Lyophilized cells

Scheme 9.3 Biooxidation of diols by lyophilized cells of R. ruber.

In another approach, the alcohol moiety, formed by an enzymatic hydrolysis of an ester, can act as a nucleophile. In their synthesis of pityol (8-37a), a pheromone of the elm bark beetle, Faber and coworkers [17] used an enzyme-triggered reaction of the diastereomeric mixture of ( )-epoxy ester 8-35 employing an immobilized enzyme preparation (Novo SP 409) or whole lyophilized cells of Rhodococcus erythro-polis NCIMB 11540 (Scheme 8.9). As an intermediate, the enantiopure alcohol 8-36 is formed via kinetic resolution as a mixture ofdiastereomers, which leads to the diastereomeric THF derivatives pityol (8-37a) and 8-37b as a separable mixture with a... 

The reduction of several ketones, which were transformed by the wild-type lyophilized cells of Rhodococcus ruber DSM 44541 with moderate stereoselectivity, was reinvestigated employing lyophilized cells of Escherichia coli containing the overexpressed alcohol dehydrogenase (ADH- A ) from Rhodococcus ruber DSM 44541. The recombinant whole-cell biocatalyst significantly increased the activity and enantioselectivity [41]. For example, the enantiomeric excess of (R)-2-chloro-l-phenylethanol increased from 43 to >99%. This study clearly demonstrated the advantages of the recombinant whole cell biocatalysts over the wild-type whole cells. 

PHA content and composition in the lyophilized cell material were determined using gas chromatography (GC) and nuclear magnetic resonance (NMR) analyses. For GC analysis [17], approximately 15 mg of lyophihzed cell was subjected to methanolysis in the presence of methanol and sulfuric acid [85% 15% (v/v)]. The reaction mixture was incubated at 100°C for 3 hours. The organic layer containing the reaction products was separated, dried over Na SO, and analyzed by GC. For... 

To facilitate its application in organic synthesis, we developed a lyophilized cell powder of Sphingomonas sp. HXN-200 as a biohydroxylation catalyst. Hydro-xylation of A-benzyl-piperidine with such catalyst powder showed 85% of the activity of a similar hydroxylation with frozen/thawed cells, shown in Figure 15.6. The fact that rehydrated lyophilized cells are able to carry out such a reduced nicotinamide adenine dinucleotide (NADH)-dependent hydroxylation indicates that these cells are capable of retaining and regenerating NADH at rates equal to or exceeding the rate of hydroxylation. To our knowledge, this is the first example of the use of lyophilized cells for a cofactor-dependent hydroxylation. 

P% frozenAhawed cells S% frozenAhawed cells P% Lyophilized cells S% Lyophilized cells... 

Figure 15.6 Hydroxylation of iV-benzylpiperidine (5 mM) with lyophilized cell powder and frozen/thawed cells of Sphingomonas sp. HXN-200 at cell concentration of 4.0 g cdw/L.

Cells were harvested by centrifugation for 20 min at 4 °C and 8000 rpm, washed with 50 mM pH 7.5 phosphate buffer and lyophilized. Lyophilized cells were stored at 4 °C. 

Although the use of an epoxide hydrolase was already claimed for the industrial synthesis of L- and meso-tartaric acid in 1969 [51], it was only recently that applications to asymmetric synthesis appeared in the hterature. This fact can be attributed to the limited availabihty of these biocatalysts from sources such as mammals or plants. Since the production of large amounts of crude enzyme is now feasible, preparative-scale applications are within reach of the synthetic chemist. For instance, fermentation of Nocardia EHl on a 701-scale afforded > 700 g of lyophilized cells [100]). 

Finally, natural (i )-(-)-mevalonolactone, a key intermediate from a broad spectrum of cellular biological processes and their regulation, was synthesised via eight steps in 55% overall yield and > 99% ee (Scheme 19). In the key step, the aforementioned enantioconvergent chemoenzymatic deracemization route was applied. Thus, 2-methyl-2-benzyl-oxirane ( )-2 g was deracemized on a large scale (10 g) using lyophilized cells of Nocardia EHl and sulfuric... 

