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Biotransformation of benzaldehyde and pyruvate to ( R ) -Phenylacetyl carbinol by Hansenula polymorpha was investigated in two-phase aqueous-organic reaction media. With octonol as organic dissolver, maximal biotransformation activity was observed with a wet content of 10 % . Of the organic dissolvers tested, highest biotransformation activities were observed with octonol and hexadecane, and lowest activities occurred with trichloromethane and methylbenzene. Biocatalyst samples from biphasic media incorporating octonol, decane and methylbenzene manifested no evident cell structural harm when examined utilizing scanning negatron microscopy. In contrast, cellular biocatalyst recovered from two-phase systems incorporating trichloromethane, butylacetate and ethylacetate exhibited harm in the signifier of cell puncturing after different incubation periods. Phospholipids were detected in reaction media from biocatalytic systems which exhibited cell harm in negatron micrographs. Phospholipid release was much lower in the two-phase systems incorporating methylbenzene or octonol or in 100 % aqueous biocatalytic system.

Baker ‘s barm has attracted significant attending as a accelerator for biotransformation procedures, particularly affecting carbon-carbon bond formation and oxidization decrease reactions, because barm is so cheap and easy to obtain [ 1 ] . Yeast induced carbon-carbon condensation of benzaldehyde to acetaldehyde in the production of ( R ) – Phenylacetyl carbinol ( PAC ) , a precursor of ephedrine, was one of the first microbic biotransformation processes to be commercialised [ 2 ] . During production of PAC, some of the benzaldehyde is besides reduced to benzyl intoxicant [ 3 ] . In our research lab, biotransformation procedures for production of PAC and benzyl intoxicant have been investigated in item in aqueous systems with a position to characterizing factors act uponing these reactions [ 3,4 ] . We have demonstrated that a broad scope of substituted aromatic aldehydes can be converted to the corresponding substituted carbinol merchandises and so substituted aromatic intoxicant byproducts [ 5 ] . While the transition of benzaldehyde to benzyl intoxicant can be catalysed by barm intoxicant dehydrogenase, we have shown that mutant strains missing the chief ADH isoenzymes manifest similar capacity to wild-type strains to bring forth benzyl intoxicant [ 6 ] . The capacity of baker ‘s barm to transport out asymmetric reductive biotransformations of carbonyl compounds in aqueous media has been widely recognised. Therefore, conventionally, yeast has been used as a biocatalyst for organic synthesis in aqueous conditions [ 1, 7 ] .

However, the bulk of organic chemicals are ailing H2O soluble but extremely soluble in organic dissolvers and the execution of biotransformation systems in organic solvent-containing media offers possible to increase the solubility of ailing H2O soluble substrates [ 10 ] . While some probes of biotransformations utilizing stray barm enzymes in micro-aqueous solvent systems and two stage systems have been documented [ 11-14 ] , small accent has been placed on whole cell systems. Whole cell biotransformations have advantages over stray enzymes in that costs due to catalyst extraction and purification are avoided, an chance to recycle the enzyme incorporating cells is provided, and the biotransforming enzyme may be stabilized in the intra-cellular surroundings [ 4, 6, 15 ] .

Merely a little sum of H2O is required by enzymes to keep catalytic activity [ 7 ] . Where enzyme reactions have been carried out in organic dissolvers holding a really low H2O content ( & A ; lt ; 0.02 % ) the system has been described as ‘micro-aqueous ‘ [ 13,8 ] . Where organic and aqueous stages are present in surplus of common impregnation degrees, the reaction medium is described as ‘biphasic ‘ [ 16,17 ] . Whole cell biocatalysts tend to necessitate more H2O than isolated enzymes and appear to by and large necessitate biphasic systems for biotransformation of ailing H2O soluble substrates [ 18 ] . We have late reported the capacity of wild-type and mutant strains of Hansenula polymorpha to transport out reductive biotransformations in aqueous-organic solvent biphasic systems [ 19 ] . We now report a survey of the biotransformation of benzaldehyde and pyruvate to PAC in two-phase aqueous-organic dissolver systems with accent on the consequence of dissolver on merchandise formation and cell construction.

