A.E.M. Janssen

Thermozymes and their applications

Enzymes from thermophilic microorganisms, thermozymes, have unique characteristics such as temperature, chemical, and pH stability. They can be used in several industrial processes, in which they replace mesophilic enzymes or chemicals. Thermozymes are often used when the enzymatic process is compatible with existing (high-temperature) process conditions. The main advantages of performing processes at higher temperatures are reduced risk of microbial contamination, lower viscosity, improved transfer rates, and improved solubility of substrates. However, cofactors, substrates, or products might be unstable or other side reactions may occur. Recent developments show that thermophiles are a good source of novel catalysts that are of great industrial interest. Thermostable polymer-degrading enzymes such as amylases, pullulanases, xylanases, proteases, and cellulases are expected to play an important role in food, chemical, pharmaceutical, paper, pulp, and waste-treatment industries. Considerable research efforts have been made to better understand the stability of thermozymes. There are no major conformational differences with mesophilic enzymes, and a small number of extra salt bridges, hydrophobic interactions, or hydrogen bounds seem to confer the extra degree of stabilization. Currently, overexpression of thermozymes in standard Escherichia coli allows the production of much larger quantities of enzymes, which are easy to purify by heat treatment. With wider availability and lower cost, thermophilic enzymes will see more application in industry.

Effect of temperature and enzyme origin on the enzymatic synthesis of oligosaccharides

The aim of this research is to quantify the effect of temperature and enzyme origin on the enzymatic synthesis of oligosaccharides. Quantification of these effects is important because temperature and enzyme origin are important process parameters. A kinetic model was used to describe the concentrations in time. The kinetic parameters were determined by using data obtained in batch experiments at various temperatures (20, 30, 40, and 50 degrees C) and by using beta-galactosidases from Bacillus circulans, Aspergillus oryzae, Kluyveromyces lactis, and Kluyveromyces fragilis. The effect of temperature on the kinetic parameters could be described with the Arrhenius equation, except for the inhibition parameter. Slightly higher oligosaccharide yields were found at higher temperatures. However, the influence of the initial lactose concentration was much larger. The higher yield at higher temperatures is an additional advantage when operating at high initial lactose concentrations and consequently elevated temperatures. Clear differences between the beta-galactosidases were found concerning amount, size, and type of oligosaccharides produced. The beta-galactosidase from B. circulans produced the most abundant amount, the most different, and largest-sized oligosaccharides. The beta-galactosidases from Kluyveromyces spp. produced mainly trisaccharides. The kinetic parameters for the different enzymes were determined and differences were discussed.

Water activity fails to predict critical hydration level for enzyme activity in polar organic solvents: Interconversion of water concentrations and activities

Abstract When studying biocatalysts in organic media, it is useful to be able to convert between water concentration and thermodynamic water activity ( a w ). We have obtained relationships for this purpose based on published vapor-liquid equilibrium data for a range of commonly used polar solvents. Their use is illustrated by the reanalysis of literature data for the activity of an immobilized laccase. Kinetic constants measured as a function of water concentration can thus be related to a w . We show that the critical water content for significant V m values is characterized by a similar a w (about 0.4) in five different alcohols; however, the critical a w becomes progressively higher in other water-miscible solvents in the order: dioxane, acetonitrile, tetrahydrofuran, acetone (critical a w about 0.8). This indicates that such polar solvents probably have important direct effects on the enzyme.

Kinetics of lipase-catalyzed esterification in organic media : correct model and solvent effects on parameters

The Ping-Pong model (incl. alcohol inhibition) is not the correct model to describe the kinetics of a lipase-catalyzed esterification reaction. The first product, water, is always present at the start of the reaction. This leads to an equation with one extra parameter. This new equation fits our experimental data on the esterification of sulcatol and fatty acids in toluene, catalyzed by Candida rugosa lipase. The new model does not significantly improve the mean square of the fit; however, using a model which can be expected to be more correct, results in the conclusion that a larger part of the differences can be explained by substrate solvation. For comparison of the kinetic constants in different solvents, it is essential to make corrections for solvation. The deviation from the average corrected kinetic constant shows to what extent differences can be explained by substrate solvation and an effect on the enzyme. We have made corrections for solvation with the new model for the esterification in toluene, hexane, trichloroethane, and diisopropyl ether. This has resulted in kinetic constants that deviate less from the average value.

