M. D. Lilly

The isolation and kinetics of penicillin amidase from Escherichia coli

Abstract 1. 1. A penicillin amidase (penicillin amidohydrolase, EC 3.5.1.11) was extracted from Escherichia coli NCIB 8743A and purified by precipitation first with (NH 4 ) 2 SO 4 and then polyethyleneglycol, followed by chromatography on DEAE-cellulose. 2. 2. The enzyme was shown to be inhibited by excess substrate, benzylpenicillin, and by both of the products of hydrolysis. The inhibition by phenylacetic acid was found to be competitive and by 6-aminopenicillanic acid to be non-competitive. 3. 3. The kinetic and inhibition constants for the enzyme were measured over the range of pH, 7.0–8.5, at 37 °C and at pH 8.0 at 27 °C.

Porous glass as a solid support for immobilisation or affinity chromatography of enzymes

Abstract Chymotrypsin (EC 3.4.4.5) and β-galactosidase (gb-D-galactoside galactohydrolase, EC 3.2.1.23) were immobilised by attachment with glutaraldehyde to aminoalkylsilyl glass. The preparations contained 16 and 12 mg protein/g glass respectively. The retention of activity on immobilisation for chymotrypsin was 50% and for β-galactosidase was 36%. Glycyl-D-phenylalanine was immobilised in the same way and used for purification of carboxypeptidase A (peptidyl-L-amino-acid hydrolase, EC 3.4.1.2) by affinity chromatography.

Effect of cycling dissolved oxygen concentrations on product formation in penicillin fermentations

SummaryLimitations in mass and momentum transfer coupled with high hydrostatic pressures create significant spatial variations in dissolved gas concentrations in large fermenters. Microorganisms are subjected to fluctuating environmental conditions as they pass through the zones in a stirred vessel or along a closed loop fermenter.A 7-litre fermenter was modified to simulate the dissolved gas and hydrostatic pressure gradients in large vessels.The effect of cycling dissolved oxygen tension (DOT) on penicillin production by Penicillium chrysogenum P1 was investigated. The fermentation was affected by evironmental conditions such as medium composition, pH, size of inoculum, stirrer speed and DOT. Inoculum size below 10% (v/v) and stirrer speeds above 850 rpm caused significant reductions in specific prenicillin production rates (qpen). qpen values were measured at different constant DOT levels. Below 30% air saturation qpen decreased sharply and no production was observed at 10%. Penicillin synthesis was impaired irreversibly below 10% DOT. The same profile was observed at higher stirrer speeds and air flow rates indicating that the effect was a physiological one. Oxygen uptake of the culture was affected significantly below 7% DOT, demonstrating that the critical DOT values for penicillin production and oxygen uptake are two distinct parameters. Carrying out the fermentation at one atmosphere over pressure was found to have no effect. When the dissolved oxygen concentration of the culture medium was cycled around the critical DOT for penicillin production, a considerable decrease in the specific penicillin production rate was observed. The effect was reversible but not transient, indicating a shift in cell metabolism.These results demonstrate the unfavourable effect of fluctuating environmental conditions on culture performance in stirred tanks. They suggest that these effects should be accounted for during strain selection, process development and scale up stages of an industrial process if the productivities in small scale vessels are to be obtained.

The enzymatic transformation of water‐insoluble reactants in nonaqueous solvents. Conversion of cholesterol to cholest‐4‐ene‐3‐one by a Nocardia sp.

The rapid conversion of cholesterol to cholestenone by Nocardia in the presence of high proportions of water‐immiscible solvent has been demonstrated. At high agitator speeds, the reaction rate was not limited by the rates of transfer of oxygen or cholesterol to the microorganisms. Using 100 g of thawed cells in 200 ml of carbon tetrachloride containing 16% (w/v) cholesterol, at 20°C cholestenone was formed at 7 g/hr. Cells could be separated easily from the organic solvent and reused. After 7 runs (69 hr) the reaction rate had fallen only to half the value for the first run.

Enzymic interesterification of fats: factors influencing the choice of support for immobilized lipase

Abstract Three grades of diatomaceous earth (Celite 560, Filtercel and Hyflo Supercel) and a controlled-pore silica have been examined for their suitability as support materials for lipase (triacyglycerol acylhydrolase, EC 3.1.1.3) catalysing the interesterification of fats. The controlled-pore silica gave a preparation with a low activity. Although all three Celites gave preparations with similar lipolytic activities, Hyflo Supercel gave the highest interesterification activity. The distribution of enzyme protein in Hyflo Supercel was examined by transmission electron microscopy.

