Proposed kinetic mechanism of the production of biodiesel from palm oil using lipase

Experimental determination of the separate effects of palm oil and methanol concentrations on the rate of their enzymatic transesterification was used to propose suitable mechanismic steps and to test the generated kinetic model. The reaction took place in n-hexane organic medium and the lipase used was from Mucor miehei. At a constant methanol concentration of 300 mol m⁻³, it was found that, initially as the palm oil concentration increased, the initial reaction rate increased. However, the initial rate dropped sharply at substrate concentrations larger than 1250 mol m⁻³. Similar behaviour was observed for methanol concentration effect, where at a constant substrate concentration of 1000 mol m⁻³, the initial rate of reaction dropped at methanol concentrations larger than 3000 mol m⁻³. Ping Pong Bi Bi mechanism with inhibition by both reactants was adopted as it best explains the experimental findings. A mathematical model was developed from a proposed kinetic mechanism and was used to identify the regions where the effect of inhibition by both substrates arised. The proposed model equation is essential for predicting the rate of methanolysis of palm oil in a batch or a continuous reactor and for determining the optimal conditions for biodiesel production.     

Immobilization of porcine pancreatic lipase on poly-hydroxybutyrate particles for the production of ethyl esters from macaw palm oils and pineapple flavor

Poly-hydroxybutyrate particles (PHB) were used as support to immobilize porcine pancreatic lipase (PPL). The biocatalysts prepared were tested in the synthesis of pineapple flavor by esterification of butanol and butyric acid in heptane medium, and in the synthesis of ethyl esters by transesterification of macaw palm pulp (MPPO) and macaw palm kernel (MPKO) oils with ethanol in solvent-free systems. The effect of protein loading on the biocatalyst activity was assessed in olive oil hydrolysis. Maximum hydrolytic activity of 292.8 ± 8.60 IU/g was observed. Langmuir isotherm model was applicable to the adsorption of PPL on PHB particles. Maximum immobilized protein amount was 24.3 ± 1.70 mg/g. The optimal pH and temperature in hydrolysis reaction for the immobilized PPL were at pH 8.5 and 50 °C, while for the crude PPL extract were at pH 8.0 and 45 °C. Immobilized PPL exhibited full hydrolytic activity after 2 h of incubation in non-polar solvents. In esterification reaction, optimal conversion was around 93% after 2 h of reaction. After six esterification cycles, the biocatalyst retained 63% of its initial activity. The biocatalyst prepared attained transesterification yield of 50% after 48 h of reaction for MPKO and 35% after 96 h of reaction for MPPO.     

Comparison of batch,fed-batch and continuous well-mixed reactors for enzymatic hydrolysis of orange peel wastes

An alternative potential feedstock for bioethanol in the automotive sector is citrus peel waste (CPW), which can be processed through enzymatic hydrolysis and fermentation. The present work considers mathematical modeling of orange peel wastes (OPW) hydrolysis with the use of free enzymes and compares the performance of batch, fed-batch and continuous well-mixed reactors after introducing appropriate rate equations in dynamic mass balances. MATLAB^® was used for model implementation.Following the Michaelis–Menten approach, the authors used their own kinetic parameters for the pectin hydrolysis rate equation. The parameters were generated in an apposite experimental program for OPW hydrolysis to galacturonic acid with consideration of product inhibition; the corresponding values were obtained after Lineweaver–Burk linearization and are: r_max = 0.28 g/(L min), K_m = 19.80 g/L and K_IGA = 6.96 g/L, respectively. Vice-versa, the authors adopted the Kadam's group kinetic schemes and parameters for cellulose hydrolysis to cellobiose and glucose. The mathematical model of a well-mixed batch reactor was perfectly validated against the experimental results of OPW hydrolysis to galacturonic acid. In the case of a continuous well-mixed reactor, high dilution rates determine low conversion of OPW. The increased complication of fed-batch operation does not add advantages when compared to batch processing.     