A simplified procedure for detecting the M. aeruginosa toxins was devised. A 20.0 mg sample of lyophilized cells was extracted for 1 hr with 1 ml of 38% ethanol, 5% n-butanol, 50 mM ammonium acetate pH 6.0, shaking the suspension manually for a few seconds several times. The suspension was then centrifuged (5 min at 12,000 Xg). 

Bacterial cells, wet packed, 2 to 3 g. Use B. subtilis, Escherichia coli, or Clostridium wekhii. If lyophilized cells are available, use 0.5 g. Saline-EDTA, 0.15 M NaCl plus 0.1 M ethylenediaminetetraacetate, pH 8... 

Suspend 2 to 3 g of wet packed bacterial cells or 0.5 g of lyophilized cells in 25 mL of saline-EDTA solution in a 125-mL Erlenmeyer flask. Add 1.0 mL of the lysozyme solution (10 mg of lysozyme) and incubate the mixture at 37°C for 30 to 45 minutes. To bring about complete cell lysis, add 2.0 mL of 25% SDS solution and heat the mixture in a 60°C water bath for 10 minutes. To avoid excessive foaming, stir the mixture very gently. As the nucleic acid is released from the lysed cells, the solution will show an increase in viscosity and a decrease in cloudiness. 

From 20 g of lyophilized cells. Experimental details are given in the text. Recoveries in ultrafiltration and dialysis were essentially quantitative, and these steps are omitted in the table. Overall purification of approximately 2000-fold with 15% recovery. 

Implantation of cerebral cells 1949 First injection of lyophilized cells... 

Kinetic and thermodynamic studies of geranyl acetate production by direct geraniol acetylation with lyophilized cells of A. oryzae MIM were carried out in n-heptane [13-15]. Batch tests were performed varying the starting substrates equimolar level from 25 to 150 mM, cell concentration from 5 to 30g/l, and temperature from 30 to 95 °C. The initial rates at different initial substrate concentrations were measured and an apparent Michaelis constant KJ of 62mM and a fee value of 0.88 mmol/g/h were estimated by direct fitting of the initial rates against the initial substrate concentrations <75 mM [15]. 

Table 6.3 Apparent thermodynamic parameters of geraniol and ethanol acetylations by lyophilized cells of A. oryzae MIM estimated by the Arrhenius model under different conditions, with reference temperature 50°C.

Figure 6.1 Time course of (R)-2-octylbutanoate production catalyzed by lyophilized cells (30g/l) of R. oryzae CBS 112.07 in n-heptane at 30°C. Alcohol concentration = l. Og/l, with an equimolar concentration of butanoic acid as the acylating agent.

Figure 6.2 Profile of the e.e. of the esterification of (R,S)-IPG with butyric acid under optimized conditions T = 30°C, the concentration of the mycelia = 30g/l, the concentration of (R, S)-iPG = 3g/l, an equimolar concentration of butyric acid, solvent n-heptane, = 0.75, and biocatalyst lyophilized cells of A. oryzae MIM.

Kurosu J, Sato T, Yoshida K, Tsugane T, Shimura S, Kir-imura K, Kino K, Usami S. Enzymatic synthesis of alpha-arbutin by alpha-anomer-selective glucosylation of hydroquinone using lyophilized cells of Xanthomonas campestris WU-9701. J. Biosci. Bioeng. 2002 93 328-330. 

The PCR method that we have described is able to detect cell cultures containing contaminating mycoplasma species with great sensitivity (1.10 cfu/10 p.1) and seems to be more sensitive than the other techniques available. Also, PCR analysis can be performed from lyophilized cell cultures, which facilitates the transport of samples. Moreover, sample preparation is very simple and does not require any DNA extraction, so the results are obtained in 1 day. 

A few synthetic applications for obtaining biologically active compounds have been described, based on the use of these bacterial enzymes. For instance, the pheromone (S)-frontalin was synthesized in five steps in 94% ee (but rather low overall yield) via a chemoenzymatic route implying epoxide resolution using lyophilized cells oi Rhodococcus equi [189] (Fig. 16). 

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