11beta-Hydroxylation of Cortexolone (Reichstein Compound S) to Hydrocortisone by Curvularia lunata Entrapped in Photo-Cross-Linked Resin Gels

Spores of Curvularia lunata were immobilized by entrapment with photo-cross-linkable resin prepolymers and incubated to form mycelium in potato dextrose broth containing cortexolone (Reichstein compound S) as an inducer of steroid 11beta-hydroxylase. In a buffer system containing 2.5% dimethyl sulfoxide, this immobilized mycelium hydroxylated cortexolone to hydrocortisone. The activity of this mycelium was comparable to the activity of free mycelium. Dimethyl sulfoxide did not inhibit hydroxylase activity at the concentration used and was effective in dissolving the product. Of the various photo-cross-linkable resin prepolymers examined, use of ENT-4000, whose main chain was polyethylene glycol 4000 (chain length, approximately 40 nm), resulted in maximum hydroxylation activity of the entrapped mycelium. The chain length of prepolymers affected markedly mycelial growth in the gels and, subsequently, the activity of the entrapped mycelium. The immobilized hydroxylation system was more stable than the system in free mycelium and could be reactivated by incubation of the entrapped mycelium in potato dextrose broth containing cortexolone. The system was tested 50 times during 100 days of operation and was found to carry out the desired transformation with overall yields of 60%.     

11α-Hydroxylation of progesterone by gel-entrapped living Rhizopus stolonifer mycelia

Summary Spores of Rhizopus stolonifer were immobilized aseptically by entrapment with photo-crosslinkable resin prepolymers, urethane prepolymers or several kinds of polysaccharides. The entrapped spores were allowed to germinate and develop in situ. The immobilized living mycelia so obtained were induced for the steroid 11-hydroxylation system and examined for their activity to hydroxylate progesterone at 11-position in a buffer system containing 2.5% of organic cosolvent. Of various water-miscible organic cosolvents, methanol was found to be most effective in terms of the activity of the entrapped mycelia and the solubility of the product, 11-hydroxyprogesterone. Though all the living mycelia entrapped in different gels exhibited the hydroxylation activity, mycelia entrapped in photo-crosslinked gels showed the maximum activity which was rather higher than that of the free mycelia. The net-work size of the photo-crosslinked resins, namely the chain length of the photo-crosslinkable resin prepolymers, affected markedly the mycelial growth in gels, and subsequently, the hydroxylation activity of the entrapped mycelia. Entrapment significantly enhanced the operational activity and stability of the 11-hydroxylation system in the mycelia, and permitted the intermittent reactivation of the system by incubating the entrapped mycelia in potato-dextrose broth.     

Effect of high-cell-density fermentation of Candida utilis on kinetic parameters and the shift to respiro-fermentative metabolism

In this study, biodesulfurization (BDS) was carried out using immobilized Rhodococcus erythropolis KA2-5-1 in n-tetradecane containing dibenzothiophene (DBT) as a model oil (n-tetradecane/immobilized cell biphasic system). The cells were immobilized by entrapping them with calcium alginate, agar, photo-crosslinkable resin prepolymers (ENT-4000 and ENTP-4000), and urethane prepolymers (PU-3 and PU-6); and it was found that ENT-4000-immobilized cells had the highest DBT desulfurization activity in the model oil system without leakage of cells from the support. Furthermore, ENT4000-immobilized cells could catalyze BDS repeatedly in this system for more than 900 h with reactivation; and recovery of both the biocatalyst and the desulfurized model oil was easy. This study would give a solution to the problems in BDS, such as the troublesome process of recovering desulfurized oil and the short life of BDS biocatalysts.     

Application of Urethane Prepolymers to Immobilization of Biocatalysts: Δ1Dehydrogenation of Hydrocortisone by Arthrobacter simplex Cells Entrapped with Urethane Prepolymers

Acetone-dried cells of Arthrobacter simplex having appreciable steroid Δ¹-dehydrogenase activity were immobilized by mixing the cell suspension with water-miscible urethane prepolymers synthesized from toluene diisocyanate and polyether diols. The entrapped cell activity in the transformation of hydrocortisone to prednisolone was affected by the properties of urethane prepolymers, such as the isocyanate group content in prepolymers, the molecular weight of polyether diols and the ethylene oxide content in diols. The addition of 10% of organic solvents, such as methanol and glycols, to the aqueous reaction mixture enhanced the solubility of the substrate greatly and the reaction rate of the immobilized cells. The activity of immobilized cells remained high even in the system containing 30% of methanol, which drastically inhibited the activity of free cells. The presence of an electron acceptor, phenazine methosulfate or 2,6-dichlorophenolindophenol, significantly stimulated the steroid conversion with entrapped cells, as well as free cells. The stability of the cells over repeated reactions was greatly improved by immobilization.     

