Annikka Mustranta

Use of lipases in the resolution of racemic ibuprofen

SummaryResolution of (R,S)-ibuprofen enantiomers by esterification in different organic solvents was studied using Candida cylindracea lipase. This enzyme preparation had high enantiospecificity for S(+)-ibuprofen in the esterification reaction of a racemic ibuprofen with primary alcohols. The esterification yields of secondary alcohols were much lower than those of primary alcohols. Esterification with tertiary alcohols was not observed. The synthesis of esters was profoundly affected by the amount of water in the reaction mixture. C. cylindracea lipase was active only in very hydrophobic solvents. The esterification activity of the lipase was reduced significantly by addition of water. The R- and S-enantiomers of ibuprofen were determined without derivatization by HPLC using a chiral column.

Reactivity of Trametes laccases with fatty and resin acids.

Abstract. Lipophilic extractives commonly referred to as wood pitch or wood resin can have a negative impact on paper machine runnability and product quality. The lipophilic extractives are composed mainly of fatty acids, resin acids, sterols, steryl esters and triglycerides. In this work, the suitability of laccases for the modification of fatty and resin acids was studied, using two model fractions. In the treatments, resin and fatty acid dispersions were treated with two different laccases, i.e. laccases from Trametes hirsuta and T. villosa. Different chromatographic methods were used to elucidate the effects of laccase treatments on the chemistry of the fatty and resin acids. Both laccases were able to modify the fatty and resin acids to some extent. In the case of fatty acids, a decrease in the amount of linoleic, oleic and pinolenic acids was observed, whereas the modification of resin acids resulted in a reduced amount of conjugated resin acids.

Comparison of lipases and phospholipases in the hydrolysis of phospholipids

Abstract Two commercial fungal lipase preparations from Aspergillus niger and Penicillium cyclopium , two fungal phospholipases from A. niger (A 1 and A 2 ) and one phospholipase from porcine pancreas (A 2 , Lecitase) were compared in the hydrolysis of soybean phospholipids. The isoelectric points, pH and temperature optima and temperature stabilities of the phospholipases in these enzyme preparations were also determined. Lipase preparations which also contained phospholipase and lysophospholipase activities were more efficient than phospholipase preparations in the hydrolysis of soybean phospholipids. Phospholipase A 1 and lipase preparations released fatty acids from both positions of the substrate molecule and as a result glyceryl-phosphoryl-choline was formed. Both the A 2 phospholipase preparations tested produced lysophospholipids through partial selective hydrolysis of phospholipids.

Modification of spruce lignans with Trametes hirsuta laccase

Summary The effect of Trametes hirsuta laccase on isolated spruce wood lignans was evaluated. Lignans were isolated from the heartwood of spruce branches and treated with different laccase dosages and treatment times. The effect of the treatment was monitored by gas chromatography, size exclusion chromatography and ionization difference UV spectroscopy. Lignans were efficiently oxidized by T. hirsuta laccase. About half of the phenolic groups present in lignans remained intact during the treatment. The oxidation of phenolic groups in lignans produced oligomeric structures containing approximately 4–5 lignan units (i.e., 8–10 phenyl propane units). Precipitation of the formed oligomeric structures probably prevented further polymerization.

Transesterification of soy lecithin by lipase and phospholipase

Soy lecithin was modified by enzymatic transesterification in a solvent-free system. 1,3-SpecificRhizomucor miehei lipase was found to be efficient in the transesterification with lauric acid and oleic acid, where oleic acid was more incorporated into soy lecithin. Phospholipase A2 incorporated lauric acid hardly at all, but it hydrolyzed lecithin efficiently. The mixture of lipase and phospholipase A2 (1:1, w/w) incorporated lauric acid to the same extent as did 1,3-specific lipase alone at the same total enzyme concentration. The main fatty acids replaced were palmitic and linoleic acids by 1,3-specific lipase and its mixture with phospholipase A2, and linoleic and linolenic acids by phospholipase A2 alone, suggesting an improved oxidative stability of the resulting product. Hydrolysis could not be prevented, but it could be regulated by incubation time and by enzyme dosage. The minimal water content for significant incorporation of lauric acid into lecithin was below 0.5% of the weight of the reaction mixture.

