Altered acyl chain length specificity of Rhizopus delemar lipase through mutagenesis and molecular modeling

The acyl binding site of Rhizopus delemar prolipase and mature lipase was altered through site-directed mutagenesis to improve lipase specificity for short-or medium-chain length fatty acids. Computer-generated structural models of R. delemar lipase were used in mutant protein design and in the interpretation of the catalytic properties of the resulting recombinant enzymes. Molecular dynamics simulations of the double mutant, val209trp+phe112trp, predicted that the introduction of trp112 and trp209 in the acyl binding groove would sterically hinder the docking of fatty acids longer than butyric acid. Assayed against a mixture of triacylglycerol substrates, the val209trp+phe112trp mature lipase mutant showed an 80-fold increase in the hydrolysis of tributyrin relative to the hydrolysis of tricaprylin while no triolein hydrolysis was detected. By comparison, the val94Trp mutant, predicted to pose steric or geometric constraints for docking fatty acids longer than caprylic acid in the acyl binding groove, resulted in a modest 1.4-fold increase in tricaprylin hydrolysis relative to the hydrolysis of tributyrin. Molecular models of the double mutant phe95asp+phe214arg indicated the creation of a salt bridge between asp95 and arg214 across the distal end of the acyl binding groove. When challenged with a mixture of triacylglycerols, the phe95asp+phe214arg substitutions resulted in an enzyme with 3-fold enhanced relative activity for tricaprylin compared to triolein, suggesting that structural determinants for medium-chain length specificity may reside in the distal end of the acyl binding groove. Attempts to introduce a salt bridge within 8 Å of the active site by the double mutation leu146lys+ser115asp destroyed catalytic activity entirely. Similarly, the substitution of polar Gln at the rim of the acyl binding groove for phe 112 largely eliminated catalytic activity of the lipase.     

Optimization of critical medium components using response surface methodology for lipase production by Rhizopus delemar

The production process of a 1,3-position specific lipase from Rhizopus delemar was optimized by response surface methodology (RSM) and a Box–Behnken experimental design was used to study the interactive effects of fermentation medium components on lipase activity and microorganism growth. Preliminary batch tests were employed to obtain the favorable conditions for lipase activity analysis and found that sucrose, molasses, yeast extract, sunflower oil, tween-80 have significant influences on the lipase production and microorganism growth. The concentrations of five fermentation medium components were optimized. Among five variables, molasses sucrose and yeast extract were identified as less significant variables for lipase production. The optimum fermentation medium composition for lipase production by R. delemar was sucrose concentration 4.19 g/L, molasses sucrose 1.32 g/L, yeast extract 0.53 g/L, sunflower oil 1.11% (v/v), and tween-80 1.80% (v/v). In these conditions, the biomass concentration of 4.52 g/L with a lipolytic activity of 1585 μmol/L min was reached.     

Purification and characterization of an extracellular lipase from the fungusRhizopus delemar

The complete purification and characterization of an extracellular lipase (acylglycerol acylhydrolase, EC 3.1.1.3) fromR. delemar is described. The final product was homogeneous as judged by electrophoresis in denaturing polyacrylamide gels and by isoelectric focusing, and was shown by means of an activity stain to be lipolytic. The purified enzyme had a monomer molecular weight of 30,300, an isoelectric point of 8.6, and approximately one monosaccharide moiety per molecule.N-Terminal sequence data (28 residues) and the amino acid composition of the lipase indicated that it corresponds to the product of a lipase-encoding cDNA previously isolated fromR. delemar. Optimal activity occurred between pH 8.0 and 8.5. The activity and stability of the enzyme were maximum at 30°C. Divalent cations were required for activity, with barium, calcium and manganese conferring maximum activity. Activation by calcium was maximal at and above 10 mM. The lipase was not inactivated by reducing agents, sodium fluoride or phenylmethlsufonyl fluoride. It was resistant toN-ethylmaleimide, and inactivated byp-chloromercuribenzoic acid in a manner which was not reversed by cysteine. Mention of brand or firm names does not constitute an endorsement by the U.S. Department of Agriculture over others of a similar nature not mentioned.     

