathways in the kinetic sequence of the dihydrofolate reductase from Mycobacterium tuberculosis

Dihydrofolate reductase from Mycobacterium tuberculosis (MtDHFR) catalyzes the NAD(P)H-dependent reduction of dihydrofolate, yielding NAD(P)(+) and tetrahydrofolate, the primary one-carbon unit carrier in biology. Tetrahydrofolate needs to be recycled so that reactions involved in dTMP synthesis and purine metabolism can be maintained. Previously, steady-state studies revealed that the chemical step significantly contributes to the steady-state turnover number, but that a step after the chemical step was likely limiting the reaction rate. Here, we report the first pre-steady-state investigation of the kinetic sequence of the MtDHFR aiming to identify kinetic intermediates, and the identity of the rate-limiting steps. This kinetic analysis suggests a kinetic sequence comprising two parallel pathways with a rate-determining product release. Although product release is likely occurring in a random fashion, there is a slight preference for the release of THF first, a kinetic sequence never observed for a wild-type dihydrofolate reductase of any organism studied to date. Temperature studies were conducted to determine the magnitude of the energetic barrier posed by the chemical step, and the pH dependence of the chemical step was studied, demonstrating an acidic shift from the pK(a) observed at the steady state. The rate constants obtained here were combined with the activation energy for the chemical step to compare energy profiles for each kinetic sequence. The two parallel pathways are discussed, as well as their implications for the catalytic cycle of this enzyme.     

A reexamination of the nucleotide incorporation fidelity of DNA polymerases

Intensive study has been devoted to understanding the kinetic and structural bases underlying the exceptionally high fidelity (low error frequencies) of the typical DNA polymerase. Commonly proposed explanations have included (i) the concept of fidelity check points, in which the correctness of a nascent base pair match is tested at multiple points along the reaction pathway, and (ii) an induced-fit fidelity enhancement mechanism based on a rate-limiting, substrate-induced conformational change. In this article, we consider the evidence and theoretical framework for the involvement of such mechanisms in fidelity enhancement. We suggest that a "simplified" model, in which fidelity is derived fundamentally from differential substrate binding at the transition state of a rate-limiting chemical step, is consistent with known data and sufficient to explain the substrate selectivity of these enzymes.     

Parameter identification of a dynamic model of CHO cell cultures: an experimental case study

In this paper, we address the problem of parameter identification in dynamic models of animal cultures, and we propose a step-by-step procedure, which gradually considers more detailed models. This procedure allows subsets of parameters to be estimated at each step, which can be used in the initialization of the next identification step. Finally, the full parameter set can be re-estimated starting from the results of the last step. The efficiency of the procedure is illustrated with a simulation case study and with the identification of a dynamic model from experimental data collected in CHO cell culture.     

Synergistic effect of laccase mediators on pentachlorophenol removal by Ganoderma lucidum laccase

Laccases have low redox potentials limiting their environmental and industrial applications. The use of laccase mediators has proven to be an effective approach for overcoming the low redox potentials. However, knowledge about the role played by the mediator cocktails in such a laccase-mediator system (LMS) is scarce. Here, we assembled different dual-agent mediator cocktails containing 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS), vanillin, and/or acetovanillone, and compared their mediating capabilities with those of each individual mediator alone in oxidation of pentachlorophenol (PCP) by Ganoderma lucidum laccase. Cocktails containing ABTS and either vanillin or acetovanillone strongly promoted PCP removal compared to the use of each mediator alone. The removal enhancement was correlated with mediator molar ratios of the cocktails and incubation times. Analysis of the kinetic constants for each mediator compound showed that G. lucidum laccase was very prone to react with ABTS rather than vanillin and acetovanillone in the cocktails. Moreover, the presence of the ABTS radical (ABTS^+•) and vanillin or acetovanillone significantly enhanced PCP removal concomitant with electron transfer from vanillin or acetovanillone to ABTS^+•. These results strongly suggest that vanillin and acetovanillone mediate the reaction between ABTS and PCP via multiple sequential electron transfers among laccase and its mediators.     

PCR扩增试验的动力学数学模型

PCR技术已日趋成熟,但因为影响因素较多、反应过程比较复杂,直到目前PCR技术已创立近二十年,尚未能给出较好的描述PCR 反应的数学方法。我们根据它的基本原理提出了能够描述其反应过程的动力学方程：Wamp=［Ntarg×(1+P)n1+0.5×Cenz×U×P×Ceactiv×(n-n1)-Ntarg× (1+n×P)］×Cu×M,准确地描述了PCR反应的产物积累规律,建立了PCR反应的动力学数学模型。用动力学数学模型预测的PE 7700仪器的CT值与仪器的实际数值一致。动力学数学模型配合适当的监测设备可以构成自动化的PCR 定量仪器。PE 7700 仪器使用本动力学模型处理、分析数据,定量结果的准确性会更好。各实验室可根据各自的实验条件,由模型估算PCR产物数量,为PCR后产物继续处理提供较准确的数量信息。本模型阐明了PCR反应在多次循环后必然由指数扩增转变为线性扩增的分子基础,为定量PCR 提供了准确的计算方法。 Abstract:The PCR technique has been set up for nearly twenty years and is becoming more and more ripe.But because of the multiple influencing factors and complicated reaction procedures,no mathematical method that can describe the PCR reaction has been given.On the basis of its elementary principle,we suggested a kinetic equation to describe the reaction procedure,Wamp=［Ntarg×(1+P)n1+0.5×Cenz×U×P×Ceactive×(n-nl)-Ntarg×(1+n×P)］×Cu×M.This equation can describe correctly the accumulation rule of PCR product and thus build up the kinetic-mathematical model of PCR reaction.The predicted CT value of PE 7700 by the kinetic-mathematical model was in accordance with the real value detected by the machine.This kinetic-mathematical model accompanied by proper detecting equipment and computer could make an automatic PCR instrument,which would produce much better result.A laboratory can predict the amount of PCR product by this model and provide accurate information for further handling of PCR product according to its own condition.In this model,the molecular basis that PCR reaction is doomed to change from exponential amplification to linear amplification had been clarified.     

