Characterization of <Emphasis Type="Italic">Mucor miehei</Emphasis> lipase immobilized on polysiloxane-polyvinyl alcohol magnetic particles

Mucor miehei lipase was immobilized on magnetic polysiloxane-polyvinyl alcohol particles by covalent binding. The resulting immobilized biocatalyst was recycled by seven assays, with a retained activity around 10% of its initial activity. K_m and V_max were respectively 228.3 M and 36.1 U mg of protein^–1 for immobilized enzyme. Whereas the optimum temperature remained the same for both soluble and immobilized lipase (45 °C), there was a shift in pH profiles after immobilization. Optimum pH for the immobilized lipase was 8.0. Immobilized enzyme showed to be more resistant than soluble lipase when assays were performed out of the optimum temperature or pH.     

Incorporation of omega-3 polyunsaturated fatty acids into soybean lecithin: effect of amines and divalent cations on transesterification by lipases

The transesterification of soybean lecithin with methyl esters of EPA and DHA in an organic solvent (hexane) using various commercially available lipases was studied. Lipases produced by Candida antarctica, Pseudomonas fluorescens, Burkholderia cepacia, Mucor miehei, Thermomyces lanuginosus and Rhizomucor miehei were compared, in the absence or presence of histidine, arginine, urea, Ca²⁺, Mg²⁺, or a combination of urea and divalent cations (additives at 5 % of the total lipid mass). Transesterification using the R. miehei enzyme reached 11.32 and 12.30 % in the presence of Ca²⁺ or Mg²⁺ respectively, and 8.58 and 9.31 % when urea was also added. These were the greatest degrees of transesterification obtained. The results suggest the potential use of this immobilized lipase as a catalyst for interesterification reactions in organic solvent systems with low water content.     

Enzymatic synthesis of an acylated phloroglucinol derivative with phloroglucinol and vinyl octanoate in acetonitrile

Monooctanoyl phloroglucinol with anti-microbial activities was synthesized from phloroglucinol (50 mM) and vinyl octanoate (150 mM) with agitation at 40 °C using 10 mg lipase AK (Pseudomonas fluorescens) in acetonitrile (1 ml, aw=0.07). The yield was approximately 70% as conversion rate of phloroglucinol over 3 days. The product, at 205 g ml^–1, was bacteriocidal against Escherichia coli and Staphylococcus epidermidis.     

PARTIAL PURIFICATION AND CHARACTERIZATION OF THE ORGANIC SOLVENT-TOLERANT LIPASE PRODUCED BY Pseudomonas fluorescens RB02-3 ISOLATED FROM MILK

Eighty-five putative Pseudomonas isolates were obtained from various raw milk and pasteurized milk samples using Pseudomonas CFC agar. Among them, 36 isolates were identified as Pseudomonas fluorescens, and one isolate was identified as Pseudomonas putida. Lipase activity of the strains was quantitatively measured by the spectrophotometric method using p-nitrophenyl palmitate (p-NPP) as substrate. Detected lipase activity of the strains was between 10.03 U/mL and 22.16 U/mL. Pseudomonas fluorescens RB02-3 possessed the highest lipase activity. The extracellular lipase of P. fluorescens RB02-3 strain was homogeneously purified using a combination of ammonium sulfate precipitation, dialysis, and gel filtration column chromatography. This purification procedure resulted in 2.97-fold purification with 20.3% recovery. The enzyme was characterized, and exhibited maximum activity at pH 7.0 and 50°C; after it was incubated for 1 h it was activated in the presence of hexane, ethyl acetate, isopropanol, and ethanol and remained stable after the incubation was extended for 2 hr. The lipase was slightly inhibited in the presence of Zn²⁺, Co²⁺, Cu²⁺, Ni²⁺ salts, and ethylenediamine tetraacetic acid (EDTA), whereas Cd²⁺, sodium dodecyl sulfate (SDS), and Tween-80 had no effect on its activity.     