Additive effects of acyl-binding site mutations on the fatty acid selectivity of Rhizopus delemar lipase

The fatty acid specificity and pH dependence of triacylglycerol hydrolysis by the Rhizopus delemar lipase acylbinding site mutant Val206Thr+Phe95Asp (Val, valine; Thr, threonine; Phe, phenylalanine; Asp, aspartic acid) were characterized. The activity of the double mutant prolipase was reduced by as much as 10-fold, compared to the wild-type prolipase. However, the fatty acid specificity profile of the enzyme was markedly sharpened and was dependent on the pH of the substrate emulsion. At neutral pH, strong preference (10-fold or greater) for hydrolysis of triacylglycerols of medium-chainlength fatty acids (C_8:0 to C_14:0) was displayed by the variant prolipase, with no hydrolysis of triacylglycerols of short-chain fatty acids (C_4:0 to C_6:0) and little activity manifested toward fatty acids with 16 or more carbons. At acidic pH values, the fatty acid selectivity profile of the double mutant prolipase expanded to include short-chain triacylglycerols (C_4:0, C_6:0). When assayed against a triacylglycerol mixture of tributyrin, tricaprylin and triolein, the Val206Thr+Phe95Asp prolipase displayed a high selectivity for caprylic acid and released this fatty acid at least 25-fold more efficiently than the others present in the substrate mixture. When presented a mixture of nine fatty acid methyl esters, the wild-type prolipase showed a broad substrate specificity profile, hydrolyzing the various methyl esters to a similar extent. Contrastingly, the double mutant prolipase displayed a narrowed substrate specificity profile, hydrolyzing caprylic methyl ester at nearly wild-type levels, while its activity against the other methyl esters examined was 2.5- to 5-fold lower then that observed for the wild-type enzyme.     

Purification of docosahexaenoic acid by selective esterification of fatty acids from tuna oil with Rhizopus delemar lipase

To purify docosahexaenoic acid (DHA), we attempted the selective esterification of fatty acids originating from tuna oil with lipases. Tuna oil was hydrolyzed in NaOH-ethanol solution, and the resulting fatty acid mixture [DHA, 23.2%; named tuna-free fatty acid (FFA)] was used as a starting material. Rhizopus delemar which acted lightly on DHA, was a suitable catalyst for the selective esterification of tuna-FFA, and lauryl alcohol was the best substrate. The reaction proceeded most effectively when a mixture of 2.4 g lauryl alcohol/tuna-FFA (2:1, mol/mol), 0.6 g water, and 600 U Rhizopus lipase was incubated at 30°C for 20 h with stirring at 500 rpm. Under these conditions 72% of tuna-FFA was esterified, and 84% of DHA was recovered in the unesterified fatty acid fraction. The DHA content in the fatty acid fraction rose from 23 to 73% with this reaction. To further elevate the DHA content, the unesterified fatty acids were extracted, and then esterified again under the same conditions. By this repeated esterification, DHA was purified to 89% with a recovery of 71% of its initial content.     

Production of structured lipids containing essential fatty acids by immobilizedRhizopus delemar lipase

