Gérald André

Lipase-catalyzed production of wax esters

The lipase (triacylglycerol acylhydrolase, E.C. 3.1.1.3) catalyzed synthesis of wax esters has been investigated via two different approaches. All studies were performed using an immobilized 1,3-specific lipase [Lipozyme from Novo Industries (Montréal, Québec, Canada)]. The first approach involves reacting stoichiometric amounts of a fatty acid and stearyl alcohol in the presence of lipase. The medium is solvent-free, which allows for high substrate concentrations (1.55 M) and use of 5% (w/w) Lipozyme. In this reaction, maximum wax ester synthesis was found to be dependent upon the efficient removal of the water produced by the reaction. Under optimal conditions, yields of 100% were routinely reached after only 2 hr. The medium was then exclusively composed of the wax and the enzyme, no purification was required. The second method involves alcoholysis of a triglyceride, in this case triolein, with stearyl alcohol to produce 1,2-diolein, 2-monoolein and the wax ester of oleic acid. Again, no organic solvent was used. The wax ester yield was found to be directly dependent upon the alcohol concentration that was used to modulate the outcome of the reaction towards either the wax or the partial glycerides. The process was applied to the synthesis of waxes from high erucic acid rapeseed oil.

Production of poly-β-hydroxybutyrate from methanol: characterization of a new isolate of Methylobacterium extorquens

SummaryPoly-β-hydroxybutyric acid (PHB) and similar bacterial polyesters are promising candidates for the development of environment-friendly, totally biodegradable plastics. The use of methanol, one of the cheapest noble substrates available, may help to reduce the cost of producing such bioplastics. As a first step, a culture collection of 118 putative methylotrophic microorganisms was obtained from various soil samples without any laboratory enrichment step to favour culture diversity. The most promising culture was selected based on rapidity of growth and PHB accumulation and later identified as Methylobacterium extorquens. This isolate was obtained from soml contaminated regularly with used oil products for some 40 years. Concentrations of methanol greater than 8 g/l affected growth significantly and the methanol concentration was optimal at 1.7 g/l. PHB concentrations averaged 25–30% (w/v) of dry weight under non-optimized conditions. Controlling methanol concentration, using an open-loop configuration, led to biomass levels of 9–10 g/l containing 30–33% PHB while preventing methanol accumulation. The new isolate was also able to produce the co-polymer PHB/poly-β-hydroxyvalerate (PHV) using the mixture methanol + valerate. The PHV-to-PHB ratio was about 0.2 at the end of the fermentation. An average molecular mass varying between 2 and 3 × 105 Da was obtained for three PHB samples using two different measurement methods.

Solvent free triglyceride synthesis using lipozymeTM IM-20

SummaryThe synthesis of triglycerides using LipozymeTM IM-20 (Mucor miehei lipase immobilized on weak anion exchange resins by Novo) is described. Use of pure substrates in stoichiometric amounts in the absence of any organic solvent enables high conversion to be obtained with the addition of molecular sieves to remove water produced by the reaction.

Simple high performance liquid chromatography methods for monitoring lipase reactions

This paper describes three simple high performance liquid chromatography methods to separate mixtures of free fatty acids, mixtures of different triglycerides and mixtures of all fat classes (monoglycerides, diglycerides, triglycerides and free fatty acids). It is possible with our methods to identify and quantify each peak of the chromatogram. These methods have been designed to monitor lipase reactions. Using a first set of conditions, we have been able to separate five fatty acids: linolenic, linoleic, palmitic, oleic and stearic, without any specific preparation of the samples. With a second set of conditions, we showed that the same mobile phase and the same column could separate both triglyceride species and fat classes. However, in the latter case, a flow gradient was used.

Use of lipases in multiphasic systems solely composed of substrates

Three lipase-catalyzed reactions have been investigated in relation to specificity and water dependence. The reactions in question include: the synthetic reaction between oleic acid and glycerol; the enzymatic hydrolysis of triolein; and alcoholysis/glycerolysis transesterification reactions. All reactions were carried out under solventfree conditions. In each case, the medium composition and reaction conditions were optimized in order to work at elevated substrate concentrations and to minimize the production of by-products. Different lipase preparations have been tested in each reaction. In the synthetic reaction, the effective removal of produced water was found to be vital for the production of triolein. With water removal and glycerol amounts not higher than required by the stoichiometry of the reaction, 95% of the available oleic acid was converted to triolein in 48 hr. The production of triolein was also found to be dependent on the availability of the 1,2-diglyceride to react with oleic acid. In the hydrolysis reaction, best conversion yields of triolein towards monoolein, diolein and free fatty acid were obtained when water was considered simply as a substrate of the reaction. In glycerolysis reactions, the reaction of triolein to give monoolein and diolein followed much the same pattern as for hydrolysis, when water was replaced by glycerol. It was shown again that near stoichiometric amounts of substrates led to the best conversion to mono- and diglycerides. A small excess of glycerol was found to be very inhibitory to the reaction. All possible isomers were formed during the reaction. Conversely, in alcoholysis reactions between triolein and stearyl alcohol the specificity of the lipase was upheld. Excess alcohol in this instance was found to be beneficial.