Assessment of a chemostat-coupled modified Robbins device to study biofilms

The combination of a modified Robbins device (MRD) attached to the effluent line of a continuous cultivation vessel was assessed by the adhesion of planktonic bacteria maintained at a controlled growth rate. This combination of a chemostat and an MRD provides a large number of sample surfaces for monitoring both the formation and control of biofilms over extended periods of time. This apparatus was used to monitor the colonization of two soil isolates,Pseudomonas fluorescens (EX101) andPseudomonas putida (EX102) onto silastic rubber surfaces. At a similar _rel, both bacteria attached to the silastic, howeverP. fluorescens formed confluent, dense biofilms in less than 24 h, whereasP. putida adhered as single cells or microcolonies after the same period. The metabolic activity, measured by INT-formazan formation, was similar for both organisms with a peak at 6 h of colonization and a subsequent decrease after 24 h. Long term colonization studies ofP. fluorescens produced a population of greater than 9.5 log cfu cm^–2 at 28 days demonstrating the advantages of the chemostat-MRD association. This technique proved to be successful for studying bacterial adhesion and biofilm formation in tubular devices by bacterial populations at controlled and low growth rates.     

Construction of a new host-vector system inArthrobacter sp. and cloning of the lipase gene

Arthrobacter sp. strain MIS38 was transformed with a shuttle vector containing the kanamycin resistant genekan (derived from Tn5) by an electroporation method. This shuttle vector is fromBrevibacterium lactofermentum andEscherichia coli, pULRS8: - The following optimal condition of electroporation was determined. A square wave pulse of 1 kV/cm electric field strength for 0.5 ms duration yielded 3 × 10⁵ transformants/,g plasmid DNA. The number of transformants increased with the amount of DNA over the range 0.01-5 g. This host-vector system was then used successfully to clone and express a lipase gene fromArthrobacter sp. strain MIS38 into bothArthrobacter sp. MIS38 and E. coli JM109.     

Ester synthesis from α-substituted carboxylic acid catalyzed by polyethylene glycol-modified lipase fromCandida cylindracea in benzene

Summary Lipase fromCandida cylindracea was coupled with polyethylene glycol(PEG). In contrast to the previously used lipase fromPseudomonas fluorescens, the modified lipase catalyzed the ester synthesis in benzene at 25°C from short-chain alcohols and - or -substituted carboxylic acid. Using the modified lipase, following esters were synthesized; pentyl -methylpentanate, methyl benzoate, and methyl retinoate.     

Variables that Affect Immobilization of Mucor Miehei Lipase on Nylon Membrane

Nylon membrane was used to immobilize Mucor miehei lipase. Variables that affect this immobilization procedure were studied by experimental design. A 2³ full factorial design was employed for this purpose. The protein retention and hydrolytic activity of the immobilized lipase were used as response variables. The rapid loss of enzyme activity was the main problem during repetitive use. Two strategies were used to improve the low operational stability: nylon treated with HCl and nylon coated with polyvinyl alcohol (PVA). Lipase-nylon-PVA was the best enzyme derivative, allowing performance of five consecutive assays, with a retained activity of 0.5 U mg of protein⁻¹ g of support⁻¹.     

Synthesis of monoglyceride containing omega-3 fatty acids by microbial lipase in organic solvent

Summary An enzymatic method for synthesis of monoglyceride from 1,2-isopropylidene glycerol and n-3 polyunsaturated fatty acid concentrate was investigated in organic solvent. Optimal reaction conditions for monoglyceride synthesis by lipase were established. Lipase IM-60 fromMucor miehei produced yields of monoglyceride of up to 80% in this system. The resultant monoglyceride contained 76.2% n-3 polyunsaturated fatty acid (eicosapentaenoic acid 43.3%; docosahexaenoic acid, 32.7%). Isooctane and hexane were suitable organic solvents for monoglyceride synthesis and optimal initial water content was 2.5%. Lipase IM-60 was relatively stable in organic solvent and is easily recovered for reuse.     