The effect of organic solvents on the equilibrium position of enzymatic acylglycerol synthesis

The effect of organic solvents on the equilibrium position of lipase‐catalyzed esterification of glycerol and decanoic acid has been investigated. The reaction is carried out in an aqueous‐organic two‐phase system. In polar solvents, high mole fractions of monoacylglycerol and low mole fractions of triacylglycerol and measured, while in nonpolar solvents, the measured differences in the mole fractions of monodi‐, and triacylglycerols are less. There is a good correlation between the ester mole fractions at equilibrium and the log P of the solvent (partition coefficient in n‐octanolwater), however, only if the group of tertiary alcohols is excluded. In the plot of the easter mole fractions as a function of the logarithm of hte solubility of water in the organic solvent, the tertiary alcohols can be included; however, in this case other deviations appear.

Modelling and parameter estimation of the enzymatic synthesis of oligosaccharides by beta-galactosidase from bacillus circulans

The aim of this research is to develop a model to describe oligosaccharide synthesis and simultaneously lactose hydrolysis. Model A (engineering approach) and model B (biochemical approach) were used to describe the data obtained in batch experiments with beta-galactosidase from Bacillus circulans at various initial lactose concentrations (from 0.19 to 0.59 mol.kg(-1)). A procedure was developed to fit the model parameters and to select the most suitable model. The procedure can also be used for other kinetically controlled reactions. Each experiment was considered as an independent estimation of the model parameters, and consequently, model parameters were fitted to each experiment separately. Estimation of the parameters per experiment preserved the time dependence of the measurements and yielded independent sets of parameters. The next step was to study by ordinary regression methods whether parameters were constant under the altering conditions examined. Throughout all experiments, the parameters of model B did not show a trend upon the initial lactose concentration when inhibition was included. Therefore model B, a galactosyl-enzyme complex-based model, was chosen to describe the oligosaccharide synthesis, and one parameter set was determined for various initial lactose concentrations. Copyright 1999 John Wiley & Sons, Inc.

Thermodynamically controlled synthesis of β-lactam antibiotics. Equilibrium concentrations and side-chain properties

For the enzymatic synthesis of the antibiotic cephalexin, an activated acyl donor is generally used as one of the substrates (kinetically controlled approach); however, the thermodynamically controlled approach might be of interest since there is no need for activation of the acyl donor and less waste is produced. If the synthesis reaction can be combined with an effective product removal step, the thermodynamic approach can be beneficial. The thermodynamically controlled synthesis of cephalexin was studied at various pH values, solvent concentrations, and temperatures. With direct synthesis in water, only small amounts of cephalexin were formed (0.1 mm from 20 mm starting material by the Xanthomonas citri enzyme). Addition of water-miscible organic solvent had a positive effect on synthesis (by the Escherichia coli enzyme); the equilibrium concentration of cephalexin, however, was at best increased by a factor of 2-3 (in methanol and triglyme). The equilibrium antibiotic concentrations reported in this study were notably lower than the values reported in the literature. These differences originate from the improved analytical methods that are available nowadays. Low product concentrations were also found for other side-chains with an amino group at the α-position. Side-chains without this group can be coupled and give acceptable product concentrations. For these antibiotics, a thermodynamically controlled process may be an alternative to kinetically controlled coupling. Copyright (C) 1999 Elsevier Science Inc. All rights reserved. | For the enzymatic synthesis of the antibiotic cephalexin, an activated acyl donor is generally used as one of the substrates (kinetically controlled approach); however, the thermodynamically controlled approach might be of interest since there is no need for activation of the acyl donor and less waste is produced. If the synthesis reaction can be combined with an effective product removal step, the thermodynamic approach can be beneficial. The thermodynamically controlled synthesis of cephalexin was studied at various pH values, solvent concentrations, and temperatures. With direct synthesis in water, only small amounts of cephalexin were formed (0.1 mM from 20 mM starting material by the Xanthomonas citri enzyme). Addition of water-miscible organic solvent had a positive effect on synthesis (by the Escherichia coli enzyme); the equilibrium concentration of cephalexin, however, was at best increased by a factor of 2-3 (in methanol and triglyme). The equilibrium antibiotic concentrations reported in this study were notably lower than the values reported in the literature. These differences originate from the improved analytical methods that are available nowadays. Low product concentrations were also found for other side-chains with an amino group at the α-position. Side-chains without this group can be coupled and give acceptable product concentrations. For these antibiotics, a thermodynamically controlled process may be an alternative to kinetically controlled coupling.