Kinetics modeling of the acidolysis with immobilized Rhizomucor miehei lipases for production of structured lipids from sunflower oil

Sunflower oil modification for production of semisolid fats was carried out via acidolysis using palmitic and stearic acids (P + St), hexane and a developed biocatalyst from Rhizomucor miehei lipases. Its kinetic behavior was studied by employing three mathematical models proposed in the literature. Furthermore, a new model was proposed to describe not only the variation of triacylglycerols (TAG), diacylglycerols (DAG), and free fatty acids groups but also the acyl migration reaction occurrence. The effect of the reaction temperature on the kinetic and equilibrium parameters, as well as TAG and reaction intermediates profiles was analyzed. Increasing reaction temperature generated major changes in the overall composition of acylglycerols and gave rise to the highest composition of P + St in the obtained structured lipids (58%, 70 h, 60 °C). P + St incorporation was successfully adjusted by an empirical model (Model I) and a lumped parameter model (Model II) for all the studied reaction times, while the model based on a Ping Pong Bi Bi mechanism (Model III) was only able to describe the kinetics behavior (through the variation of reactant saturated fatty acids concentration) until 24 h. Experimental data were fit satisfactorily by the proposed model (Model IV), showing that the increment in the disaturated TAG formation achieved by the increment in temperature was principally related to the favored DAG formation from triunsaturated TAG.     

Kinetic study on enzymatic esterification of tuna fish oil fatty acids with butanol

Docosahexaenoic acid (DHA) is an important polyunsatured fatty acid (PUFA) which can be purified from tuna fish oil fatty acids by selective enzymatic esterification. The present paper investigates the kinetic study for selective esterification of tuna fish oil fatty acids with butanol catalyzed by Rhizopus oryzae lipase (ROL) in biphasic solvent system. Under the most suitable reaction conditions, 76.2% esterification was achieved in 24 h. Different kinetic models for esterification given by Segel [1], Oliveira et al. [2], Gogoi et al. [3], and Kraai et al. [4] were tested for fitting the esterification data and the model given by Oliveira et al. [2] was found to be most suitable. The model given by Prazeres et al. [5] for hydrolysis was also tested for esterification and the model with second order product inhibition was found to provide better match between the predicted and experimental values than that of model by Oliveira et al. [2]. The kinetic model was fitted using MATLAB^® to determine the best kinetic parameters. The average value of kinetic constants using the model given by Prazeres et al. were estimated as K_m = 23.6 μmoles FFA/ml, K_i1 = 4.6 × 10⁻⁵ μmoles FFA/mg enzyme h, K_i2 = 0.0062 μmoles FFA/mg enzyme h and K₂ = 149.5 μmoles FFA/mg enzyme h.     

Effect of process parameters on lipase production by Candida cylindracea in stirred tank bioreactor using renewable palm oil mill effluent based medium

A two-level full factorial design (FFD) was employed to determine the effects of process parameters on lipase production by Candida cylindracea ATCC 14830 in palm oil mill effluent (POME)-based medium. Ten experimental runs based on three parameters (temperature, agitation and aeration) as indicated by the FFD were carried out in a stirred-tank bioreactor. On statistical analysis of the results, the optimum temperature, aeration and agitation rates were found to be 30 °C, 1.0 vvm and 400 rpm respectively, with a maximum activity of 41.46 U/ml after 36 h of fermentation. Analysis of variance (ANOVA) showed a high coefficient of determination (R²) value of 0.999, indicating a satisfactory fit of the model with the experimental data. All the three parameters were statistically significant at p < 0.05. The validation experiment also confirmed that apart from lipase production, there was an increase in chemical oxygen demand (COD) removal throughout the fermentation period.     

Effect of enzymatic and hydrothermal treatments of rapeseeds on quality of the pressed rapeseed oils: Part I: Antioxidant capacity and antioxidant content