An attempt was made to produce structured lipids containing essential fatty acid by acidolysis with 1,3-positional specificRhizopus delemar lipase. The lipase was immobilized on a ceramic carrier by coprecipitation with acetone and then was activated by shaking for 2 d at 30°C in a mixture of 5 g safflower or linseed oil, 10 g caprylic acid, 0.3 g water and 0.6 g of the immobilized enzyme. The activated enzyme was transferred into the same amount of oil/caprylic acid mixture without water, and the mixture was shaken under the same conditions as for the activation. By this reaction, 45–50 mol% of the fatty acids in oils were exchanged for caprylic acid, and the immobilized enzyme could be reused 45 and 55 times for safflower and linseed oils, respectively, without any significant loss of activity. The triglycerides were extracted withn-hexane after the acidolysis and then were allowed to react again with caprylic acid under the same conditions as mentioned above. When acidolysis was repeated three times with safflower oil as a starting material, the only products obtained were 1,3-capryloyl-2-linoleoylglycerol and 1,3-capryloyl-2-oleoyl-glycerol, with a ratio of 86∶14 (w/w). Equally, the products from linseed oil were 1,3-capryloyl-2-α-linolenoyl-glycerol, 1,3-caprylol-2-linoleoyl-glycerol, and 1,3-capryloyl-2-oleoly-glycerol (60∶22∶18, w/w/w). All fatty acids at the 1,3-positions in the original oils were exchanged for caprylic acid by the repeated acidolyses, and the positional specificity ofRhizopus lipase was also confirmed to be strict.     

转盘反应器固定根霉重复批次发酵生产脂肪酶

以聚氨酯海绵为载体将根霉固定于转盘表面,利用转盘反应器实现脂肪酶的固定化发酵。考察了通气量、转速、装液量及转盘个数等操作参数对发酵过程的影响,得到了优化的反应操作条件,即通气量4 L·min^-1,转速100 r·min^-1,装液量3.5L,采用三个转盘。在上述条件下进行重复批次实验,可重复5批次, 单位时间产率10931.1 U·h^-1, 是批次反应的3.5倍。固定化细胞连续发酵,大大缩短了发酵的时间, 酶的平均活力和时间产率获得大幅提高。     

Biochemical and structural characterization of triacylglycerol lipase from Penicillium cyclopium

An extracellular lipase, active on water-insoluble triacylglycerols, has been isolated from Penicillium cyclopium. The purified enzyme has a molecular mass of 29 kDa by gel filtration and SDS-polyacrylamide gel electrophoresis. It hydrolyzes emulsions of tributyrin, trioctanoin, and olive oil at the same rate as pancreatic lipase and shows very low activity against partial acylglycerols (monooctanoin and dioctanoin) and methyl esters. It is stable at 35°C for 60 min and has maximal activity in a pH range of 8–10. Hydrolysis of triacylglycerols by P. cyclopium lipase is inhibited by detergents such as Triton X-100. Comparison of the sequence of the 20 first amino acid residues of P. cyclopium triacylglycerol lipase with other Penicillium lipases indicates a high homology with previously characterized lipases produced by P. expansum and P. solitum which are enzymes of comparable size and substrate specificity. Conversely, homology between P. cyclopium lipase and P. simplicissimum lipase, a nonspecific lipolytic enzyme, is low. Penicillium cyclopium triacylglycerol lipase shows no homology with P. camembertii lipase which is specific to monoacylglycerol and diacylglycerol.     

Alcoholysis of palm oil mid-fraction by lipase from Rhizopus rhizopodiformis

A mycelial lipase from Rhizopus rhizopodiformis was prepared in fragment form. The lipase was examined to catalyze the alcoholysis of palm oil mid-fraction (PMF) in organic solvents. High percentage conversions of PMF to alkyl esters were achieved when methanol or propanol was used as acyl acceptor. Of the two most prevalent fatty acids in PMF, palmitic acid seemed to be preferred over oleic acid in the formation of methyl and propyl esters. The optimal ratio of oil to methanol in the alcoholysis reaction is 1 to 2 moles. The lipase exhibited high alcoholysis activities in nonpolar solvents (log P>2), such as hexane, benzene, toluene, and heptane. The enzyme showed exceptionally high thermostability.     

