The application of flow microcalorimetry to the study of enzyme kinetics

The use of flow microcalorimetry to measure velocities for enzyme-catalyzed reactions is described. Studies are presented involving the enzymes chymotrypsin, trypsin, and ribonuclease A. In determining the Michaelis-Menten parameters, V_m and K_m, for these systems complications arise due to substrate depletion and product inhibition. By employing an integrated Michaelis-Menten equation, V_m and K_m can be determined.     

Effect of biocatalyst swelling on the operation of packed-bed immobilized enzyme bioreactor

A general mathematical model was developed for predicting the performance and simulation of a packed-bed immobilized enzyme reactor performing a reaction that follows Michaelis–Menten kinetics with competitive product inhibition. The performance of a packed-bed immobilized enzyme reactor was analyzed taking into account the effect of bed swelling on various diffusional phenomena such as axial dispersion, internal and external mass transfer limitations. The numerical solutions were compared with experimental data obtained for a packed-bed reactor operating with β-galactosidase entrapped in Ca-alginate-K-κ-carrageenan gels for lactose hydrolysis.     

Modelling of enzyme hydrolysis of maltose in a single pellet of immobilized biocatalyst

The reversible hydrolysis of maltose to glucose by immobilized glucoamylase entrapped in spherical solid particles is studied theoretically. For this purpose a known kinetic model taking into account these reversible reactions and the competitive synthesis of iso-maltose was adopted. The mass transfer limitations in the bulk liquid and in the pores of the particles containing the enzyme are considered, using Fick's law. On the basis of mathematical modelling the optimum conditions for biocatalyst performance are established. An appropriate combination of particle size and initial substrate concentration may lead to reduction of undesirable mass transfer resistance and therefore product inhibition and to an improved selectivity of the biocatalyst with respect of glucose formation.List of Symbols C _i kmoles/m³ current concentration ofi-th component along the radius - C _oi kmoles/m³ bulk concentration ofi-th component - C _i ^* kmoles/m³ concentrations ofi-th component on the pellet surface - D _si ,D _i m²/s internal and molecular diffusion coefficient ofi-th component - W _M kmoles/m³·s reaction rate of maltose hydrolysis - W _IM kmoles/m³·s reaction rate of iso-maltose formation - W _G kmoles/m³·s reaction rate of glucose production - R ₀ m pellet radius - r m current radius of the pellet - t s time coordinate - r ₀ ratio of the time step to the square of the radial coordinate - Re Reynolds number =w·R/v - Sc Schmidt number =v/D - Bi Biot number = R/D - A _j ,B, C _j coefficients in the system of linear equations, Eq. (8) - X _i dimensionless degree of transformation - NR number of independent reactions - N number of division sections of the pellet radius - G kmoles/m³ concentration of glucose - M kmoles/m³ concentration of maltose - IM kmoles/m³ concentration of isomaltose - K _m kmoles/m³ Michaelis constant - V _max kmoles/m³·s maximum reaction rate in Eq. (6) - K _i kmoles/m³ inhibition constant - K _1eq ,K _2eq equilibrium constants in Eq. (6) - , h steps along the time and radial coordinate in the pellet - m/s mass transfer coefficient - dimensionless radius of the pellet - computation accuracy Indices i number of reaction component - j index along the radius of the pellet - k index along the time coordinate This work was accomplished with thanks to the financial support of the Bulgarian National Fund for Scientific Investigations —Grant No. MU-1-BE/93.     

Application of differential flow microcalorimetry for study of drug interactions in the blood system

A compact differential flow microcalorimeter has been developed to investigate biomolecular reactions, especially drug interactions in the blood system. The calorimeter is an adiabatic type and consists of a twin-cell structure, each mixing part having a volume of 60 microliters. Both the precision and accuracy of the instrument have been evaluated by dilution of sucrose solutions to be 0.1-0.5% at a heat effect of 100-10 microW. The resolution is approximately 0.5 microW (less than 10(-3) Torr). The heat produced in erythrocyte hemolysis induced by chlorpromazine hydrochloride (CPZ) and the binding heat of CPZ to human blood components viz., intact erythrocytes, erythrocyte membranes, serum albumin and plasma were measured. The heat effect of hemolysis was endothermic and related to the quantity of free hemoglobin released from erythrocytes. The overall binding of CPZ to blood components was, however, an exothermic process. The thermodynamic and binding parameters were computed directly from the calorimetric data by use of a nonlinear least squares regression method, assuming a one-class binding model, and the stoichiometry of the binding reaction was determined.     