Response surface methodology was used to evaluate the quantitative effects of three independent variables: rapeseed moisture content, concentration of the added enzymes and conditioning temperature, on the antioxidant capacity and total phenolic, tocopherol, and phospholipid contents in the enzyme-treated rapeseed oils. The highest antioxidant capacity (1220.0, 964.8 μmol TE/100 g) total phenolic (83.3, 74.0 mg SA/100 g) and phospholipid (12,532, 12,376 mg/kg) contents reveal two rapeseed oils extruded from seeds contained 11% moisture, treated with cellulolytic and pectolytic enzymes (0.05%), respectively, and heated at 120 °C. However, the highest content of total tocopherols was determined in rapeseed oils pressed from seeds with 7% moisture, after addition of cellulolytic (0.05%) and pectolytic (0.1%) enzymes, heated at 90 and 105 °C, respectively. Total phenolic and phospholipid contents in the enzyme-treated rapeseed oils correlated significantly (p < 0.0000001) with antioxidant capacities of oils (R² = 0.8710 and 0.6581, respectively). Experimental results of the antioxidant capacity, total phenolic, tocopherol and phospholipid contents were close to the predicted values calculated from the polynomial response surface models equations (R² = 0.9727, 0.9870, 0.8390 and 0.9706 for the cellulolytic enzyme-assisted rapeseed oils and R² = 0.9148, 0.9489, 0.9426 and 0.9479 for the pectolytic enzyme-assisted rapeseed oils). The optimum rapeseed moisture content, enzyme concentration and conditioning temperature for the cellulolytic and pectolytic enzyme-treated rapeseed oils were 11% and 9.7%, 0.08% and 0.1%, and 120 °C, respectively.     

Evaluation of sap flow methods to determine water use by cultivated palms

Sustainable management of water resources allocated to palm crops requires reliable tools for measuring palm water use. Thermometric sap flow methods developed for woody dicot plants hold great potential for use in palms, but there have been few investigations to determine whether such methods can be used successfully in ‘woody’ monocots. Here, we evaluate two sap flow methods for measuring whole-plant water use by potted cocos palms (Syagrus romanzoffiana): the Heat Ratio Method (HRM) and the Compensation Heat Pulse Method (CHPM). Measurements of whole-plant water use from the HRM and the CHPM were compared to gravimetric measurements acquired from an electronic balance. Of the two methods, the HRM gave the most accurate results and it most precisely described patterns of transpiration with respect to environmental conditions and leaf morphology. Estimates of total daily water use from the HRM and the balance were highly correlated (R² = 0.92; P < 0.0001) and very near to a 1:1 relationship—an excellent result given the potential for error associated with each method. As expected, the CHPM was seriously limited at low flow rates, but it agreed well with the HRM at higher flow rates (heat pulse velocity >4 cm h⁻¹). Anatomical investigations revealed that vascular bundles in measured palm fronds were evenly distributed and the distance between bundles was comparable in scale to the diameter of probes of HRM sensors, and most likely an order of magnitude smaller than the probable zone of thermal influence for HRM measurements. This contention was supported by results of mathematical modeling suggesting that HRM heat pulse velocities in palm-like sapwood remain largely unaffected by increases in heterogeneity caused by larger xylem vessels and wider interstitial tissues. Although wounding models for heat pulse sap flow methods have not historically catered for the effects of discrete vascular bundles, they are sufficiently approximate given our empirical validations, and we conclude that palm sapwood is thermally homogenous enough for the HRM and the CHPM to be used without modification. Overall, we present strong evidence that heat pulse methods such as the HRM and the CHPM can be used successfully in woody monocots. Finally, we note that the CHPM is likely to perform better in cases of very high flow rates, while the HRM provides more comprehensive monitoring of the usual range of flow rates including at night, early morning and during water deficit.     

Lipase-catalyzed synthesis of palmitanilide: Kinetic model and antimicrobial activity study

Enzymatic syntheses of fatty acid anilides are important owing to their wide range of industrial applications in detergents, shampoo, cosmetics, and surfactant formulations. The amidation reaction of Mucor miehei lipase Lipozyme IM20 was investigated for direct amidation of triacylglycerol in organic solvents. The process parameters (reaction temperature, substrate molar ratio, enzyme amount) were optimized to achieve the highest yield of anilide. The maximum yield of palmitanilide (88.9%) was achieved after 24 h of reaction at 40 °C at an enzyme concentration of 1.4% (70 mg). Kinetics of lipase-catalyzed amidation of aniline with tripalmitin has been investigated. The reaction rate could be described in terms of the Michaelis–Menten equation with a Ping–Pong Bi–Bi mechanism and competitive inhibition by both the substrates. The kinetic constants were estimated by using non-linear regression method using enzyme kinetic modules. The enzyme operational stability study showed that Lipozyme IM20 retained 38.1% of the initial activity for the synthesis of palmitanilide (even after repeated use for 48 h). Palmitanilide, a fatty acid amide, exhibited potent antimicrobial activity toward Bacillus cereus.     

