Candida hydrolysate

Candida utilis is grown on sulfite waste Hquor in Western Europe and North America, on sugar cane molasses in Cuba and Taiwan and on ceUulose acid hydrolysates in Eastern Europe and the former Soviet Union. C. ///i/if utilizes hexoses, pentoses, and many organic acids. Sulfite Hquor from hardwoods contains 2—3% fermentable sugars of which 20% are hexoses and 80% pentoses in softwood Hquors the proportions are reversed. The SO2 must be stripped out to allow yeast growth, which is carried out in large, highly-aerated fermentors. Eor continuous fermentations, carried out at pH 4 and 30°C, the dilution rate is 0.27—0.30 (34). 

In contrast to nonionic surfactants, ionic surfactants build up a high zeta-po-tential at the water-oil interface which can also can influence the enzyme activity. Most investigated systems used AOT as the surfactant because its phase behaviour is well understood. However, AOT is often not very suitable, because it can totally inhibit enzymes (e.g. the formate dehydrogenase from Candida bodinii). The usage of lipases in AOT-based microemulsions is generally unfavourable as AOT is an ester that is hydrolysed itself. 

Candida utilis is grown to high biomass concentrations and the extracted RNA is subsequently hydrolysed into the four 5 nucleotides adenosine 5 -monophosphate (AMP), GMP, cytidine and uridine 5 -monophosphate by crude nuclease PI from Penicillium the desired nucleotides are isolated by ion-exchange chromatography and AMP is converted to IMP by adenyl deaminase from Aspergillus [22, 36]. 

Enantioselective enzymatic transesterifications have been used as a complementary method to enantioselective enzymatic ester hydrolyses. The first example of this particular type of biotransformation is the synthesis of the optically active 2-acetoxy-l-silacyclohexane (5 )-78 (Scheme 19). This compound was obtained by an enantioselective transesterification of the racemic l-silacyclohexan-2-ol rac-43 with triacetin (acetate source) in isooctane, catalyzed by a crude lipase preparation from Candida cylindracea (CCL, E.C. 3.1.1.3)62. After terminating the reaction at 52% conversion (relative to total amount of substrate rac-43), the product (S)-78 was separated from the nonreacted substrate by column chromatography on silica gel and isolated in 92% yield (relative to total amount of converted rac-43) with an enantiomeric purity of 95% ee. The remaining l-silacyclohexan-2-ol (/ )-43 was obtained in 76% yield (relative to total amount of nonconverted rac-43) with an enantiomeric purity of 96% ee. Repeated recrystallization of (R)-43 led to an improvement of enantiomeric purity by up to >98% ee. Compound (R)-43 has already earlier been prepared by an enantioselective microbial reduction of the l-silacyclohexan-2-one 42 (see Scheme 8)53. The l-silacyclohexan-2-ol (R)-43 is the antipode of compound (.S j-43 which was obtained by a kinetic enzymatic resolution of the racemic 2-acetoxy-l-silacyclohexane rac-78 (see Scheme 15)62. For further enantioselective enzymatic transesterifications of racemic organosilicon substrates, with a carbon atom as the center of chirality, see References 64 and 70-72. 

Perego et al. (20) found that during the fermentation of hemicellu-lose hydrolysates by Candida shehatae, the addition of 50 mM acetone resulted in ethanol yields comparable with those obtained under micro-aerophilic conditions. Thus, the addition of 50 mM acetone to the culture... 

Biocatalysts such as Candida cylindracea lipase enzymes have recently been used in enantioseleetive hydrolyses of racemic esters by Fowler, Maefarlane and Roberts.15 The (R,R) diastereoisomer was the main product (91 +/- 0.5 %) as determined by NMR spectroscopic analysis of the products. These authors point out the difficulty of measuring such diastereoisomer ratios by using NMR methods, however, their results show a clear selectivity towards the (R,R) diastereoisomer. 

Candida rugosa lipase (CRL) hydrolysed the other enantiomer selectively (Scheme 6.11). This proves once again that in most cases enzymes with the desired stereoselectivity are available. 

Utilising Candida cylindracea lipase (CCL) a chiral propionic acid was resolved by DSM [34, 46]. Only the undesired enantiomer of the ester was hydrolysed and at a conversion of 64% the remaining desired ester had an ee of 98% (Scheme 6.12). Although this means that the yield of the enantiopure ester is below 40% it did enable a new access to enantiopure captopril. 

Scheme 10.2 Stereochemistry of hydrolyses by Candida rugosa esterase.

While it is a powerful solvent, DMSO is undesirable for industrial use. Instead, the surfactant Triton X-100 can be an effective replacement for DMSO in enzymatic hydrolyses [14], and in our case, worked very well. Triton X-100 forms micelles when mixed with water and would solubilize low concentrations of ester for hydrolysis. However, increasing the surfactant charge did not accelerate hydrolysis. In an attempt to reduce the somewhat long reaction time, we screened the enzymes Amano PS Lipase XIII, Pseudomonas jluorescens and Candida cylindracea versus Amano PS-30. PS-30 was still considered the best choice of enzyme when... 