Transesterification of phospholipids in different reaction conditions

Transesterification of synthetic dimyristoyl phosphatidylcholine with oleic acid by commercial lipase preparations fromAspergillus niger andRhizomucor miehei was studied in the presence and absence of solvent. A high-performance liquid chromatography method for determination of the modified phosphatidylcholine was developed. Under solvent-free conditions, transesterification could be carried out as efficiently as in toluene, and the degree of hydrolysis was lower than in toluene. Transesterification was influenced by the water content as well as by the fatty acid concentration in the reaction mixture. The optimum water content for transesterification in solvent-free reaction medium was higher than in toluene with both lipases. The yield of modified phosphatidylcholine increased, and the degree of hydrolysis decreased with increasing fatty acid concentration. The maximum yield of modified phospha-tidylcholine, 35% of the original phospholipid, was obtained withR. miehei lipase.

Enzymatic Hydrolysis of Oat and Soya Lecithin: Effects on Functional Properties

Enzymatic hydrolysis of oat and soy lecithins and its effects on the functional properties of lecithins were investigated. The phospholipase used was most efficient at low enzyme and substrate concentrations. More fatty acids were released from soy lecithin than from oat lecithin. The maximum degree of hydrolysis was 760 μmol free fatty acids per gram soy lecithin and 170 μmol free fatty acids per gram oat lecithin. On the basis of the total carbohydrate and phosphorus contents in the polar fractions of the lecithins, oat lecithin contained more glycolipids and less phospholipids than soy lecithin. With regard to functional properties, the stability of oil-in-water emulsions was enhanced by hydrolyzed soy lecithin and by crude and hydrolyzed oat lecithins, but only hydrolyzed soy lecithin prevented the recrystallization of barley starch. The dissociation enthalpy of amylose-lipid-complex (AML-complex) was significantly higher when hydrolyzed soy lecithin was present. Hydrolyzed oat lecithin slightly affected the dissociation enthalpy of AML-complex. The other lecithins had no effect on recrystallization or dissociation enthalpies in the barley-starch matrix.

Enzymatic Hydrolysis of Barley Starch Lipids

ABSTRACT The efficiency of phospholipase and lipase preparations in the hydrolysis of lysophospholipids of native and gelatinized barley starch was examined. The degree of hydrolysis was analyzed by determination of the amount of released fatty acids by an enzymatic method. Thermal and structural properties of the enzyme-treated starch were studied by differential scanning calorimetry and light microscopy. Lysophospholipids of the gelatinized barley starch were easily hydrolyzed, in contrast to the lipids of the granular starch. The maximum degree of hydrolysis achieved for the gelatinized starch was 80% and for the native starch ≈20%. Gelatinization enthalpies and micrographs indicated that even though the amount of the released fatty acids from the native starch was small, formation of free fatty acids inhibited swelling and gelatinization of starch granules.

Production of peroxidase by Inonotus weirii

SummaryThe white rot fungus Inonotus weirii produced an extracellular peroxidase which was excreted in association with cell growth and in the absence of an inducer. Production of peroxidase was greatly influenced by the carbon and nitrogen source. The highest activities were obtained on glucose-and xylose-based media containing a combination of ammonium nitrate, yeast extract and distillers spent grain as nitrogen source. The enzyme produced had a molecular weight of 42000, was stable in the pH range 3–8 at room temperature and had optimal activity at pH 3. The fungus Inonotus weirii could be a potential producer of peroxidase for industrial applications in spite of its rather slow production rate.

Acid Hydrolysis of Sunflower Seed Husks for Production of Single Cell Protein

SummarySunflower seed husks were chosen as a typical lignocellulosic waste product of low value. This model substrate was hydrolyzed with sulphuric acid at 120°C. The hydrolysis was carried out in two steps: hydrolysis of the pentosan fraction and subsequent hydrolysis of the cellulose fraction. The pentosan fraction was nearly quantitatively hydrolyzed. For the cellulose hydrolysis the yield was 79% of the theoretical yield. The hydrolyzates were neutralized to pH 5 with solid calcium hydroxide and used for preparation of growth media forCandida yeasts andPaecilomyces variotii. For the pentosan hydrolyzates the yields of yeast biomass were 35–36 g per 100 g available reducing sugars (supplied to the medium). In cellulose hydrolyzates the corresponding yields were 45–48 g withCandida utilis andC. tropicalis and about 30 g withC. pseudotropicalis. P. variotii was noticeably superior to the yeasts. In pentosan hydrolyzates it produced 63 g dry mycelium from 100 g reducing sugars supplied; in cellulose hydrolyzates, 94 g. This suggests that it must be an effective utilizer of a wide range of compounds, for example, organic acids in the medium.