MATERIALS AND METHODS

Yeast civilization: Hansenula polymorpha strain was maintained on civilization medium containing ( g/l ) glucose 20, yeast extract 3.0, ( NH4 ) 2SO4 2.0, KH2PO4 1.0, MgSO4.7H2O 1.0, agar 1.5 with an initial pH of 6.0. For its growing and subsequent immobilisation, this strain was cultivated in a agitation medium consisting of ( g/l ) glucose 60, yeast extract 10, ( NH4 ) 2SO4 10, KH2PO4 3.0, Na 2HPO4.12H2O 2.0, MgSO4.7H2O 1.0, CaCl2 0.05, FeSO4 0.05, Mn SO4.4H2O 0.05 at an initial pH of 5.0 and temperature of 250C.

Preparation of biocatalysts for biotransformation: Fresh Hansenula polymorpha cells, was resuspended in 0.05 M Na citrate buffer ( pH 6.0 ) . The suspension was lyophilised and stored at 40 for usage in biotransformation experiments.

Preparation of organic dissolvers: All organic dissolvers were obtained in anhydrous signifier or with the lowest H2O content possible. Prior to utilize the dissolvers were saturated with 0.05 M Na citrate buffer ( pH 6.0 ) . To fix two stage systems incorporating different wet degrees, different proportions of pre-saturated dissolvers and 0.05 M Na citrate buffer ( pH 6.0 ) were assorted. For illustration, to fix a 10 % wet degree, 10 milliliter of buffer was assorted with 90 milliliters of presaturated dissolver.

Biotransformation conditions: Lyophilised biocatalysts, 1.3 g ( 1 g support stuff and 300 milligrams dried cells ) was added to a 250-ml Erlenmeyer flask incorporating 30 milliliter of biotransformation medium: aqueous or matching aqueous-organic solvent two-phase system. Sodium pyruvate ( 50 g/l ) was used as substrate. The reaction was initiated by the add-on of the cosubstrate benzaldehyde ( 6 g/l ) . Biotransformations were conducted on an orbital shaker set at 280C and 300 revolutions per minute. The advancement of merchandise formation was monitored utilizing gas chromatography. Samples were besides taken at 0, 2, 6 and 26 H to analyze the consequence of the organic dissolvers.

Gas chromatography ( GC ) : Political action committee, benzaldehyde and benzyl intoxicant concentrations were determined by GC analysis utilizing a Shimadzu GC 14A gas chromatograph equipped with a fire ionisation sensor and connected to a Shimadzu Chromatopac CR6A planimeter. The gap column was a amalgamate silicon oxide megabore 30 m long and 0.52 millimeters internal diameter coated with 1 & A ; Acirc ; µm thickness of 25 % cyanopropyl, 25 % phenyl, 50 % methyl polysiloxane. Analytic conditions were: injection and column temperature 1500C and sensor temperature 200 0C Helium gas was used as the bearer and cyclo hexanone was used as internal criterion. Aqueous stage samples were extracted twice with an equal volume of diethyl quintessence. PAC, benzyl intoxicant and benzaldehyde were used as criterions.

Thin-layer chromatograph: ( TLC ) . Cells, incubated in the aqueous stage and two-phase systems for 26 H, were separated from the medium. The supernatants from the two-phase systems were evaporated utilizing a rotary vacuity evaporator and the residues were kept for TLC analysis. The aqueous stage sample was freeze dried. The residue was dissolved in a 6 ml mixture of chloroform/ methanol/water ( 1:2:0.8 ) , vortexed for 1 rain and centrifuged at 5000 ten g for 5 min and the supernatant was separated. The extraction process was repeated and the two supernatants were combined. To the infusion, 8 milliliter of H2O and 3 milliliter of trichloromethane was added and the mixture vortexed for 1 min and centrifuged at 5000 ten g for 5 min.