The simple furan alcohol 131 is successfully resolved32 with a lipase from Candida cyclindracea and you should note that the same enzyme is used to form the octanoate 132 and, under different conditions, to hydrolyse it to the pure alcohol (+)-(R)-131. 

However, the closely related amino acid 133 was not a substrate for either lipase (from pigs or Candida) but could be resolved with the proteolytic enzyme papain. This acted as an esterase, hydrolysing the methyl ester rather than the amide. Note that this kinetic resolution produces a single enantiomer of the carboxylic acid rather than the alcohol and that separation of 134 from 133 is very easy as the free acid can be extracted from organic solvents by aqueous base in which it is soluble as the anion. 

In some cases two different enzymes may show opposite enantioselectivities. The lipase from Candida cylindraceae (CCL) and the esterase from pig liver (PLE) do so with racemic cyanohydrins9 such as 20. Note that the acetate is hydrolysed rather than the ethyl ester. Each enantiomer may be reduced in high yield to a single enantiomer of the amine 21. The literature value for the rotation of this compound is [a]D 20.8 and the measured value for (S)-2 was 20.9 and for (7 )-21 was +20.6. Though this is not usually a reliable method of assessing enantiomeric purity, it is quite convincing here. Later in this chapter you will meet an alternative way to make the same sorts of compounds using different enzymes. 

The Candida cylindracea lipase-catalyzed, Candida rugosa lipase-catalyzed and cholesterol esterase-catalyzed hydrolyses of acetates 88b-102b are examples of the utilization of a remote phenolic ester group as the site of enzymatic attack. For such cases, cholesterol esterase seems to be particularly well suited. 

The action of j8-D-fructofuranosidase from Candida utilis on various derivatives of sucrose (1) and methyl j8-D-fructofuranoside (2), as well as on compounds with related structures has been examined.None of the compounds in Table 1 was hydrolysed by the enzyme, even though many of them are only slightly different structurally from sucrose or methyl S-D-fructofuranoside. It is clear that minor changes at the 1 -, 4 -, or 6 -position cause loss of substrate capability, and it was concluded that ]8-D-fructofuranosidase from Candida utilis is a highly specific enzyme. 

Table 1 Compounds not hydrolysed by -n-fructofuranosidase (from Candida utilis)...

C J l ratio, 67 caldum defidency, 142 caldum gluconate, 142,145 production of, 1 caldum hydroxide, 135 GOTdttii, 126,137,334 Candida brumptii, 138 Candida guilliermondii, 87 Candida lipolytica, 85 Candida utdis, 76,78,83,87 capital equipment, 26 capital expenditure, 26 capital investment, 21 caramelisation, 254 caraway, 238 caibapenems, 151,152 carbohydrates, 202 carbon balance, 256 carbon limited cultures, 50,56 carbojqrpeptidase A, 283 carrageenan, 287 carvone, 238 casein hydrolysate, 204 catabolism, 121 catalase, 143... 

There are numerous examples of enantioselective hydrolyses of the types described in Schemes 3.2 and 3.3, catalysed by lipases and esterases. The selective hydrolysis of amino acid derivatives has been an important part of this field of study. For example, the hydrolase enzyme a-chymotrypsin catalyses the enantioselective hydrolysis of JV-acetyl-DL-phenylalanine methyl ester (5) to give optically pure (L)-acid (6) in 40% yield (Scheme 3.4). The lipase from the fungus Candida cylindracea (ccl) has been shown to hydrolyse octyl 2-chloropropionate (7) with high stereoselectivity on a large scale, giving the (/ )-acid (8) in 46% yield (96% e.e.) and the (S)-ester (45% yield) (Scheme 3.5). 

The predictability of the outcomes of such hydrolyses (i.e. the stereochemistry of the more rapidly hydrolysed enantiomer) has been considerably advanced in recent years by the availability of active-site models for some of the more commonly used enzymes (e.g. pig liver esterase, porcine pancreatic lipase and Candida cylindracea lipase). These models have been constructed by analysing the stereoselectivity of the hydrolysis for a wide range of substrates. Recently, X-ray crystallographic data have been reported for two lipases, and such information obviously will lead to more accurate information on the active sites for these enzymes. 

Cholesterol esterase (CE) and lipase from Candida rugosa (CRL) were very efficient in this kinetic resolution, even on a preparative scale. The best result was obtained with CRL 1 g of racemic 138 yielded 0.48 g of unreacted substrate (5)-138 in 90% ee and 0.39 g of deacetylated product (i )-139 in 88% ee. (iS)-138 was hydrolysed with NaOH, producing (iS)-139 in 75% yield and in almost optically pure form after recrystallisation. Both enantiomers of 139 were subjected to further chemical transformations. For example, the alcohol in (5)-139 was methylated giving 75% yield of enantiomerically pure (5)-140, which was finally reduced by trichlorosilane/triethylamine to give phosphine (5)-141 in 96% ee and 72% yield. This last step occurs with inversion of configuration but some erosion of enantiomeric purity was observed. 

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