Coordination of the dynamics of yeast sphingolipid metabolism during the diauxic shift

Background

During the development of an enantioselective synthesis using the lipase from Mucor miehei an unusual reaction course was observed, which was analyzed precisely. For the first time an allosteric modulation of a lipase changing its selectivity was shown.

Theory

Considering the biological relevance of the discovered regulation mechanism we developed a theory that describes the regulation of energy homeostasis and fat metabolism.

Conclusion

This theory represents a new approach to explain the cause of the metabolic syndrome and provides an innovative basis for further research activity.     

Thermophilic mycoflora of cigarettes and cured tobacco leaves

Nine species of thermophilic fungi were obtained from retailed packets of imported and locally manufactured brands of cigarettes and from cured tobacco leaves. They include known human pathogens such asThermoascus aurantiacus Miehesensu Apinis,Mucor pusillus Lindt andMucor miehei Cooney and Emerson. Chaetomium thermophile La Touche,Humicola insolens Cooney and Emerson,Thermoascus crustaeus (Apinis and Chesters) Stolk andMucor miehei have not been previously reported on tobacco products.Fewer species were obtained from foreign cigarettes than from locally manufactured brands. A mean moisture content of 19% was recorded for imported cigarettes and 37% for Nigerian brands. The potential health hazards posed to man by the unrestricted use of tobacco products are discussed.     

Immobilisation ofCandida cylindracea lipase on a new range of ceramic supports

Summary Lipase fromC. cylindracea was covalently immobilised to a number of surface-treated ceramic supports (3–10 mg. (g dry wt support)^–1). At room temperature, the immobilised lipase could convert R,S-citronellol and butyric acid to citronellyl butyrate at rates in the range 7–51 mol. (mg lipase.min)^–1. The lipase maintained 90–100% of its initial activity over a period of 150 days.     

Overexpression of a thermostable lipase gene from Pseudomonas fluorescens in Escherichia coli

Summary A thermostable lipase gene from Pseudomonas fluorescens SIK W1 was overexpressed in Escherichia coli BL21 using expression vector pTTY2. The amount of lipase produced by E. coli BL21 with pTTY2 was more than 40% of the total cell proteins when induced with isopropyl--d-thiogalactopyranoside. The lipase was produced as inclusion bodies in the cytoplasm of E. coli. They were solubilized by 8 m urea and refolded into biologically active form. The refolded lipase showed high thermostability; the time required for 90% inactivation of the enzyme (D-value) was 4 h at 95°C and the increment of temperature to reduce heating times by 90% (z _d value) was 76°C.Offprint requests to: J. S. Rhee     

Significance of algal extracellular products to bacteria in lakes and in cultures

In simulated diurnal experiments withChlorella pyrenoidosa andPseudomonas fluorescens, bacterial growth was virtually confined to the daylight period and occurred at the expense of glycolate, the predominant extracellular product of the alga. Both glycolate levels and¹⁴C-DOC excretion rates were much lower in mixed algal-bacterial than in axenicChlorella cultures.This close coupling of bacterial growth to algal photosynthesis and extracellular release was also observed in Jack's Lake, but not in Lake Erie. Experimental enrichment with lake water particulates >30m suppressed the daytime growth of bacteria in Jack's Lake, but increased it dramatically in Lake Erie. Daylight doubling times for bacteria in lakewater ranged from 2 to 19 days. In mixed culture withChlorella, Pseudomonas had a doubling time of about 2 hours in the light.     

Comparison of lipase-catalysed esterification in supercritical carbon dioxide and in n-hexane

Summary Oleic acid esterification by ethanol has been performed by an immobilized lipase fromMucor miehei in supercritical carbon dioxide and in n-hexane as solvents. In both media, determination of apparent kinetic constants has been achieved and influence of water content has been shown to be different due to various rates of water solubilities. Stability of the lipase has been proved to be correct and similar in both solvents. Inhibition by ethanol excess has been found but is greater in n-hexane. That can explain the higher initial velocities obtained in supercritical carbon dioxide for the highest ethanol concentrations.     