Application of microcalorimetry to the study of lipid-protein interaction.

The enthalpy changes associated with protein-lipid binding were measured in an isothermal microcalorimeter. This technique was applied to the study of the association between albumin with fatty acid and lysolecithin, and between the plasma apolipoproteins and phospholipids.Microcalorimetry enables determination of the maximal enthalpy of binding and of complex stoichiometry in systems where one ligand, such fatty acid or a phospholipid is bound to either BSA or an apolipoprotein. The competition between 2 ligands, such as fatty acid and a dye for albumin can also be followed.The binding of synthetic phosphatidylcholines to the isolated major apoproteins of HDL and VLDL lipoprotein classes was compared by this technique. The maximal enthalpies decrease in the order $\text{apoA-II}\text{?}\text{apoC-III}\text{～}\text{apoA-C}\text{>}\text{apoA-I}$, in agreement with the reported affinities of these peptides for phospholipids.Interapoprotein association between the two major apoproteins of apoHDL was demonstrated in the presence of phospholipids and compared to the behaviour of the whole apoHDL.Finally the association of two phosholipids naturally occurring in the HDL molecule was demonstrated.The interpretation and relevance of these various types of application of microcalorimetry to the study of protein-lipid systems is discussed and evaluated.     

Eupergit C250L固定化D-海因酶及其催化性质

D-海因酶是海因酶法制备D-氨基酸的关键酶。利用Burkholderic cepecia1003菌发酵产酶,所得海因酶纯化后,以Eupergit C250L为载体进行共价固定化。分别考察了酶液蛋白浓度、固定化时间对蛋白固定量和酶活回收率的影响以及固定化前后海因酶催化性质的变化。结果表明:较高的酶液蛋白浓度和较长的固定化时间均有助于改善海因酶的固定化效果;固定化可显著提高海因酶的最适作用温度,但对其最适作用pH影响不大;固定化后海因酶对D,L-BH和MH的米氏常数均有较大幅度的降低。固定化酶反应器的实验表明:40℃下,底物(D,L-BH)1.0 g.L-1,体积流速1.0 mL.min-1,经21 h转化,产物N-Phe质量浓度可达0.47 g.L-1,转化率达43.21%。     

Immobilization of oxalate decarboxylase to Eupergit and properties of the immobilized enzyme

Oxalate decarboxylase, an oxalate degradation enzyme used for medical diagnosis and decreasing the oxalate level in the food or paper industry, was covalently immobilized to Eupergit C. Different immobilization parameters, including ratio of enzyme to support, ammonia sulfate concentration, pH, and incubation time, were optimized. Under the condition of enzyme/support ratio at 1:20, pH 9, with 1.5?mol/L (NH(4))(2)SO(4), room temperature, and shaking at 30?rpm for 24?hr, activity recovery of immobilized Oxdc reached 90% with an apparent specific activity of 0.44?U/mg support. The enzymatic properties of immobilized Oxdc were investigated and compared with those of the soluble enzyme. Both shared a similar profile of optimum conditions; the optimum pH and temperature for soluble and immobilized Oxdc were 3.5 and 50°C, respectively. The immobilized enzyme was more stable at lower pH and higher temperatures. The kinetic parameters for soluble and immobilized enzyme were also determined.     

