Elsevier

Journal of Biotechnology

Volume 79, Issue 1, 14 April 2000, Pages 13-26
Journal of Biotechnology

d-malate production by permeabilized Pseudomonas pseudoalcaligenes; optimization of conversion and biocatalyst productivity

https://doi.org/10.1016/S0168-1656(00)00220-0Get rights and content

Abstract

For the development of a continuous process for the production of solid d-malate from a Ca-maleate suspension by permeabilized Pseudomonas pseudoalcaligenes, it is important to understand the effect of appropriate process parameters on the stability and activity of the biocatalyst. Previously, we quantified the effect of product (d-malate2−) concentration on both the first-order biocatalyst inactivation rate and on the biocatalytic conversion rate. The effects of the remaining process parameters (ionic strength, and substrate and Ca2+ concentration) on biocatalyst activity are reported here. At (common) ionic strengths below 2 M, biocatalyst activity was unaffected. At high substrate concentrations, inhibition occurred. Ca2+ concentration did not affect biocatalyst activity. The kinetic parameters (both for conversion and inactivation) were determined as a function of temperature by fitting the complete kinetic model, featuring substrate inhibition, competitive product inhibition and first-order irreversible biocatalyst inactivation, at different temperatures simultaneously through three extended data sets of substrate concentration versus time. Temperature affected both the conversion and inactivation parameters. The final model was used to calculate the substrate and biocatalyst costs per mmol of product in a continuous system with biocatalyst replenishment and biocatalyst recycling. Despite the effect of temperature on each kinetic parameter separately, the overall effect of temperature on the costs was found to be negligible (between 293 and 308 K). Within pertinent ranges, the sum of the substrate and biocatalyst costs per mmol of product was calculated to decrease with the influent substrate concentration and the residence time. The sum of the costs showed a minimum as a function of the influent biocatalyst concentration.

Introduction

One of the major application areas in biotechnology is the production of optically active compounds for the fine-chemicals industry (Elferink et al., 1991). d-malate is an optically active α-hydroxy acid with potential commercial applications. It can be used as a chiral synthon (Seebach and Hungerbühler, 1980, Crosby, 1992), as a resolving agent (Clarke et al., 1978, Shiozawa et al., 1988), or as a ligand in asymmetric synthesis (Tanabe et al., 1973, Heil et al., 1985).

Van der Werf et al. (1992) showed that enzymatic production of d-malate with maleate hydratase from Pseudomonas pseudoalcaligenes is promising, since the substrate maleate is a cheap bulk chemical that is converted into d-malate in a simple one-step bioconversion. In addition, the process does not require cofactor regeneration (Van der Werf et al., 1993), while the enantiomeric purity of such d-malate is 99.97% (Van der Werf et al., 1992). However, purification of maleate hydratase is complicated and yields a rather unstable enzyme (Van der Werf et al., 1993). As an alternative, permeabilized P. pseudoalcaligenes may be used as a more robust biocatalyst (Van der Werf et al., 1992, Michielsen et al., 1998a, Michielsen et al., 1999). Michielsen et al. (1999) reported that in that case stability improved, although inactivation still occurred within a few hours at 308 K. Inactivation was found to decrease with decreasing temperature and increasing product concentration (Michielsen et al., 1999). Furthermore, production of d-malate was found to be inhibited by substrate and product (this work; Van der Werf et al., 1993, Michielsen et al., 1998a).

We aim at developing a continuous process for the production of d-malate with permeabilized P. pseudoalcaligenes. To minimize substrate and product inhibition and thus maximize the production rate per unit volume, a liquid–solid–solid three-phase system with a Ca-maleate suspension as a feed will be used. The reactions involved are shown in Fig. 1.

In this liquid–solid–solid three-phase system, production and downstream processing can be integrated and downstream processing costs can be reduced (as the product accumulates in the solid phase).

In order to complete the biokinetic model, the work presented here focussed on the effects of the remaining process parameters (ionic strength, (high) substrate and Ca2+ concentration) on biocatalyst activity. With respect to the effect of Ca2+ concentration. Van der Werf et al. (1995) reported that free Ca2+ can influence the activity of maleate hydratase either by decreasing the availability of the substrate maleate2−, or by direct enzyme inhibition. The latter was investigated in the work presented here.