Covalent immobilization and characterization of Rhizopus oryzae lipase on core-shell cobalt ferrite nanoparticles for biodiesel production

Rhizopus oryzae lipase (ROL) was immobilized on the surface of silica coated amino modified CoFe2O4 nanoparticles and applied for biodiesel production.The results indicated more affinity of the ROL toward its substrate upon immobilization,as revealed by a lower Km value for the immobilized ROL compared to its free counterpart.Intrinsic fluorescence spectroscopy indicated a lower intensity for ROL immobilized on CoFe2O4 nanoparticles.Besides,immobilized ROL steady state anisotropy measurements presented lower values,which implied assembly of ROL molecules on magnetic nanoparticles upon immobilization as well as their restricted rotation upon covalent attachment.Thermal stability analysis revealed improved activity at higher temperatures for the immobilized enzyme compared to its free counterpart.Accordingly,Pace analysis to determine protein thermal stability revealed preservation of the protein conformation in the presence of increasing temperatures upon immobilization on nanoparticles.Finally,ROL immobilized on CoFe2O4 nanoparticles exhibited improved efficiency of biodiesel production in agreement with thermal activity profile.Therefore,the authors suggest application of the lipase mole-cules immobilized on CoFe2O4 nanoparticles for more efficient biodiesel production.     

Lactic acid production by immobilized cells of the fungus Rhizopus oryzae with simultaneous product extraction

It is shown that is possible to obtain up to 15 g/l lactic acid in the organic phase within 25 h using Rhizopus oryzae fungus cells immobilized in a polyvinyl alcohol cryogel with simultaneous product extraction into octane with trioctylmethylammonium chloride as a lactate ion carrier.     

Preparation and characterization of PLT thick-films produced by a chemical route

Thick-films of lanthanum modified lead titanate ceramic (Pb_1−xLa_x)TiO₃ with x=0.10, 0.15 and 0.20 were prepared by a co-precipitation chemical process where oxide powders were used as precursors. The co-precipitated particles were dispersed in an organic vehicle in order to prepare the material for deposition. The films were deposited by dip coating on corning 7059 glass and chrome/nickel alloy substrates. The initially deposited films were amorphous and a heat treatment was necessary to crystallize them. Initially, the heat treatment was for volatilizing the organic material present in the films and in a second stage to crystallize them at higher temperatures. The crystallization of the films was monitored through X-ray diffraction analysis. The dielectric constant was measured at 1 kHz and the observed values were compared with those of bulk ceramic. The phase transition temperature of the films with x=0.20 was measured and the value observed agreed with those of bulk ceramic.     

Synthesis and characterization of porous media produced by a sol-gel method

Mesoporous silica materials were synthesized by sol-gel method using tetraethoxysilane (TEOS) as precursors and surfactants i.e., cetyltrimethyl ammonium bromide (CTAB), sodium dodecyl sulfate (SDS), and polyoxyethylene cetyl ether (Brij 56) as templates. Surfactant templates were completely removed by calcination to form mesoporous structure. The effects of type and amount of surfactants on the characteristics of samples were studied. The textural characteristics such as surface area, pore volume, pore size, and pore size distribution were determined by nitrogen sorption isotherms. Fourier transform infrared (FTIR) spectroscopy was employed to qualitatively identify the chemical functionality and to confirm the removal of surfactant template. Scanning electron microscope (SEM) and transmission electron microscope (TEM) were used to directly observe surface morphology and mesoporous structure, respectively. The adsorption capacity of the synthesized adsorbent for toluene vapor was examined. We found that the pore volume and pore size of mesoporous materials affected the adsorption capacity. The sample prepared with high content of CTAB under basic condition (pH ∼7) yielded large pore volumes and pore sizes and subsequently possessed the high adsorption capacity for toluene vapor.     