Stability of immobilized laccase on Luffa Cylindrica fibers and assessment of synthetic hormone degradation

In this work were studied the pH, thermal, and storage stability of free and immobilized laccases. Enzymes were produced by Pleurotus ostreatus on potato dextrose (PD) broth and potato dextrose modified (PDM) broth, and immobilized using Luffa cylindrica fibers as support. Both free and immobilized enzymes were assessed on their respective enzymatic activities and for 17α-ethinylestradiol (EE2) degradation. The optimum pH conditions concerning laccase activity ranged from 3.6 to 4.6, while temperature ranged between 30?°C and 50?°C for both free and immobilized enzyme. Laccase produced using PD broth presented greater storage stability and thermal stability than that of PDM. Best EE2 removals were of 79.22% and 75.00% for the free and immobilized enzymes, respectively. Removal rates were assessed during 8?h at pH 5. The removal of 17α-ethinylestradiol was stabilized in the fourth cycle of use. Results imply that immobilization promoted stability towards pH and temperature variations, although media played a decisive role in the enzymatic activity. Both free and immobilized laccases of P. ostreatus were able to degrade EE2, whereas immobilized laccase in PDM medium presented possible reuse applicability, albeit removal was not optimal when compared to other reports.     

Application of photo-crosslinkable resin prepolymers to entrap microbial cells. Effects of increased cell-entrapping gel hydrophobicity on the hydrocortisone Δ1-Dehydrogenation

Summary Acetone-dried cells of Arthrobacter simplex, whose steroid ¹ activity had been previously induced, were entrapped by the use of photo-crosslinkable resin prepolymers. When the hydrophobicity of the cell-entrapping gel was increased by mixing a hydrophobic prepolymer (main chain component; polypropyleneglycol) with a hydrophilic prepolymer (main chain component; polypropyleneglycol) with a hydrophilic prepolymer (main chain component; polyethyleneglycol) (up to 30%), the hydrocortisone to prednisolone conversion rate of the immobilized cells increased significantly, attaining approximately 20% of that of the free cells. A 10% addition of organic solvents, such as methanol, to the aqueous reaction mixture enhanced the solubility of the substrate greatly and to a lesser degree the reaction rate of the immobilized cells. The presence of an electron acceptor, phenazine methosulfate or 2,6-dichlorophenolindophenol, stimulated the steroid conversion of the entrapped as well as the free cells. The stability of the entrapped cells over repeated reactions was improved by immobilization.     

Production of nikkomycin by immobilized Streptomyces cells — Physiological properties

Summary The antibiotic nikkomycin can be produced by calcium alginate immobilized mycelium of Streptomyces tendae Tü 901 in batch and continuous culture.Scanning electron micrographs show the porous structure of the matrix and the distribution of the cells in the gel.Some physiological properties of free and immobilized mycelia were compared. Immobilization does not change the relative amounts of nikkomycin compounds in the culture broth. DNA and protein content were the same in free and immobilized cells. The specific activity of fructosediphosphate aldolase dropped during fermentation and was lower for entrapped than for free cells. The specific activity of mannitol dehydrogenase increased up to the end of the fermentation and was the same for free and immobilized mycelium.In continuous culture the relative amount of mannitol consumed decreased with increasing flow rate. When the medium was supplemented with amino acids mannitol consumption increased significantly.Dedicated to Prof. Dr. L. Acker on the occasion of his 70th birthday     

Application of immobilized lipase to regio-specific interesterification of triglyceride in organic solvent

Summary Lipase from Rhizopus delemar was immobilized by entrapment with photo-crosslinkable resin prepolymers or urethane prepolymers or by binding to various types of porous silica beads. The immobilized lipase preparations thus obtained were examined for their activity in converting olive oil to an interesterified fat (cacao butter-like fat), whose oleic acid moieties at 1- and 3-positions were replaced with stearic acid moieties, in the reaction solvent n-hexane. Although all of the immobilized preparations exhibited some activity, lipase adsorbed on Celite and then entrapped with a hydrophobic photo-crosslinkable resin prepolymer showed the highest activity, about 75% of that of lipase simply adsorbed onto Celite. Entrapment markedly enhanced the operational stability of lipase.Dedicated to Professor H. Holzer, Freiburg University, on his 60th birthday (June 13, 1981)     

Protease production by the ontjom fungus,Neurospora sitophila

Summary The production of protease and mycelium byNeurospora sitophila cultured on solid and liquid potato dextrose media was studied. Maximal activity of protease extracted from 4-day-old cultures occurred at pH 6.5 when an unfractionated peanut (groundnut) protein substrate was used. The greatest protease activity and mycelium production occurred during the first day of the 4-day test period. Potato dextrose media containing more than 0.2 M NaCl resulted in decreased protease and mycelium production, while tapioca starch was without affect at concentrations up to 1.4%. Addition of up to 0.3 M sucrose to growth media greatly stimulated protease production and mycelial growth. Maximal proteolytic activity was observed in extracts from mycelium cultured in potato dextrose media adjusted from pH 6.0 to 7.5. Activity was greater when soluble peanut protein was used as a substrate, compared to unfractionated or globulin protein substrates.     

