Simple alkyl ester derivatives of restaurant grease were prepared using immobilized lipases as biocatalysts. The lipases studied included those of Thermomyces lanuginosa and Candida antarctica supported on granulated silica (gran- T.l. and gran- C.a., respectively), C. antarctica supported on a macroporous acrylic resin (SP435) and Pseudomonas cepacia immobilized within a phyllosilicate sol-gel matrix (IM PS-30). All alcoholysis reactions were carried out in solvent-free media employing a one-step addition of the alcohol to the reaction system. Of the lipases studied, IM PS-30 was found to be the most effective in catalysing the methanolysis and ethanolysis of grease. The processes catalysed by gran- T.l. and gran- C.a. lipases gave poor conversions to esters, and the SP435-catalysed reactions gave intermediate yields of ethyl and methyl esters. Water activity (a(w)) was an important factor in the methanolysis reactions; reaction media with a(w)<0.5 resulted in the highest conversions to methyl esters. Molecular sieves also improved methyl ester yields by as much as 20% in transesterification reactions catalysed by IM PS-30. The immobilized lipases also were evaluated for their ability to produce alkyl esters of grease with several additional normal and branched-chain alcohols.

Ascorbic acid (AA), dehydroascorbic acid (dehydroAA), isoascorbic acid (isoAA), ascorbic acid-Z-phosphate @A-2-PO& and ascorbic acid-2-sulfate (AA-2-SOJ were tested as inhibitors of mushroom polyphenoloxidase (PPO). Kinetic analysis indicated that AA and isoAA were more effective than dehydroAA. The half times (tr,J that decreased 50% of PPO activity for AA, isoAA and dehydroAA were 2.5,3.1, and 1.9 hr, respectively, and the concentrations that inhibited half of PPO activity were as follows: ascorbic acid, 0.04 mM, isoAA, 0.25 mM; and dehydroAA, 7.5 mM. Electron spin resonance studies demonstrated that the Cuz+ of PPO was reduced to Cu+ by AA. AA-2-PO, and AA-2-SO4 were not inhibitors for PPO. However, the digestion of AA-2-PO4 with acid phosphatase yielded AA to inhibit PPO activity. AA-2-SO4 was found to be a poor substrate for sulfatase.

The lipase-catalyzed synthesis of alkyl esters from tallow and grease using Pseudomonas cepacia lipase (PS-30) immobilized within a phyllosilicate sol-gel matrix was investigated. The effects of the presence of alcohol and of the amount of enzyme used were studied. The matrix-immobilized PS-30 lipase effectively converted grease and tallow to ethyl esters in greater than 95% yield when using ethanol. The final conversion of grease or tallow to alkyl esters was aided by the addition of molecular sieves (0.4 wt% of substrates) to the reaction mixture. The matrix-immobilized PS-30 enzyme was easily recovered and could be reused at least five times without losing its activity. Accordingly, the phyllosilicate sol-gel immobilized PS-30 lipase is potentially useful for the economic production of biodiesel fuel.Paper no. J9788 in JAOCS 78, 585-588 (June 2001). FIG. 4. Reusability of free and matrix-immobilized lipase PS-30 in the synthesis of alkyl esters of grease. Reactions were conducted with grease (0.2 mmol), 95% ethanol (0.8 mmol), and matrix-immobilized lipase PS-30 (150 mg) or free lipase (10 mg) at 50°C for 18 h. At the end of each cycle, the enzyme was recovered, washed with hexane, and dried. Fresh substrates were added to start the next reaction: free lipase PS-30 (I), immobilized lipase PS-30 (I I). For manufacturer see Figure 1.

Incubation of mouse myeloma microsomes with GDP-[14Cjmannose results in the biosynthesis of ['4Clmannose phosphoryl dolichol [Baynes, J. W., Hsu, A.-F. & Heath, E. C. (1973) J. Biol. Chem. 248, and a The mouse myeloma tumor, MOPC-46B, synthesizes a kappatype immunoglobulin light chain. This light chain is a glycoprotein of approximately 25,000 molecular weight with a single carbohydrate side chain attached to the peptide chain by an N-glycosidic linkage between N-acetylglucosamine (GlcNAc) and asparagine residue 34 of the protein (1). We recently reported (2) that microsomal preparations from this myeloma tumor catalyze the biosynthesis of mannosylphosphoryl-dolichol (Man-P-Dol), and that this mannolipid serves as a donor of mannosyl residues in the glycosylation of

A novel procedure is described for immobilizing a lipase from Pseudomonas cepacia (PS-30) within a phyllosilicate sol-gel matrix. The method is based on cross-linking a phyllosilicate clay with silicate polymers produced by the controlled hydrolysis of tetramethyl orthosilicate (TMOS). The activity of the phyllosilicate sol-gel-immobilized lipase was dependent upon the type of alkylammonium salt, inorganic catalyst and volume ratio of phyllosilicate clay to TMOS used. Lipase PS-30 immobilized in this way was more stable and had higher activity compared with the free lipase. Studies on the lipase-catalysed esterification of lauric acid with octan-1-ol in iso-octane showed that under controlled water activity conditions the phyllosilicate sol-gel-immobilized lipase had better activity compared with other supported lipase preparations. In addition, the phyllosilicate sol-gel-immobilized lipase was reusable for at least five esterification cycles without significant loss of activity.