Stereoselective biotransformation of phenylglycine nitrile by heterogeneous biocatalyst based on immobilized bacterial cells and enzyme preparation

We studied the effect of a heterogeneous environment on the stereoselectivity of transformation of racemic phenylglycine nitrile. Immobilized biocatalysts were prepared by adhesion of Pseudomonas fluorescens C2 cells on carbon-containing supports and covalent crosslinking of nitrile hydratase and amidase of Rhodococcus rhodochrous 4–1 to activated chitosan as well as by the method of cross-linked aggregates. At a reaction duration of 20 h, the ratio of phenylglycine stereoisomers changes depending on the presence of support in medium. The highest optical purity of the product (enantiomeric excess of L-phenylglycine solution, 98%) is achieved when enzyme aggregates of nitrile hydratase and amidase cross-linked with 0.1% glutaraldehyde are used as a biocatalyst.     

Immobilization on Eupergit C of cyclodextrin glucosyltransferase (CGTase) and properties of the immobilized biocatalyst

The extreme thermophilic cyclodextrin glucanotransferase (CGTase) from Thermoanaerobacter sp. was covalently attached to Eupergit C. Different immobilization parameters (incubation time, ionic strength, pH, ratio enzyme/support, etc.) were optimized. The maximum yield of bound protein was around 80% (8.1 mg/g support), although the recovery of β-cyclodextrin cyclization activity was not higher than 11%. The catalytic efficiency was lower than 15%. Results were compared with previous studies on covalent immobilization of CGTase.

The enzymatic properties of immobilized CGTase were investigated and compared with those of the soluble enzyme. Soluble and immobilized CGTases showed similar optimum temperature (80–85 °C) and pH (5.5) values, but the pH profile of the immobilized CGTase was broader at higher pH values. The thermoinactivation of the CGTase coupled to Eupergit C was slower than the observed with the native enzyme. The half-life of the immobilized enzyme at 95 °C was five times higher than that of the soluble enzyme. The immobilized CGTase maintained 40% of its initial activity after 10 cycles of 24 h each. After immobilization, the selectivity of CGTase (determined by the ratio CDs/oligosaccharides) was notably shifted towards oligosaccharide production.     

An extension of the pressure-test technique for immobilized biocatalyst preparations

Based on an analysis of variables in the Ergun equation for pressure drop in granular beds, the technique of pressuretesting of immobilized biocatalyst preparations is reexamined from the point of view of evaluation of biocatalyst mechanical properties in packed bed reactors. The variation of biocatalyst particle size in the course of bioreactor performance was found to be a convenient estimate of mechanical strength for the biocatalyst particles. Based on this finding, an extension of the pressure-test technique with particle size analysis was proposed as an opportunity to carry out differential analysis of biocatalyst structure, regardless of time and position. An example of bed destruction is given to support the suggested characterization analysis.     

A new biocatalyst: Penicillin G acylase immobilized in sol-gel micro-particles with magnetic properties

The present work focuses on the development and basic characterization of a new magnetic biocatalyst, namely penicillin G acylase (PGA), immobilized in sol-gel matrices with magnetic properties, ultimately aimed for application in cephalexin (CEX) synthesis. A mechanically stable carrier, based on porous xerogels silica matrixes starting from tetramethoxysilane (TMOS), was prepared leading to micro-carriers with medium sized particles of 30 μm, as determined by scanning electron microscopy. An immobilization yield of 95–100% and a recovered activity of 50–65% at 37°C, as determined by penicillin G (PG) hydrolysis (pH STAT method), were observed. These results clearly exceed those reported in a previous work on PGA immobilization in sol-gel, where only 10% of activity was recovered. The values of activity were kept constant for 6 months. Immobilized PGA (682 U/g_{dry weight}) retained high specific activity throughout ten consecutive runs for PG hydrolysis, suggesting adequate biocatalyst stability. The CEX synthesis was performed at 14°C, using the free and immobilized PGA in aqueous medium. Phenylglycine methyl ester was used as acyl donor at 90 mM and 7-aminodeacetoxycephalosporanic acid was the limiting substrate at 30 mM. The CEX stoichiometric yield after 1-h reaction was close to 68% (23 mM CEX/h) and 65% (19 mM CEX/h), respectively.     