Sequential conversion of cortexolone to prednisolone by immobilized mycelia of Curvularia lunata and immobilized cells of Arthrobacter simplex

Summary Two-step bioconversion of cortexolone (Reichstein's Compound S) to its ¹-dehaydro-11-hydroxy derivative, prednisolone, was successfully performed by the combined use of immobilized Curvularia lunata mycelia and immobilized Arthrobacter simplex cells. Immobilized living mycelia of C. lunata having a high 11-hydroxylation activity were prepared by in situ germination of spores entrapped in photo-crosslinked resin gels of a suitable net-work structure. Acetone-dried cells of A. simplex having an induced steroid ¹-dehydrogenase activity were also entrapped with photo-crosslinkable resin prepolymers and used for - dehydrogenation of hydrocortisone to prednisolone. For the production of prednisolone from cortexolone, the combination of sequential steps, 11-hydroxylation and subsequent ¹-dehydrogenation, was found to be suitable. Each immobilized microbial cell system was stable and could be used for the sequential reactions repeatedly (operational period, 25 days).     

Production of adenine arabinoside by gel-entrapped cells of Enterobacter aerogenes in water-organic cosolvent system

Summary Gel-entrapped whole cells of Enterobacter aerogenes, which has a transglycosylation activity, were used to produce adenine arabinoside from uracil arabinoside and adenine, in an appropriate water-organic cosolvent system. Cells of E. aerogenes entrapped with a hydrophilic photo-crosslinkable resin prepolymer, ENT-4000, or a urethane prepolymer, PU-6, had a high and stable transglycosylation activity. To improve the poor solubility in water of the substrate (adenine) and product (adenine arabinoside), dimethyl sulfoxide was selected as the cosolvent based on the criteria of operational stability of the immobilized biocatalyst and solubility of both substrate and product. Addition of 40% dimethyl sulfoxide to the reaction mixture permitted use of a high substrate concentration range which gave high productivity under homogeneous reaction conditions. The immobilized cells of E. aerogenes exhibited a markedly improved operational stability, retaining their initial level of activity during repeated use for at least 35 days at 60°C in 40% dimethyl sulfoxide. When the reaction was carried out with 150 mM uracil arabinoside and 50 mM adenine as the substrates, the yield of adenine arabinoside was maintained at 100% based on the molar ratio of adenine, throughout the reaction.Abbreviations used AraA adenine arabinoside - AraU uracil arabinoside     

Hydroxylation of indolyl-3-acetic acid by immobilized mycelium of Aspergillus niger

Summary The immobilization in polyacrylamide gel (PAAG) of the Aspergillus niger mycelium, which has the activity of hydroxylating indolyl-3-acetic acid (IAA) at 4-, 5-, and 6-positions of the indole nucleus, was studied. To preserve the hydroxylating activity, the immobilization should be performed at 5°C–10°C for 5–10 min. The hydroxylating activity of the A. niger mycelium entrapped in PAAG attained 70%–80% of that of free cells. The IAA transformation in the presence of sodium desoxycholate, polyethylene-glycol-400 (PEG-400), Span-60 or preincubation of granules entrapping mycelia in the presence of Tween-80 or PEG-400 not only double the hydroxylation rate but stabilize the activity as well. Gels entrapping mycelia may be used five or six times without altering activity. Incubation of gels with mycelium in the nutrient medium also increases and stabilizes the hydroxylating activity. In aerated columns, it is possible to obtain continuous hydroxylation of IAA, at a concentration of 0.5 g/l, by the immobilized mycelium of A. niger. The yield of hydroxy derivatives reached 70%, the activity remaining unaltered during 15 days' operation of the column.     