Immobilization of penicillin G acylase onto chemically grafted nylon particles

Abstract Nylon particles, grafted with diethylene glycol dimethacrylate (DGDA) using potassium persulphate as initiator, were treated with hexamethylene diamine (HMDA). The aminoalkylated particles were activated with glutaraldehyde and finally, penicillin G acylase (PA) was immobilized to these activated particles. Both the conditions of the aminoalkylation- and the immobilization process were optimized. The hydrolysis of cephalexin was used as model conversion. The retention of activity of the immobilized enzyme was 12%. This value improved to 30% by adding phenyl acetic acid (PAA), as active-site protecting agent, to the enzyme solution. The results suggest formation of multi-point attachment between the enzyme and the matrix.

Enzymatic synthesis of carbohydrate esters in 2-pyrrolidone.

Abstract The lipase-mediated esterification of sorbitol and fatty acid was investigated in a two-phase system with 2-pyrrolidone as cosolvent for sorbitol. The lipase from Chromobacterium viscosum showed an initial esterification rate of 1.4 mmol g −1 h −1 , and after 74 h, 80% of the initial sorbitol content was converted into sorbitol esters. With fructose or glucose as a substrate, initial esterification rates were 0.2 and 0.04 mmol g −1 h −1 , respectively; disaccharides were not reactive at all. The effects of the sorbitol, fatty acid, water, and 2-pyrrolidone concentrations on esterification activity were studied. An excess of fatty acid and a water concentration around 1 m were found to be necessary for optimum ester production. The polar organic cosolvent 2-pyrrolidone can inactivate the lipase. It is a suitable cosolvent for carbohydrates, provided that its concentration is low. Esterification was also studied in a two-phase membrane reactor. The value of the enzyme-based initial reaction rate was half of the reaction rate in an emulsion system. The water activity in the membrane system was relatively high, which resulted in low product yields.

Enzymatic synthesis of oligosaccharides: product removal during a kinetically controlled reaction.

In this article, the enzymatic production of oligosaccharides, which is an example of a kinetically controlled reaction, is studied. The aim is to show that the product yield can be enhanced by continuous removal of oligosaccharides from the reaction mixture. The oligosaccharides were removed by adsorption on activated carbon. The absorption could be described by the multicomponent Langmuir isotherm with different maximum saturation constants for mono-, di-, and trisaccharides. The affinity for trisaccharides was larger (k(tri) = 3.52 l/g) than for di- (k(di) = 0.94 l/g) and monosaccharides (k(mono) = 0.11 l/g). A model combining kinetics, adsorption on activated carbon, and mass transfer in an adsorption column was developed. Model calculations for the batch process with removal showed a yield improvement of 23% compared to the batch process without removal. Experimentally, a yield improvement of 30% was obtained. Model calculations for the continuous process studied did not result in an increase of the yield. The advantages of removal were masked by the negative influence of recirculation and the relative large time between formation and removal.