Valorization of olive stones for xylitol and ethanol production from dilute acid pretreatment via enzymatic hydrolysis and fermentation by Pachysolen tannophilus

Olive stones are an agro-industrial by-product abundant in the Mediterranean area that is regarded as a potential lignocellulosic feedstock for sugar production. Statistical modeling of dilute-sulphuric acid hydrolysis of olive stones has been performed using a response surface methodology, with treatment temperature and process time as factors, to optimize the hydrolysis conditions aiming to attain maximum d-xylose extraction from hemicelluloses. Thus, solid yield and composition of solid and liquid phases were assessed by empirical modeling. The highest yield of d-xylose was found at a temperature of 195 °C for 5 min. Under these conditions, 89.7% of the total d-xylose was recovered from raw material. The resulting solids from optimal conditions were assayed as substrate for enzymatic hydrolysis, while fermentability of hemicellulosic hydrolysates was tested using the d-xylose-fermenting yeast Pachysolen tannophilus. Both bioprocesses were considerably influenced by enzyme loading and inoculum size. In the enzymatic hydrolysis step, about 56% of cellulose was converted into d-glucose by using an enzyme/solid ratio of 40 FPU g⁻¹, while in the fermentation carried out with a cell concentration of 2 g L⁻¹ a yield of 0.44 g xylitol/g d-xylose and a global volumetric productivity of 0.11 g L⁻¹ h⁻¹ were achieved.     

Inulin hydrolysis and citric acid production from inulin using the surface-engineered Yarrowia lipolytica displaying inulinase

The INU1 gene encoding exo-inulinase cloned from Kluyveromyces marxianus CBS 6556 was ligated into the surface display plasmid and expressed in the cells of the marine-derived yeast Yarrowia lipolytica which can produce citric acid. The expressed inulinase was immobilized on the yeast cells. The activity of the immobilized inulinase with 6 × His tag was found to be 22.6 U mg^?1 of cell dry weight after cell growth for 96 h. The optimal pH and temperature of the displayed inulinase were 4.5 and 50 °C, respectively and the inulinase was stable in the pH range of 3–8 and in the temperature range of 0–50 °C. During the inulin hydrolysis, the optimal inulin concentration was 12.0% and the optimal amount of added inulinase was 181.6 U g^?1 of inulin. Under such conditions, over 77.9% of inulin was hydrolyzed within 10 h and the hydrolysate contained main monosaccharides and disaccharides, and minor trisaccharides. During the citric acid production in the flask level, the recombinant yeast could produce 77.9 g L^?1 citric acid and 5.3 g L^?1 iso-citric acid from inulin while 68.9 g L^?1 of citric acid and 4.1 g L^?1 iso-citric acid in the fermented medium were attained within 312 h of the 2-L fermentation, respectively.     

Optimization and modeling of lactose hydrolysis in a packed bed system using immobilized β-galactosidase from Aspergillus oryzae

This work studied the hydrolysis of lactose using β-galactosidase from Aspergillus oryzae immobilized with a combination of adsorption and glutaraldehyde cross-linking onto the ion exchange resin Duolite A568 as a carrier. A central composite design (CCD) was used to study the effects of lactose concentration and feed flow rate on the average hydrolysis reaction rate and lactose conversion in a fixed bed reactor operating continuously with an upflow at a temperature of 35 ± 1 °C. The optimal conditions for the average hydrolysis reaction rate and the lactose conversion included a lactose concentration of 50 g/L and a feed flow rate of 6 mL/min. The average reaction rate and conversion reached 2074 U and 65%, respectively. The immobilized enzyme activity was maintained during the 30 days of operation in a fixed bed reactor with a 0.3 mL/min feed flow rate of a 50 g/L lactose solution at room temperature. Feed flows ranging from 0.6 to 12 mL/min were used to determine the distribution of residence times and the kinetics of the fixed bed reactor. A non-ideal flow pattern with the formation of a bypass flow in the fixed bed reactor was identified. The conditions used for the kinetics study included a lactose solution concentration of 50 g/L at pH 4.5 and a temperature of 35 ± 1 °C. Kinetic models using a PFR and axial dispersion methods were used to describe the lactose hydrolysis in the fixed bed reactor, thus accounting for the competitive inhibition by galactose. To increase the lactose conversion, experiments were performed for two fixed bed reactors in series, operating in continuous duty with upflow, with the optimal conditions determined using the CCD for a fixed bed reactor. The total conversion for the two reactors in series was 82%.     