Characterization of membrane-bound lipase from a thermophilic Rhizopus oryzae isolated from palm oil mill effluent

The characteristics of the membrane-bound lipase from a thermophilic Rhizopus oryzae were studied. The pH and temperature optima for lipase activity were at 7.0 and 37°C, respectively. The enzyme was stable and acidic conditions, retaining more than 80% of its initial activity at pH 4.0 after 30 min incubation. It was stable up to 50°C with 70% of initial activity retained after 3 h incubation. The enzyme is 1,3 specific and exhibits substrate preference. Monoacid triglyceride substrates were hydrolyzed better than methyl esters, polyoxysorbitan and sorbitan substrates.     

The effect of immobilization on the hydrolytic/interesterification activity of lipase from Rhizopus niveus

Lipase from Rhizopus niveus was immobilized by physical adsorption on Celite 545 and glass beads. The results showed that the highest immobilization efficiency and specific hydrolytic activity of 96% and 9.2 meq/mg protein/min, respectively, were obtained with Celite as the carrier. However, the specific hydrolytic activity of lipase adsorbed on glass beads by acetone precipitation was similar to that obtained by the Celite carrier, although the protein loading capacity was relatively low. The results showed that lipase immobilized on glass beads exhibited similar activity profiles with respect to reaction time, different enzyme concentrations, and water content, using trimyristin and tripalmitin as substrates, to those obtained with the free enzyme. In contrast, the immobilized lipase on Celite exhibited a considerably lower hydrolytic activity. However, the results also showed that the lipase activities of the free enzyme and the immobilized Celite enzyme were similar when the more hydrophilic triolein was used as the substrate. The interesterification of a mixture of tripalmitin and trimyristin or triolein was carried out using both the free and immobilized enzymes. The results indicated that the hydrolytic activity of lipase was similar in both cases for the first 24 h, after which it decreased dramatically. These findings suggest that at this late stage an equilibrium between the hydrolytic and interesterification reactions was reached.     

Purification and characterization of a lipase fromNeurospora sp. TT-241

An extracellular lipase, which is produced by theNeurospora sp. TT-241 strain, grown on wheat bran at 30°C for 4 d, was purified 370-fold with an overall yield of 16%. The molecular weight was determined to be 55 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimal pH at 30°C and optimal temperature at pH 6.5 were 7 and 45°C, respectively. The lipase was stable in the pH range of 5 to 8, and it was temperature-sensitive. It was active on a wide range of natural substrates of either vegetable or animal origins and towardp-nitrophenyl esters, greatly favoring those containing C₄ acyl groups. It cleaved all of the ester bonds of triolein; however, the 1- or 3-ester bond was the preferred target. A complete inhibition by diisopropyl fluorophosphate suggested the presence of a serine residue at the active site. Partial inhibition was shown by either Hg²⁺ or chloramine T. Enzyme activity persisted in nonionic surfactants, a water-miscible solvent (dimethylsulfoxide), and a water-immiscible solvent (hexane).     

Synthesis and characterization of calcium zirconate nanofibers produced by electrospinning

Calcium zirconate fibers were produced by electrospinning and characterized in this work. The solution was prepared from zirconium and calcium salts, using polyvinyl-pyrrolidone (PVP) as processing aid. The decomposition of the organic fraction and crystallization of calcium zirconate were followed by thermogravimetry and differential scanning calorimetry (TG/DSC). Raman Spectroscopy was used to measure the vibrational modes in the green as well as in the calcined fibers. The final phase composition was studied by means of X-ray diffraction (XRD). The fiber morphology was investigated by confocal laser scanning microscopy (CLS) and scanning electron microscopy (SEM). The formation reaction of calcium zirconate was observed at about 740 °C. Highly crystalline fibers were obtained already at 800 °C, but the crystallinity and calcium zirconate yield improved when the temperature was increased to 1000 °C.     

工程菌生物强化处理含酚焦化废水的研究

经过适当采样,采用现代生物技术分离、筛选出多株优良脱酚菌,然后选择适当的工艺将优良工程菌与活性污泥混合处理焦化废水.研究了曝气时间、进水酚浓度、曝气量等因素对脱酚效果的影响.