Lipase catalyzed polytransesterification in an organic solvent

Summary Lipase-catalyzed polytransesterification ofbis(2,2,2-trifluoroethyl) dodecanedioate with aliphatic diols (from 1,2-ethanediol to 1,6-hexanediol) was studied with 4 enzymes and a number of solvents. The effects of experimental parameters were investigated with the purpose of obtaining a polyester of as high as possible average molar mass. The highest mass average molar mass (M _w) of 34,750 g mol^-1 (DP = 122) was obtained under vacuum with 1,4-butanediol,Mucor miehei lipase, and diphenyl ether as solvent.     

Lipase-catalyzed esterification of hydrophilic diols in organic solvents

Summary The direct, lipase-catalyzed esterification of hydrophilic diols in organic solvents was achieved by first adsorbing the hydrophilic, solvent immiscible substrate onto a solid support with high internal surface, namely silica gel and reacting the solid mixture with fatty acid vinyl esters in an appropriate organic solvent and in presence of an immobilized lipase fromMucor miehei (Lipozyme). Quantitative conversions of the acyl donors and very high reaction rates were observed in these transformations. Furthermore, mono- or diesters of these diols could be selectively produced by this method.     

Convenient lipase-assisted preparation of both enantiomers of suprofen,a non-steroidal anti-inflammatory drug

The resolution of rac-suprofen (1) catalysed by lipase in organic solvents was investigated. Direct esterification of rac-1 with methanol in dichorometane catalysed by Novozym® 435 furnished the pharmacologically active (+)-(S)-suprofen as unreacted product with excellent enantiomeric excess. The same procedure in toluene using Mucor miehei lipase adsorbed in Celite as catalyst afforded (−)-(R)-suprofen with good optical purity. © 1996 Wiley-Liss, Inc.     

Products of the oxidation ofn-decane byPseudomonas aeruginosa andMycobacterium rhodochrous

Pseudomonas aeruginosa andMycobacterium rhodochrous (both soil isolates) have been grown in a mineral salts medium containing hydrocarbon as the only carbon source. The fermentation products from decane withPseudomonas aeruginosa were 1-, 2-, 3-, and 4- and or 5-decanol and the corresponding ketones (no decanal was found) and decanoic, nonanoic, octanoic and heptanoic acid. This indicates that non-specific first oxidation occurs followed by seission of the decanone at the keto group. The products fromMycobacterium rhodochrous were 1-decanol,n-decanal, decanoic, octanoic, hexanoic, butyric and acetic acids, indicating that initial terminal oxidation is followed by-oxidation.     

Chromate resistance and reduction in Pseudomonas fluorescens strain LB300

Pseudomonas fluorescens LB300 is a chromateresistant strain isolated from chromium-contaminated river sediment. Chromate resistance is conferred by the plasmid pLHB1. Strain LB300 grew in minimal salts medium with as much as 1000 g of K₂CrO₄ ml^–1, and actively reduced chromate to Cr(III) while growing aerobically on a variety of substrates. Chromate was also reduced during anaerobic growth on acetate, the chromate serving as terminal electron acceptor. P. fluorescens LB303, a plasmidless, chromatesensitive variant of P. fluorescens LB300, did not grow in minimal salts medium with more than 10 g of K₂CrO₄ ml^–1. However, resting cells of strain LB303 grown without chromate reduced chromate as well as strain LB300 cells grown under the same conditions. Furthermore, resting cells of chromate-sensitive Pseudomonas putida strain AC10, also catalyzed chromate reduction. Evidently chromate resistance and chromate reduction in these organisms are unrelated. Comparison of the rates of chromate reduction by chromate grown cells and cells grown without chromate indicated that the chromate reductase activity is constitutive. Studies with cell-free extracts show that the reductase is membrane-associated and can mediate the transfer of electrons from NADH to chromate.