The influence of mechanical forces on the morphology and penicillin production of Penicillium chrysogenum

The influence of mechanical forces resulting from the rotation of (multiple) turbine impellers on the morphology and penicillin production of Penicillium chrysogenum Panlabs P-1 was investigated in batch fermentations using semi-defined media. Experiments were carried out at three different scales of fermentation, 5 dm3,100 dm3 and 1000 dm3 working volume, with the impeller tip speed ranging from 2.5 to 6.3 m/s. Throughout all fermentations, the dissolved oxygen concentration never fell below the critical value for maximum penicillin production. Morphological measurements using image analysis showed that the mean main hyphal length and mean hyphal growth unit increased during the rapid growth period and then decreased to a relatively constant value dependent on the agitation intensity. The specific rate of penicillin production (qpen)and the average main hyphal length during the linear penicillin production phase were lower at high agitation speed, which promoted more rapid mycelial fragmentation and a higher branching frequency. Comparison of the results from the three scales showed that impeller tip speed is a poor scale up parameter whereas a term based on mycelial circulation through the zone of high energy dissipation fitted the data well.

Turbulent breakage of filamentous microorganisms in submerged culture in mechanically stirred bioreactors

Abstract A discussion is presented suggesting that hyphal breakage in mechanically stirred fermenters is likely to occur by direct tensile stresses acting on the opposite ends of the hypha of filamentous microorganisms. These stresses originate from the dynamic pressure fluctuations of eddies with scales in the inertial convection subrange of the turbulent energy spectrum. The maximum strain energy theory of failure of an elastic material under this mode of stress is used to set up a relationship between the stable length of hyphae and some of the physical parameters affecting it, including the mean energy dissipation rate in the bioreactor. Experimental data are reported for the rate of breakage of hyphae obtained under different operating conditions during the fermentation of Penicillium chrysogenum in a 7 and 150 1 mechanically stirred bioreactor. Data on the stable size of hyphae agree well with the model based on the maximum energy criterion. The experimental data further suggest that hyphal breakage is approximately a first-order kinetic process with a rate constant which appears to be moderately dependent on the product of the mean energy dissipation rate and the reciprocal of the impeller circulation time.

The preparation of soluble high molecular weight NAD derivative active as a cofactor

Abstract 1. 1. A water soluble high molecular weight derivative of NAD has been prepared by coupling NAD to polyethyleneimine. 2. 2. The polyethyleneimine-NAD shows cofactor activity with both free and immobilised dehydrogenase. 3. 3. Kinetic data for this enzyme with free NAD is compared with succinyl-NAD and polyethyleneimine-NAD. 4. 4. The ability of polyethyleneimine-NAD to act as a cofactor in a two-enzyme system, where it is alternatively reduced and oxidased, is demonstrated.

Effect of processing parameters on the feasibility and operational stability of immobilized viable microbial cells

The use of aerated immobilized viable microorganisms despite their potential has so far been limited on the large scale except in waste treatment. This review assesses the information available on the effect of processing parameters on the behavior and operational stability of aerated immobilized viable cells being used for synthesis. It highlights the problems of adequate oxygen supply and a need to develop new reactors via a rational approach to process design and operation.

Immobilisation of α-amylase by attachment to soluble support materials

Abstract 1. 1.α-Amylase (α-1,4-glucan 4-glucanohydrolase, EC 3.2.1.1) has been immobilised by attachment to soluble amino- s -triazinyl derivatives of dextran 2000 DEAE-dextran 2000 and to CM-cellulose (Edifas B10). 2. 2.These amylase derivatives show up to 67% of the specific activity of the free enzyme. 3. 3.The pH optimum shows 0.5 unit shifts depending on the charge of the support. 4. 4.Attachment of amylase to CM-cellulose and DEAE-dextran increases the stability of the enzyme at neutral pH. Attachment to CM-cellulose increases the heat stability of the enzyme. 5. 5.The increased stability of the CM-cellulose-amylase compared with free amylase was demonstrated in the continuous hydrolysis of starch in an ultrafilter reactor.