Lorraine Katz

Purification and characterization of a novel bioconverting lipase from Pseudomonas aeruginosa MB 5001

Abstract The purification and characterization of a lipase produced by Pseudomonas aeruginosa strain MB 5001 that was selected for its unique bioconversion properties is described herein. The purified lipase bioconverts dimethyl 5-(3-(2-(7-chloroquinolin-2-yl)-ethyl)phenyl)4, 6-dithianonanedioate (diester) to its (S)-ester acid, an intermediate in the synthesis of Verlukast, a leukotriene receptor antagonist. In its native form, the enzyme exists as high-molecular-weight aggregates that are dissociated with Triton X-100. The purified enzyme has a molecular weight of 29,000 daltons, a pH optimum of 8.0, a temperature optimum of 55°C, and is stable for 1 h at 40°C. This lipase is strongly inhibited by 1 m m ZnSo 4 (94% inhibition) but is stimulated by the addition of 10 m m CaCl 2 (1.24-fold) and 200 m m taurocholic acid (1.6-fold). It is more active on short-chain versus long-chain triglycerides, and hydrolyzes C18-unsaturated fatty acid esters more efficiently than it hydrolyzes C18-saturated fatty acid esters. In light of its catalytic and physicochemical properties, this enzyme is regarded as a novel lipase .

Asymmetric bioreduction of a β-tetralone to its corresponding (S)-alcohol by the yeast Trichosporon capitatum MY 1890

The yeast Trichosporon capitatum MY 1890 was identified by microbial screening as a suitable biocatalyst for the asymmetric bioreduction of 6-bromo-β-tetralone to its corresponding (S)-alcohol (β-tetralol). This β-tetralol is a precursor to the chiral drug candidate MK-0499, a potassium channel blocker targeted for the treatment of ventricular arrhythmias. Process development studies, employing statistical exploratory designs, yielded a 10-fold increase in the rate of bioreduction and improved the (S)-β-tetralol enantiomeric excess from 71% to 99%. The (S)-β-tetralol enantiomeric excess was found to be highly dependent on glucose catabolism by T. capitatum. Elevated enantiomeric excesses were achieved when switching to a glycerol containing medium. Other process parameters such as pH, temperature and medium composition were found to mostly influence the rate of bioreduction. The developed shake flask process was scaled up to laboratory reactors (23-l scale) and supported the production of gram quantities of highly optically pure (S)-β-tetralol.

Enhancement of Lipase Production during Fed-Batch Cultivation of Pseudomonas aeruginosa MB 5001

Abstract Lipase production by Pseudomonas aeruginosa (strain MB 5001) was enhanced 6.6-fold by developing a fed-batch fermentation process. In batch culturing, lipase synthesis occurred during decelerated cell growth. Building on this information, lipase production was increased by feeding a balanced solution of glucose and ammonium chloride (carbon to nitrogen ratio of 4.13). Best performance was achieved when feeding was initiated during mid growth phase, prior to lipase biosynthesis. This lipase fermentation process was found to be unusually sensitive to dissolved oxygen tension, requiring cultivation of the microorganisms initially under dissolved oxygen limiting conditions followed by non-limiting dissolved oxygen conditions (lipase production phase). These process improvements yielded rapidly to scale up from laboratory bioreactors (23-l) to pilot plant bioreactors (1,900-l).

Optimization of lipase production byPseudomonas aeruginosa MB 5001 in batch cultivation

SummaryThe production ofPseudomonas aeruginosa MB 5001 extracellular lipase was optimized by batch cultivation employing shake flasks and 23-L bioreactors. This enzyme efficiently and selectively bioconverts dimethyl 5-(3-(2-(7-chloroquinolin-2-yl)ethyl)phenyl)4,6-dithianonanedioate (diester) to its (S)-ester acid. Process development studies focused on the identification and optimization of the physicochemical parameters required to achieve maximum lipase production. Of the media evaluated, a peptonized milk-based medium was found to support excellent lipase production and stability. Medium composition and process parameters that supported optimal lipase production were different from those supporting maximum biomass formation. Of the parameters investigated, dissolved oxygen tension had the most significant and unexpected impact on lipase production. Elevated lipase production was achieved whenP. aeruginosa MB 5001 was cultivated in a dissolved oxygen limited environment. Overall, these process development studies resulted in a 100% increase in lipase production when compared to the original shake flask process employing skim milk.

Asymmetric Bioreduction of a β-Ketoester to (R)-β-Hydroxyester by the Fungus Mortierella alpina MF 5534

Abstract Two hundred and sixty strains of microorganisms, (60 strains of yeasts, 60 strains of bacteria and 140 strains of fungi), were evaluated for their ability to stereoselectively bioreduce a β-ketoester {[2 S -(2α,4β)]-1-[(1,1-dimethylethoxy)carbonyl]-4-hydroxy-β-oxo-2-pyrrolidinepropanoic acid 1,1-dimethylethyl ester} to the corresponding ( R )-β-hydroxyester {[2 S -(2α( S ∗ ),4β)]-β,4-dihydroxy-1-[(1,1-dimethylethoxy)carbonyl]-2-pyrrolidinepropanoic acid 1,1-dimethylethyl ester}, a precursor to the β-methyl carbapenem antibiotic BO 2727. Among all the microbes evaluated, only one fungal strain, Mortierella alpina MF 5534 (ATCC 8979) was found to catalyze the desired reaction. The scaled-up bioconversion process in laboratory bioreactor (23- l scale) supported ( R )-β-hydroxyester titers of 550 mg/ l during a 250-h cultivation cycle and allowed the timely production of gram quantities of diastereomerically pure materials (diastereomeric excess>98%).

Physostigmine production byStreptomyces griseofuscus NRRL 5324: Process development and scale-up studies

A reliable and scalable fermentation process was developed for production of the acetylcholine esterase inhibitor physostigmine employingStreptomyces griseofuscus NRRL 5324. Initial fermentation in small-scale bioreactors reached physostigmine levels of approximately 60 mg L−1 after 139 h. Optimization of both process operating parameters and production medium composition rapidly yielded a seven-fold increase in physostigmine titer. The scaled up process routinely produced physostigmine titers of approximately 400 mg L−1 during a fermentation cycle of 180 h, and supported the rapid production of large amounts of physostigmine. A physostigmine production of 500 mg L−1 representing an eight-fold improvement over the original performance, was achieved using a glucose/ammonium fed-batch process.