Biotransformation of dehydroabietic acid with resting cell suspensions and calcium alginate-immobilized cells of Mortierella isabellina

Abstract

Mortierella isabeUina ATCC 38063 is a zygomycete capable of hydroxylating fish-toxic resin acids which occur in certain pulp mill effluents to nontoxic metabolites. Addition of dehydroabietic acid (1) (80 mg/liter) to a freshly inoculated culture of M. isabellina in dextrose-yeast extract broth resulted in precursor disappearance in 28 to 30 h. During growth phase, hydroxylation occurred at C-2, whereas hydroxylation at C-15 and C-16 commenced with onset of stationary phase. Alternatively, 1 added to stationary-phase culture (40 mg/liter) disappeared within 2 h and hydroxylation occurred concurrently at C-2, C-15, and C-16. Enzymatic activity of stationary-phase culture was totally cell associated and was present despite the absence of 1 during the preparatory growth phase. Resuspension of mature fungi as free mycelia or immobilized in calcium alginate beads did not diminish the effectiveness of the biotransformation, although two new metabolites, 15-hydroxy-8,9,11,12-tetradehydro-7,8-dihydroabietic acid (5) and 16-hydroxy-8,9,11,12-tetradehydro-7,8-dihydroabietic acid (7) were formed. Immobilized mycelia retained hydroxylase activity for >110 days whether or not they were challenged with fresh 1 on a regular basis. In this respect they are more long-lived than resuspended free mycelia are.

Biotransformation of Dehydroabietic, Abietic, and Isopimaric Acids by Mortierella isabellina Immobilized in Polyurethane Foam

“…Structural proof of the metabolites was performed with mass spectroscopy and 400-MHz 1H nuclear magnetic resonance spectroscopy. All data were in full agreement with previously obtained data (1,8).…”

“…Identification of two new monohydroxylated ABA metabolites substituted at C-16 or C-15 in the isopropyl side chain is easily explained because stationary-phase cells were used for the first time for an ABA transformation. Previously, it was recognized that cultures grown in the presence of DHA hydroxylate DHA at C-2 and that side chain modification does not occur until stationary phase (1). This results in hydroxylation at all C-2 sites prior to side chain hydroxylation.…”

“…Unfortunately, M. isabellina spores do not germinate in aerated pulp mill effluent, and the filamentous fungus is a weak competitor in the mixed microbial population of treatment lagoons. As hydroxylation at C-2 in the A ring and at C-1S and C-16 in the isopropyl side chain is most efficiently performed by stationary-phase mycelia (1), immobilization of this fungus may provide a means for its application.…”

“…The potential advantages of immobilization (stable environment, regeneration and reuse of the biocatalyst, increased enzyme stability, and easy adaptation to continuous flow applications) were assessed for stationary-phase M. isabellina mycelia entrapped in calcium-alginate beads (1). Compared with suspended free mycelia, the entrapped cells have a similar initial biotransformation efficiency but are superior in that they retain enzyme activity at a higher level for a longer time.…”

“…HPLC profile of DHA(1) and DHA metabolites (2 through 2) formed by foam-immobilized M. isabellina. Solvent, CH30H-H20 (0.1% CH3CO2H); flow rate, 1.5 ml min-'.-APPL.…”