The underside ( trichloromethane ) bed was dried at 36 & A ; Acirc ; µ utilizing a warming block under N watercourse for TLC analysis. The residues were dissolved in 50 & A ; Acirc ; µ1of trichloromethane and used for TLC. TLC home bases were Si 250, precoated glass home bases. The criterions ( phosphatidylethanolamine, phosphatidylcholine, phosphatidic acid, phosphatidylinositol, phosphatidylserine and phosphatidylglycerol ) . Samples ( 10 & A ; Acirc ; µ1 ) were applied to the TLC home base ( 20 centimeter x 20 centimeter ) and developed in a mixture of chloroform/ methanol/water ( 65:25:4 ) . Dittmer and Lester spray reagent [ 9 ] , which is specific for phospholipids was used for sensing. The Rf values are presented in Table 1.

Consequence

Production of PAC from benzaldehyde and pyruvate was chosen as a theoretical account system in these probes. The accelerator consisted of lyophilized barm cells adsorbed on celite. The consequence of wet content on production of PAC by cells immobilised on celite was investigated utilizing octonol as organic dissolver ( Table 2 ) . Maximal biotransformation activity was observed with a wet degree of 10 % . The consequence of solvent type on rate of production of PAC was investigated in two-phase systems incorporating 10 % wet and related to log P. The consequences are presented in Table 3. Highest biotransformation activities were observed with octonol and hexadecane and lowest activities were noted with trichloromethane and methylbenzene. Samples of the biocatalyst were withdrawn from the reaction mixture at 0, 2, 6, and 26 H, and were examined. Biocatalyst samples from biphasic media incorporating octonol, decane and methylbenzene manifested no evident harm after 26 h. Biocatalyst recovered from two-phase systems incorporating trichloromethane at nothing clip and after a 2-h incubation Note that the cells from the 2-h sample appear to be punctured. In contrast, biocatalyst recovered from butyl ethanoate incorporating media appeared integral at 2 Hs but damaged at 6 Hs, while samples from ethylacetate incorporating media appeared integral after 6 H but damaged at 26 h. The consequence of dissolver on release of phospholipids from the cell was besides investigated after 26 h incubation utilizing TLC ( Fig.1 ) . The biocatalyst was recovered by centrifugation from the reaction mixture and the latter was concentrated for TLC analysis. When the samples were subjected to TLC and tested for phospholipids, small or no phospholipid was detected from cells incubated in methylbenzene, octonol or in the 100 % aqueous system. Note that samples from decane, dodecane and hexadecane were non analyzed as these dissolvers could non be evaporated because of their high boiling points. On the other manus, samples from reaction mixtures incorporating ethylacetate, butylacetate, and trichloromethane manifested the presence of phospholipids.Thus, phospholipids were detected in samples from biocatalytic systems which exhibited cell harm in negatron micrographs.

Table 1

R degree Fahrenheit values of chromatography phospholipids separated by thin-layer

Phospholipids

R degree Fahrenheit

Phosphatidylethanolamine ( PE )

Phosphatidylinositol ( PI )

Phosphatidylcholine ( Personal computer )

Phosphatidic acid ( PA )

Phosphatidyl serine ( PS )

Phosphatidylglycerol ( PG )

0.58

0.22

0.26

0.37

0.25

0.40

Table 2 The consequence of wet content on PAC production by whole barm cells

% wet a

Activity ( mmol/h/g )

0.5

2.0

10.0

20.0

40.0

3.7 x 10-2

3.5 x 10-2

4.2 x 10-2

5.4x 10-2

4.4x 10-2

Table 3 Effect of organic dissolver on PAC production by whole barm cells

Solvent a

Log P

Activity ( mmol/h/g )

Ethylacetate

Butylacetate

Chloroform

Toluene

Octonol

Dodecane

Hexadecane

0.68

1.70

2.00

2.50

3.50

6.60

8.80

2.84 ten 10- 2

3.10x 10 – 2

1.00 ten 10- 2

0.70 x 10 – 2

6.00 x 10 – 2

4.00 x 10 – 2

5.60 ten 10- 2

Fig.1. Thin-layer chromatography of the consequence of solvent release of phospholipids from the cell. Incubation clip was 26 h. PE, PI, Personal computer, PA, PS, and PG: phospholipid criterions see Table 1 for account of abbreviations. 1-6: phospholipids released during biotransformations. 1, aqueous stage ; 2-6, two-phase systems incorporating: 2, ethylacetate ; 3, butylacetate ; 4, trichloromethane ; 5, methylbenzene ; 6, Octonol.