Investigation of the mechanism of nicotine demethylation in Nicotiana through 2H and 15N heavy isotope effects: implication of cytochrome P450 oxidase and hydroxyl ion transfer

Heavy-atom isotope effects for the N-demethylation of nicotine have been determined in vivo in static-phase biosynthetically incompetent plant cell cultures of Nicotiana species. A (2)H kinetic isotope effect of 0.587 and a (15)N kinetic isotope effect of 1.0028 were obtained. An identical (15)N kinetic isotope effect of 1.0032 was obtained for the nicotine analogue, N-methyl-2-phenylpyrrolidine. The magnitude of the (15)N heavy-atom isotope effect indicates that the fission of the CN bond is not rate limiting for demethylation. The theoretical calculation of heavy-atom isotope effects for a model of the reaction pathway based on cytochrome P450 best fits the measured kinetic isotope effect to the addition of hydroxyl ion to iminium to form N-hydroxymethyl, for which the computed (2)H- and (15)N kinetic isotope effects are 0.689 and 1.0081, respectively. This large inverse (2)H kinetic isotope effect is not compatible with the initial abstraction of the H from the methyl group playing a significant kinetic role in the overall kinetic limitation of the reaction pathway, since computed values for this step (4.54 and 0.9995, respectively) are inconsistent with the experimental data.     

Purification,characterization and gene cloning of isoeugenol-degrading enzyme from <Emphasis Type="Italic">Pseudomonas putida</Emphasis> IE27

An isoeugenol-degrading enzyme was purified to homogeneity from Pseudomonas putida IE27, an isoeugenol-assimilating bacterium. The purified enzyme was a 55 kDa monomer and catalyzed the initial step of isoeugenol degradation, the oxidative cleavage of the side chain double-bond of isoeugenol, to form vanillin. Another reaction product of isoeugenol degradation besides vanillin was identified to be acetaldehyde. The values of Km and k _cat for isoeugenol were 175 μM and 5.18 s^–1, respectively. The purified enzyme catalyzed the incorporation of an oxygen atom from either molecular oxygen or water into vanillin, suggesting that the isoeugenol-degrading enzyme is a kind of monooxygenase. The gene encoding the isoeugenol-degrading enzyme and its flanking regions were isolated from P. putida IE27. The amino acid sequence of the enzyme was similar to those of lignostilbene-α,β-dioxygenases, carotenoid monooxygenases and 9-cis-epoxycarotenoid dioxygenases.     

Selective enrichment of tryptophan-containing peptides from protein digests employing a reversible derivatization with malondialdehyde and solid-phase capture on hydrazide beads

A method for the selective enrichment of tryptophan-containing peptides from complex peptide mixtures such as protein digests is presented. It is based on the reversible reaction of tryptophan with malondialdehyde and trapping of the derivatized Trp-peptides on hydrazide beads via the free aldehyde group of the modified peptides. The peptides are subsequently recovered in their native form by specific cleavage reactions for further (mass spectrometric) analysis. The method was optimized and evaluated using a tryptic digest of a mixture of 10 model proteins, demonstrating a significant reduction in sample complexity while still allowing the identification of all proteins. The applicability of the tryptophan-specific enrichment procedure to complex biological samples is demonstrated for a total yeast cell lysate. Analysis of the processed fraction by 1D-LC-MS/MS confirms the specificity of the enrichment procedure, as more than 85% of the peptides recovered from the enrichment step contained tryptophan. The reduction in sample complexity also resulted in the identification of additional proteins in comparison to the untreated lysate.     

A kinetic approach to the evaluation of alkaline phosphatase inactivation by urea

A kinetic approach is described which enables the measurement of the enzyme inactivation rate constant during the reaction course. A mathematical analysis is presented and it is shown that a time-dependent step may be postulated to exist. Reaction kinetics follow an exponential rule with time as the independent variable and enzymatic activity as the dependent variable. A simple procedure of graphical analysis is reported and the influence on the inactivation rate constant of various conditions (temperature and inhibitor concentration) is evaluated. The method is illustrated by an experimental model: the inactivation of bovine kidney alkaline phosphatase by urea.     

Statistically optimized production and characterization of vanillin from creosol using newly isolated <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> P27

The current research study deals with the screening of a potent vanillin-producing microorganism among 96 isolated strains. Biochemical characterization and molecular identification confirmed that the isolated strain belongs to the Klebsiella pneumoniae bacteria, so it was denoted as Klebsiella pneumoniae P27. The optimization of medium components for the enhanced production of vanillin was carried out using two-stage statistical experimental designs, in which the significant medium components for vanillin production were screened using a Plackett-Burman experimental design. And the optimal levels of those noteworthy factors were determined by using central composite design. The statistical optimization of medium components resulted in increases in vanillin production and vanillyl alcohol oxidase activity of 2.05-fold and 3.055-fold, respectively. The highest vanillin production (30.88 mg/L) and vanillyl alcohol oxidase activity (0.044 U/mL) was observed after 16 h of incubation in the presence of 0.26 mL/L creosol, 8.06 g/L yeast extract and 2.77 g/L NH₄NO₃ in the production medium. The optimally produced vanillin was extracted and confirmed using FTIR and LCMS spectral analysis. The results of the current study support a statistical process optimization approach as a potential technique for the enhanced production of vanillin from creosol by using newly isolated Klebsiella pneumoniae P27 bacterial strain.