After reaching that point, all the important kinetic phenomena are known (substrate and competitive product inhibition and first-order irreversible biocatalyst inactivation). However, the kinetic parameters were determined independently in separate experiments (Michielsen et al., 1998a, Michielsen et al., 1999; this work). In each experiment, only a few kinetic parameters of the overall model were determined, assuming other kinetic phenomena not to occur significantly. In order to obtain more realistic values, all the kinetic parameters were then once more determined simultaneously, as a function of temperature. To this end, the overall model was fitted through three data sets of substrate concentration versus time (measured at the desired temperature) that featured substrate inhibition, competitive product inhibition, as well as biocatalyst inactivation.

In order to produce d-malate at the lowest costs in a continuous system, the cost-determining factors were calculated as a function of the process conditions in a continuous stirred-tank reactor (CSTR) with biocatalyst replenishment and recycling. Neglecting investment and operating costs, the potentially cost-determining factors are substrate and biocatalyst. For minimization of these costs the conversion (in mmol of product per mmol of initial or influent substrate) and biocatalyst productivity (in mmol of product per mg of biocatalyst), respectively, must be maximized (Van’t Riet and Tramper, 1991). As some process parameters (e.g. the biocatalyst concentration in the inflow) have opposed effects on the conversion and on biocatalyst productivity, a minimum with respect to the sum of the biocatalyst and substrate costs exists at certain process conditions. In this work, it is shown how to select the process conditions that correspond with this costs minimum.

Section snippets

Cultivation and permeabilization of P. pseudoalcaligenes

P. pseudoalcaligenes NCIMB 9867 (kindly supplied by the Division of Industrial Microbiology, Wageningen Agricultural University) was cultivated and permeabilized as described before (Michielsen et al., 1998a).

Effect of Ca2+ concentration on maleate–hydratase activity

In order to determine the effect of Ca2+ concentration on maleate–hydratase activity, solutions with varying concentrations of Ca2+ and a constant maleate2− concentration were made; Ca2+ concentration was set with CaCl2 · 2H2O and maleate2− with maleic acid. As in solution also Ca-maleate

Effect of Ca2+ concentration on maleate–hydratase activity

The effect of Ca2+ concentration on maleate–hydratase activity of permeabilized P. pseudoalcaligenes was found to be negligible; the average and S.D. from the average were 0.28 and 1.8×10−2 μmol min−1 mg−1, respectively, and the slope of the fitted straight line through the activity versus Ca2+ concentration data (with 95% confidence interval) was −0.14±0.36 l min−1 mg−1. Since there is no direct negative effect on biocatalyst activity itself, the Ca2+ concentration is an attractive tool to

Conclusions

The kinetics of d-malate production can be described well by a model featuring substrate inhibition and competitive product inhibition, and first-order irreversible biocatalyst inactivation. In comparison with separate independent and thereby labor-intensive determinations of kinetic parameter values, more realistic values were obtained by simultaneously fitting the complete kinetic model through only three independent substrate concentration versus time data sets (each data set with a

Nomenclature

Cconcentration (mmol l−1)
Ceactive enzyme concentration (mg l−1)
Ce(0)active enzyme concentration at t=0 (mg l−1)
$costs per mmol of product ($ mmol−1)
Cssubstrate concentration (mmol l−1)
Cs(0)substrate concentration at t=0 (mmol l−1)
ΔHactivation enthalpy (kJ mol−1)
Finflow and outflow of CSTR (m3 h−1)
Iionic strength (mol 1−1)
KmMichaelis constant (mmol 1−1)
Kpproduct inhibition constant (mmol 1−1)
KSdissociation constant of Ca-maleate (mol 1−1)
Ksisubstrate inhibition constant (mmol 1−1)
kdfirst-order

Acknowledgements

We thank S. Hartmans and M.J. van der Werf (Division of Industrial Microbiology, Wageningen Agricultural University) for supplying P. pseudoalcaligenes and for fruitful discussions on cultivation and permeabilization of P. pseudoalcaligenes. This work was financially supported by the Ministry of Economic Affairs, the Ministry of Education, Culture and Science, the Ministry of Agriculture, Nature Management and Fishery in the framework of an industrial relevant research programme of the

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