Immobilization of Aspergillus oryzae tannase and properties of the immobilized enzyme

Tannase enzyme from Aspergillus oryzae was immobilized on various carriers by different methods. The immobilized enzyme on chitosan with a bifunctional agent (glutaraldehyde) had the highest activity. The catalytic properties and stability of the immobilized tannase were compared with the corresponding free enzyme. The bound enzyme retained 20·3% of the original specific activity exhibited by the free enzyme. The optimum pH of the immobilized enzyme was shifted to a more acidic range compared with the free enzyme. The optimum temperature of the reaction was determined to be 40 °C for the free enzyme and 55 °C for the immobilized form. The stability at low pH, as well as thermal stability, were significantly improved by the immobilization process. The immobilized enzyme exhibited mass transfer limitation as reflected by a higher apparent K_m value and a lower energy of activation. The immobilized enzyme retained about 85% of the initial catalytic activity, even after being used 17 times.     

Novel immobilized biocatalyst for microbiological synthesis of pharmaceutical steroids

Applied Biochemistry and Microbiology - The steroid-transforming activity of free and immobilized cells of Pimelobacter simplex VKPM Ac-1632 entrapped in an operationally stable macroporous...     

New approaches for verification of kinetic parameters of immobilized concanavalin A: invertase preparations investigated by flow microcalorimetry

In our preceding article, we demonstrated a procedure based upon enzymic flow microcalorimetry using an enzyme thermistor (ET) to characterize the microkinetic properties of an immobilized enzyme (IME) and its further application in the screening of IMEs. To consider the ET method (single ET unit, ET system 1) as standard, it was necessary to show that the estimated relative kinetic parameter (DeltaT(max)) calorimetrically corresponds with the absolute value for the reaction rate within the whole measurement range. This article presents three experimental verification procedures. Two procedures are based on adaptation of the flow-through ET column to a mini-differential-reactor (DR) system with substrate recirculation and post-ET-column methods for determination of the concentration change of the product (spectrophotometrically in ET system 2) or the substrate (calorimetrically in ET system 3) with the IME-catalyzed enzymic hydrolysis. The third procedure is an independently operating DR system which spectrophotometrically estimates the concentration change of the product. The results obtained exhibited good correlation (r = 0.921) between the relative kinetic parameter DeltaT(max), as determined calorimetrically by ET system 1, and the absolute value for the reaction rate (r(max)) as determined by ET systems 2 and 3. These data proved that, within the whole range of experimental conditions applied in this study, the parameter DeltaT(max) instead of the true reaction rate could be employed for the IME screening. Moreover, the generality of the detection principle and the standardized configuration of the ET favor ET systems 2 and 3 for normal screening of IMEs and as miniaturized DR systems allowing dual measurements of kinetic parameters. (c) 1996 John Wiley & Sons, Inc.     

Enzymatic synthesis of cefazolin using immobilized recombinant cephalosporin-acid synthetase as the biocatalyst

Bioprocess and Biosystems Engineering - A method for the synthesis of β-lactam antibiotic cefazolin (CEZ) by enzymatic acylation of...     

A new technique for the production of immobilized biocatalyst in large quantities

A new technique is presented for the production of immobilized biocatalysts in large quantities. It consists of breaking up a jet of the biocatalyst/presupport mixture in uniform droplets by means of a resonance technique. Entrapment of yeast and plant cells in calcium alginate has been used as the model. The production capacity of the nozzles used (0.5, 0.8, and 1.1 mm exit diameters) is two orders of magnitude larger than the production capacity of the conventional techniques (maximum capacity with a 1.1-mm nozzle diameter is 24 L/h). Depending on frequency, nozzle diameter, and volumetric flow rate, the bead size varies between 1 and 2 mm, with standard deviations of 3-5% for yeast immobilization and 10-15% for plant cells. The deactivation of both yeast and plant cells is small and comparable to that found in the corresponding conventional procedures.     

Determination of the activity and concentration of immobilized and soluble enzymes by microcalorimetry

The relationship between activity and concentration of both soluble and immobilized lactate dehydrogenase and uricase was determined using the rate of heat production in a batch-type microcalorimeter. The results demonstrate the feasibility of using microcalorimetry for the determination of enzyme levels in biological fluids and for the determination of the activity of particle-bound enzymes.