Discussion

In this survey, the ability of baker ‘s barm to bring forth PAC by biotransformation of benzaldehyde and pyruvic acid in biphasic systems was demonstrated. Very small attending has been paid to execution of whole cell biotransformations in aqueous-organic media. Where whole cell biotransformations in biphasic media have been investigated, surveies, in general, focused on reactions affecting steroids, lipoids and esters. However, yeast cells holding esterase activity stereoselectively hydrolysed methyl esters in organic dissolvers [ 21 ] . We have antecedently demonstrated the decrease of benzaldehyde to benzyl intoxicant by baker ‘s barm in aqueous-organic biphasic systems [ 19 ] . As was observed above for PAC production, biotransformation activity for transition of benzaldehyde to benzyl intoxicant increased with wet content up to a value of 10 % v/v and, in both instances, the organic dissolver octonol manifested higher biotransformation activity [ 19 ] . Deetz and Rozzell [ 11 ] found an addition in activity of stray intoxicant dehydrogenase in acetonitrile up to a wet content of 10 to 15. In steroid transmutations utilizing Nocardia species, provided plenty aqueous stage was present to wholly swell the cells, alterations in the ratios of aqueous-organic stages had small consequence on reaction rate [ 22,23 ] .

An of import extension of our work will be to look into the yeast-mediated biotransformation of less water-soluble substrate and cosubstrate parallels of pyruvate and benzaldehyde to bring forth parallels of PAC holding possible biological activity. It was noted that the cell surfaces of yeast biocatalyst samples recovered from biphasic media incorporating octonol, decane and methylbenzene after a 26-h biotransformation reaction manifested no evident harm, while cell puncturing was observed after shorter biotransformation periods with the more hydrophilic dissolvers ( holding log P- & A ; lt ; 2.0 ) , trichloromethane, ethylacetate and butylacetate. Furthermore, we observed that the cell damaging dissolvers, ethylacetate, butylacetate, and trichloromethane, resulted in the extraction of phospholipids from the cells into the biphasic medium, whereas small or no phospholipids were detected from cells incubated in methylbenzene or octonol biphasic media. Phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol constitute 80-90 % of entire whole cell and cell membrane phospholipids in S. cerevisiae [ 24 ] .

Phospholipids are recognised as really of import constituents of cell membranes which influence membrane permeableness and snap [ 16 ] . Certain organic dissolvers have been shown to do rapid dislocation of the permeableness barrier map of cell membranes [ 25-29 ] . While the extent of membrane harm to bacterial suspensions appears to be related to the sum of dissolver used [ 25,28 ] , a relationship to solvent belongingss such as hydrophobicity has besides been suggested [ 26 ] . Lilly et Al. [ 30 ] have summarised the possible effects of organic dissolvers on cell morphology of microorganisms in footings of cytoplasmatic shrinking, loss of membrane administration and ultrastructural alterations.

In keeping the unity of the biocatalyst in whole cell biotransformations carried out in aqueous-solvent twophase systems, clearly choice of dissolvers which minimize cell harm requires careful consideration and experimentation [ 31 ] . Biocatalyst barm cells taken from biotransformation reactions incorporating dissolvers with log P values of & A ; gt ; 2.5 manifested no surface harm in scanning negatron micrographs. Although two of the latter three dissolvers, viz. octonol and dodecane, produced high reaction rates in biotransformations, methylbenzene did non. Therefore, there was non a perfect correlativity between evident opposition of barm cells to solvent harm and biocatalytic activity. However, the consequences indicate that octonol, which manifested optimum rates of biocatalytic activity in these biotransformations, did non look to damage cell construction, as judged from scanning negatron micrographs.

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