Improving the lactic acid production of Actinobacillus succinogenes by using a novel fermentation and separation integration system

This work describes the development of a novel integrated system for lactic acid production by Actinobacillus succinogenes. Fermentation and separation were integrated with the use of a microfiltration (MF) membrane, and lactic acid was recovered by resin adsorption following MF. The fermentation broth containing residual sugar and nutrients was then recycled back into the fermenter after lactic acid adsorption. This novel approach overcame the problem of product inhibition and extended the cell growth period from 41 h to 120 h. Production of lactic acid was improved by 23% to 183.4 g L⁻¹. The overall yield and productivity for glucose were 0.97 g g⁻¹ and 1.53 g L⁻¹ h⁻¹, respectively. These experimental results indicate that the integrated system could benefit continuous production of lactic acid at high levels.     

Continuous production of ellagic acid in a packed-bed reactor

Ellagic acid is a high-value bioactive compound that is used in the food, cosmetic and pharmaceutical industries. The aim of this work was to develop a continuous system for ellagic acid production. Ellagitannase produced by solid-state fermentation and attached to polyurethane foam particles was used as a biocatalyst in a continuous bioreactor for the hydrolysis of ellagitannins from pomegranate by-product. A packed-bed reactor containing the biocatalyst (22.22 Units per gram of dry solid, U gds⁻¹) was fed with a pomegranate ellagitannins solution (0.1%, w/v) at a flow rate of 0.27 mL min⁻¹ at 60 °C. The bioreactor completed several biotransformations while maintaining the hydrolysis rate (60%) with a half-life of 10 continuous cycles of ellagic acid production. Volumetric productivity and ellagic acid yield were 1.09 g L⁻¹ h⁻¹ and 235.89 mg g⁻¹ of pomegranate ellagitannins during the first 70 min of hydrolysis, respectively. The developed biocatalyst showed good operational and mechanical stability and may be successfully used for ellagitannin hydrolysis in a continuous system. This is the first report of high-yield continuous production of ellagic acid using an auto-immobilized enzyme.     

An organic solvent–tolerant lipase from Streptomyces sp. CS133 for enzymatic transesterification of vegetable oils in organic media

The enzymatic route for biodiesel production has been noted to be cost ineffective due to the high cost of biocatalysts. Reusing the biocatalyst for successive transesterification cycles is a potential solution to address such cost inefficiency. However, when organic solvent like methanol is used as acyl-acceptor in the reaction, the biocatalyst (lipase) gets severely inactivated due to the inhibitory effect of undissolved methanol in the reaction medium. Thus, organic solvent–tolerant lipase is highly desirable for enzymatic transesterification. In response to such desirability, a lipase (LS133) possessing aforesaid characteristic was extracted from Streptomyces sp. CS133. Relative molecular mass of the purified LS133 was estimated to be 39.8 kDa by SDS-PAGE. Lipase LS133 was stable in pH range 5.0–9.0 and at temperature lower than 50 °C while its optimum lipolytic activity was achieved at pH 7.5 and 40 °C. It showed the highest hydrolytic activity towards long chain p-nitrophenyl palmitate with K_m and V_max values of 0.152 mM and 270.2 mmol min^?1 mg^?1, respectively. It showed non-position specificity for triolein hydrolysis. The first 15 amino acid residues of its N-terminal sequence, AIPLRQTLNFQAXYQ, were noted to have partial similarity with some of the previously reported microbial lipases. Its catalytic involvement in biodiesel production process was confirmed by performing enzymatic transesterification of vegetable oils with methanol.     