具有乙酰胆碱酯酶抑制活性的蛇足石杉内生真菌分子鉴定

在马铃薯葡萄糖液体培养基(PDB)、沙氏液体培养基(SDB)和查氏液体培养基(CDB)中发酵培养蛇足石杉内生真菌菌株SF88和LF52,乙酰胆碱酯酶(AChE)抑制活性检测结果表明,菌株SF88和LF52在PDB、SDB和CDB发酵后菌体总生物碱对AChE活性的抑制率分别为45.6%和56.7%、56.3%和68.5%、40.8%和48.3%。对这两株不产孢内生真菌的DNA ITS、18S rDNA、28S rDNA、TUB和RPB进行了测序,系统发育分析表明,SF88和LF52为Phaeosphaeriaceae科中一未知种,为开发生物碱类乙酰胆碱酯酶抑制剂(AChEI)药物提供了新的潜在菌种资源。     

Production of vitamin B12 by immobilized cells of a propionic acid bacterium

Summary The ability of immobilized cells of propionic acid bacteria to form vitamin B₁₂ has been investigated. Propionibacterium arl AKU 1251 having a considerable activity to produce the vitamin was selected as a test organism among six strains of propionic acid bacteria tested. The whole cells were entrapped with urethane prepolymers, photo-crosslinkable resin prepolymers or several other materials such as -carrageenan, agar or sodium alginate, and their vitamin B₁₂ productivity was compared. Based on the criteria of the convenience of preparation and the stability of the cell-entrapping gels, a hydrophilic urethane prepolymer, PU-9, was employed as gel material. Satisfactory vitamin B₁₂ production was obtained when 5–10 g of wet cells precultured to the late exponential growth phase were entrapped with 1 g of the prepolymer. Addition of a suitable amount of cobaltous ion and of 5,6-dimethyl benzimidazole to the culture medium was effective for the production of the vitamin by the immobilized cells. The repeated use of the immobilized cells was successfully achieved when a suitable amount of cells were entrapped and allowed the proliferation of cells inside gel matrices.     

Transformation of steroids by fungal protoplasts

Summary Protoplasts of Cunninghamella elegans transformed cortexolone to the same products as did the mycelium. Transformation of the steroid by non-induced mycelium and by protoplasts released from it was almost completely inhibited by cycloheximide. However, hydroxylation of cortexolone was not affected by this antibiotic if mycelium grown in the presence of an enzyme inducer or protoplasts obtained from the induced mycelium were used. The transformation rate of protoplasts, on the basis of dry weight or protein units, was about four times higher than that of the mycelium, indicating that the mycelial cell wall was a serious rate-limiting factor in steroid bioconversion.     

Production of acid proteinases by Humicola lutea mycelium immobilized in polyurethane sponge.

Humicola lutea 120-5 spores were entrapped in polyurethane sponge cubes and were cultivated inside the carrier to form an immobilized mycelium further used for production of acid proteinases in batch mode. A carrier—spore suspension ratio of 10:0.5 (wt) should be used to obtain optimal results. The polyurethane sponge-immobilized mycelium could be applied repeatedly, the enzyme activity secreted during the first 10 cycles being about the same as that produced by free cells. The advantages of immobilizing fungal cells by germinating conidia entrapped inside the supporting material are discussed.     

Use of surface-immobilized Trichoderma in batch and fed-batch fermentations

Needle-punch polyester was shown to be an effective support material for the immobilization of Trichoderma reesei Rut C30. When used as a resident inoculum for a batch process, the immobilized Trichoderma was very stable and resulted in a reduced rate of biomass generation in the bulk liquid phase as compared to cultures inoculated with free mycelium. Fed-batch fermentations with the immobilized Trichoderma produced ca. 80% of the activity of those using free cells; however, the activity was more stable and the crude enzyme broth produced had a greatly reduced biomass concentration.     

Acid proteinases production by humicola lutea cells immobilized in polyhydroxyethylmethacrylate gel

The spores of Humicola lutea entrapped in polyhydroxyethylmethacrylate gel were precultivated in production medium for mycelial formation. The immobilized mycelium was reused in batch mode for acid proteinases production. The influence of precultivation time, initial inoculum gel volume, and gel particle size on the enzyme activity and proteinases production half-life were studied. After 70 h precultivation of the entrapped spores (10 ml initial inoculum volume, 12–27 mm³ gel particle size) maximum proteinases activity of 100–140% (compared with free cells) was registered in 15 reaction cycles. Under the same condition the half-life time was 18 cycles, while for the free cells it was 5 cycles. The main advantage of the polyhydroxyethylmethacylate immobilized H. lutea was the long acid proteinases production half-life at a low concentration of outgrowing cells in the medium.     

Adenylate energy charge and the steroid 11 alfa-hydroxylase activity in Monosporium olivaceum during growth and starvation.

During growth of Monosporium olivaceum its energy charge, E.C., (i.e. the adenylates ratio ATP + 0.5 ADP/ATP + ADP + AMP) increased from an initial value of 0.59 up to 0.85 after 25 hr of growth and then decreased to 0.51. The increase of energy charge was followed by the decrease of the activity of the 11 alpha-hydroxylase of cortexolone. This occured very clearly in the starved mycelium. Highest hydroxylation activity was observed when the lowest E.C. level (0.39-0.33) was reached.     

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

Mortierella isabellina 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 greater than 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.