Candida boidinii

In order to broaden the field of biocatalysis in ionic liquids, other enzyme classes have also been screened. Of special interest are oxidoreductases for the enan-tioselective reduction of prochiral ketones [40]. Formate dehydrogenase from Candida boidinii was found to be stable and active in mixtures of [MMIM][MeS04] with buffer (Entry 12) [41]. So far, however, we have not been able to find an alcohol dehydrogenase that is active in the presence of ionic liquids in order to make use of another advantage of ionic liquids that they increase the solubility of hydrophobic compounds in aqueous systems. On addition of 40 % v/v of [MMIM][MeS04] to water, for example, the solubility of acetophenone is increased from 20 mmol to 200 mmol L ... 

Candida boidinii - a new found producer of pectic enzymes complex. 

Candida boidinii is a further yeast producer of pectic enzymes complex. The production is induced by the presence of pectin as a C-source in the medium the primary methabolic path is the utilization of methanol and the secondary the utilization of pectate chains. The pectic enzymes were bound on the cell walls or released on the cultivation medium. The main enzyme of pectic complex, polygalacturonase, was briefly characterized and the possibility to influence the production of its multiple forms discussed. 

This study reports on the production of pectic enzymes and partial characterization of polygalacturonases produced by Candida boidinii. Candida boidinii belongs to the so called methylotrophic yeasts with famous utilization of methanol. Pectin, the natural substrate of pectic enzymes complex, can serve for microorganisms as a C - source by two different ways after deesterification with pectinesterase as methanol and after hydrolytic cleavage with... 

Candida boidinii (CCY 29-37-13) was obtained as an isolate from contaminated colunm of immobilized polygalacturonase, where 0.5% sodium pectate in 0.1 M acetate buffer, pH 4.6 was used as a substrate. Four other strains for comparison were obtained from Culture Collection of Yeasts, Institute of Chemistry (strains CCY 29-37-1, CCY 29-37-2, CCY 29-37-8, CCY 29-37-12). 

Candida boidinii was cultured at pH 3.51, 5.49 and 7.01, respectively. Czapek s Dox medium with citrus pectin (GENU Pectin, Denmark), sodium pectate or citrus pectin with 20% of D-galactopyranuronic acid (Fluka, Switzerland) as a carbon source were used. The growth curves were performed by measuring the optical density (OD) at 660 nm. 

The cultivation of this yeast strain on pectin medium showed optimal grow conditions. The behaviour of this strain was compared with that of four strains of Candida boidinii from the Culture Collection of Yeasts. The grow curves of all strains on pectin medium showed marked plateau suggesting the presence of two existing C-sources in the pectin medium, requiring two different metabolic paths (Fig. 1). 

Fig. 1. The grow curves of five strains of Candida boidinii on pectin medium.

The strain adapted to pectate showed lag phase by cultivation on this medium corresponding to the first phase of growth on pectin, but after short storage period (two weeks) on malt agar it lost completely the ability to grow on pectate (Fig. 2). There was a possibility to restore this ability to grow on pectate by primary cultivation on pectin. It seems, in conclusion, there is a primary utilization of methanol released from pectin by Candida boidinii and the secondary path, the utilization of D-galacturonate chains, required an... 

The production of individual pectic enzymes by Candida boidinii - dependence on the C - source... 

Fig. 3. Isoelectric focusing in ultrathin polyacrylamide layers (pH gradient 3 -10) of multiple forms of polygalacturonase produced by Candida boidinii under different cultivation conditions a - pectin, pH 3.51 b - pectin, pH 5.49 c -pectin, pH 7.01 d - 20% of D-galactopyranuronic acid in pectin e - pectate. A - Activity detection with print technique on colouress D-galacturonan DP 10 dyed additionally with ruthenium red ( both exo- and polygalacturonases) and B - activity detection with Ostazin Brilliant Red/D-galacturonan DP 10 agar print (polygalacturonases).

Since FDH from Candida boidinii now can be produced at pilot scale, this reaction can be generally used for NADH-regeneration. Recently, the same concept has been used for NADPH regeneration. This became possible because a NADPH-dependent FDH has been obtained by multipoint site-directed mutagenesis of the gene coding the enzyme from the bacterium sp. 101. (Seelbacheia/., 1996). 

H. Schutte, J. Flossdorf, H. Sahm, and M.-R. Kuia, Purification and properties of formaldehyde dehydrogenase and formate dehydrogenase from Candida boidinii, Eur. J. Biochem. 1976, 62, 151-160. 

H. Slusarczyk, Stabilization of NAD-depend-ent formate dehydrogenase from Candida boidinii by site-directed mutagenesis,... 

H. Slusarczyk, S. Felber, M.-R. Kula, and M. Pohl, Stabilization of NAD-dependent formate dehydrogenase from Candida boidinii by site-directed mutagenesis of cysteine residues, Eur. J. Biochem. 2000,... 

Employing heat-dried T. intermedius and heat-dried Candida boidinii SC13822 as sources of PheDH and FDH, respectively, reaction yields averaged 84% mol/mol and the e.e. was > 98% however, production of T. intermedius could not be scaled... 

For the regeneration of NADH, formate and formate dehydrogenase are most widely used. The enzyme from Candida boidinii is commercially available, formate is inexpensive, and the equilibrium favours a nearly irreversible... 

Table 16. Chiral alcohols produced by continuous enzyme-catalyzed processes. The corresponding ketones are reduced with (S)-ADH from Rhodococcus erythropolis, NADH was regenerated by simultaneous coupling with formate dehydrogenase from Candida boidinii (FDH) and formate (data from [159])...

In particular, FDH from Candida boidinii is often used for the regeneration of NADH. However, the low specific activity of this enzyme (4-6 U mg-1) is a considerable disadvantage. The KM values for NAD+ and formate are 0.09 and 13 mM, respectively. The enzyme has a broad pH optimum of 7.5-8.5 while 55°C is the optimal temperature [3]. 

Hemmann S, Blaser K, Crameri R Allergens of Aspergillus fumigatus and Candida boidinii share IgE-binding epitopes. Am J Respir Crit Care Med 1997 165 1956-1962. 

Haywood, G. W., and Large, P. J., 1981, Microbial oxidation of amines dish ibution, purification and properties of two primary amine oxidases from die yeast Candida boidinii grown on amines as sole nitrogen source, Biochem. J. 199 187n201. 

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