Design parameters and performance of a surface baffled helical ribbon impeller bioreactor for the culture of shear sensitive cells

A novel bioreactor equipped with a double helical ribbon impeller (HRI) and three surface baffles has been shown to be well adapted to shear sensitive suspension cultures. Data on power dissipation, mixing time, circulation time, mass transfer parameters (kL, KLa) were obtained at the 3 and 11 L scale. Rational design and scale-up of this type of unit are discussed. Performance of the HRI for the high density cultivation of plant cells (27g dry weight/L) and insect cells (6×106 cells/mL) are presented.

High-performance gel permeation chromatographic analysis of immunoglobulin M produced by hybridoma cell culture

Abstract A high-performance gel permeation chromatographic (HPGPC) method, using TSK-Gel SWXL 3000 and TSK-Gel SWXL 4000 columns installed in series, was developed for the analysis of immunoglobulin M (IgM) produced by hybridoma cell culture. The detection of this protein was achieved using ultraviolet absorption at 225 nm, yielding a detection limit of ca. 0.3 μg ml−1. IgM-containing samples obtained from different culture systems (T-flask, roller bottle, spinner flask, bioreactor operated in batch, fed-batch or perfusion modes) and media (Dulbeccos Modified Eagle Medium or Protein-Free Hybridoma Medium) were evaluated by this chromatographic technique and also by conventional enzyme-linked immunosorbent assay (ELISA). Concentrations of IgM in culture samples determined by both techniques were always in the same range (±10%). The main advantages of HPGPC over ELISA included improved reproducibility (relative average deviation of 1−3% compared with 10−20% for ELISA), high linearity range between the signal and the concentration [at least three decades (0.3−500 μg ml−1) compared with one decade for ELISA (25−200 ng ml−1)] and ease of operation.

Conical Bioreactor with Internal Lamella Settler for Perfusion Culture of Suspension Cells

A perfusion bioreactor configuration is presented which incorporates a conical lamella settler within a reactor vessel which is itself conical. The method used in determining dimensions for the sedimenter and in scaling up the apparatus ensures that cell residence time in the seperator is independent of reactor working volume. A benchtop-scale prototype of the reactor was tested with hybridoma cells. A 30-day perfusion culture in this vessel reached a maximum cell concentration of 6.5 million cells/mL, with a specific antibody production rate four times that seen in batch culture.

On the use of apparent kinetic parameters for immobilized enzyme with noncompetitive substrate inhibition

The use of a simple rate equation with apparent parameters to describe the kinetic behavior of an immobilized enzyme with uncompetitive [corrected] substrate inhibition was assessed. To do so, the reaction rate was calculated as a function of the interfacial substrate concentration, and the results were used to identify the apparent kinetic parameters by nonlinear regression. This procedure was repeated for different values of the diffusional constraints and of the inhibition constant. The equation using apparent parameters can describe the global kinetic behavior, provided that the diffusional and inhibitory constraints are not too high. When the constraints are high, a Michaelis-Menten equation can be used to model the kinetics for interfacial concentrations lower than the concentration leading to the maximum reaction rate.

Protein-free culture medium improvement: testing additives and their interactive effects in 96-well plates

In order to achieve a rational screening of additives suspected to improve antibody production over basal Dulbeccos Modified Eagle Medium (DMEM), we have developed a 96-well plate method for simultaneously testing individual and interactive effects of multiple additives. Cell viabilities were determined directly in each well by a colorimetric assay (MTT assay) and antibody production was measured by an enzyme-linked immunosorbent assay (ELISA) performed on well supernatants. Such as supplemented culture medium might considerably reduce production costs since commercial serum- or protein-free media are sold at serum-enriched basal medium costs. The CBM-P22 mouse cell line used in this study was shown to be sensitive to key amino acids, oxalacetic acid, ethanolamine and selenium at low cell density (<1 × 105 cells·ml−1). When these cells were inoculated in 96-well plates their antibody productivity was improved (sevenfold) by adding these additives to the basal DMEM as evidenced by ELISA absorbance readings. This improved productivity, obtained with the supplemented DMEM, named DOWSENs, is comparable to the one obtained with the commercial, but costly, protein-free hybridoma medium (PFHM II), taken here as the positive control. Results were confirmed by growing these cells in different media in standard (25 cm2) T-flask static culture. In addition, specific amino acid consumption, as analysed by HPLC, showed that asparagine and tryptophan when added to DMEM may improve antibody production, even if these amino acids are not limiting or highly consumed in PFHM II. Using this 96-well plate assay allows the assessment of a large number of different assays with a few milligrams of the product(s) tested. This is a rapid technique that gives results for additives that are not easily quantified by analytical techniques or for the study of interactive effects, which is time consuming and labour-intensive when done in individual T-flasks.