Proton transport by the nitrate-insensitive, vanadate-sensitive ATPase in KI-washed microsomes and reconstituted vesicles from maize (Zea mays L.) roots was followed by changes in acridine orange absorbance in the presence of either KNO3 or KCI. Data from such studies obeyed a kinetic model in which net proton transport by the pump is the difference between the rate of proton transport by the action of the ATPase and the leak of protons from the vesicles in the direction opposite from the pump. After establishing the steady state proton gradient, the rate of return of transported protons was found to obey first-order kinetics when the activity of the ATPase was completely and rapidly stopped. The rate of return of these protons varied with the quencher used. When the substrate Mg:ATP was depleted by the addition of either EDTA or hexokinase, the rate at which the proton gradient collapsed was faster than when vanadate was used as the quencher. These trends were independent of the anion accompanying the K and the transport assay used.Membranes from maize roots have been shown to contain at least two proton transporting ATPases (6,28). One ofthese pumps is localized on the tonoplast membrane and is similar to other vacuolar type ATPases being inhibited by NEM and nitrate, but insensitive to vanadate (7,28). The other pump is believed to be localized on the plasma membrane and similar to other E1-E2 type ATPase in forming an aspartyl phosphate intermediate, being sensitive to vanadate and utilizing Mg-ATP as substrate (1,4,6,7,25,28). Transport ATPases of the El -E2 type have been shown to exist in at least two different conformational states depending on the ligands bound to the enzyme (1, 25). These conformational states have been deduced by changes in susceptibility to proteolytic degradation ( 19 and references cited therein) and fluorescence of aromatic amino acids within the protein (14,16) and covalently bound probes (15). It has been postulated that the changes in protein structure are essential for ion movement (25), because the conformation of the E 1-E2 type ATPase is affected by binding of transported cation (14).Characterization of proton transport by the vanadate-sensitive pump from maize roots has been slowed because of difficulties in purifying plasma membranes with competent transport activities. Problems in isolating these membranes arise from the abundance of proteases in membrane fractions (8) and the presence of lipolytic activities which affect membrane integrity (3). Recent advances in purification of membranes from roots have allowed isolation of vesicles with vanadate-sensitive proton transport (6, 7, 10). Additionally, several reconstitution protocols have been developed to insert the vanadate-sensitive ATPase into liposomes (3,26).In a recent article (28), a kinetic model for describing proton transport by the tonoplast ATPase was proposed. This model quantifies the overall process of proton transport by simultaneously considering the pumping and the leakage of protons from membra...

The continuous production of ethyl esters of grease using a phyllosilicate sol-gel immobilized lipase from Burkholderias cepacia (IM BS-30) as catalyst was investigated. Enzymatic transesterification was carried out in a recirculating packed-column reactor using IM BS-30 as the stationary phase and ethanol and restaurant grease as the substrates without solvent. The bioreactor was operated at various temperatures (40-60°C), flow rates (5-50 mL/min), and times (8-48 h) to optimize ester production. Under the optimal operating conditions (flow rate, 30 mL/min; temperature, 50°C; mole ratio of substrates, 4:1 ethanol/grease; reaction time, 48 h), the ester yields were >96%. The IM BS-30 could be reused in the reactor for continuous ester production. The conversion of grease to ester was monitored by HPLC and GC, whereas free and total glycerol content in the product was determined by GC. Either water-washing or silica column chromatography reduced the free and total glycerol content of the ethyl ester preparation to acceptable levels.

The extent of enzymatic browning at cut surfaces of Atlantic potato, its siblings and parents, and Russet Burbank was investigated. Browning, measured by tristimulus colorimety, was compared with cultivar variation in polyphenol oxidase (PPO), PPO substrates, and ascorbic acid. Atlantic potato was much less subject to browning at cut and peeled surfaces than Russet Burbank. Browning in Atlantic potato could be almost eliminated by dipping in water. Belchip and Chipbelle (siblings) and Wauseon and Lenape (parents) were similar to Atlantic potato in browning behavior. The tendency to brown in these cultivars and Russet Burbank was correlated with total phenolic compounds, tyrosine, and to a lesser extent, PPO activity.