Immobilization of lipase in organic solvent in the presence of fatty acid additives

In this study porcine pancreatic lipase (PPL) was covalently immobilized on cross-linked polyvinyl alcohol (PVA) in organic media in the presence of fatty acid additives in order to improve its immobilized activity. The effects of fatty acid additions to the immobilization media were investigated choosing tributyrin hydrolysis in water and ester synthesis by immobilized PPL in n-hexane. Various fatty acids which are also the substrates of lipases in esterification reactions were used as active site protecting agents during the immobilization process in an organic solvent. The obtained results showed that covalent immobilization carried out in the presence of fatty acids as protective ligands improved the hydrolytic and esterification activity of immobilized enzyme. A remarkable increase in activity of the immobilized PPL was obtained when octanoic acid was used as an additive and the hydrolytic activity was increased from 5.2 to 19.2 μmol min⁻¹ mg⁻¹ as compared to the non-additive immobilization method. With the increase of hydrolytic activity of immobilized lipase in the presence of octanoic acid, in an analogous manner, the rate of esterification for the synthesis of butyl octanoate was also increased from 7.3 to 26.3 μmol min⁻¹ g⁻¹ immobilized protein using controlled thermodynamic water activities with saturated salt solutions. In addition, the immobilized PPL activity was maintained at levels representing 63% of its original activity value after 5 repeated uses. The proposed method could be adopted for a wide variety of other enzymes which have highly soluble substrates in organic solvent such as other lipases and esterases.     

Enzymatic hydrolysis of corncob and ethanol production from cellulosic hydrolysate

 Enzymatic hydrolysis of corncob and ethanol fermentation from cellulosic hydrolysate were investigated. After corncob was pretreated by 1% H₂SO₄ at 108 °C for 3 h, the cellulosic residue was hydrolyzed by cellulase from Trichoderma reesei ZU-02 and the hydrolysis yield was 67.5%. Poor cellobiase activity in T. reesei cellulase restricted the conversion of cellobiose to glucose, and the accumulation of cellobiose caused severe feedback inhibition to the activities of β-1,4-endoglucanase and β-1,4-exoglucanase in cellulase system. Supplementing cellobiase from Aspergillus niger ZU-07 greatly reduced the inhibitory effect caused by cellobiose, and the hydrolysis yield was improved to 83.9% with enhanced cellobiase activity of 6.5 CBU g⁻¹ substrate. Fed-batch hydrolysis process was started with a batch hydrolysis containing 100 g l⁻¹ substrate, with cellulosic residue added at 6 and 12 h twice to get a final substrate concentration of 200 g l⁻¹. After 60 h of reaction, the reducing sugar concentration reached 116.3 g l⁻¹ with a hydrolysis yield of 79.5%. Further fermentation of cellulosic hydrolysate containing 95.3 g l⁻¹ glucose was performed using Saccharomyces cerevisiae 316, and 45.7 g l⁻¹ ethanol was obtained within 18 h. The research results are meaningful in fuel ethanol production from agricultural residue instead of grain starch.     

Characterization of the lipase immobilized on Mg–Al hydrotalcite for biodiesel

Immobilization of Saccharomyces cerevisiae lipase by physical adsorption on Mg–Al hydrotalcite with a Mg/Al molar ratio of 4.0 led to a markedly improved performance of the enzyme. The immobilized lipase retained activity over wider ranges of temperature and pH than those of the free lipase. The immobilized lipase retained more than 95% relative activity at 50 °C, while the free lipase retained about 88%. The kinetic constants of the immobilized and free lipases were also determined. The apparent activation energies (E_a) of the free and immobilized lipases were estimated to be 6.96 and 2.42 kJ mol^?1, while the apparent inactivation energies (E_d) of free and immobilized lipases were 6.51 and 6.27 kJ mol^?1, respectively. So the stability of the immobilized lipase was higher than that of free lipase. The water content of the oil must be kept below 2.0 wt% and free fatty acid content of the oil must be kept below 3.5 mg KOH g [oil]^?1 in order to get the best conversion. This immobilization method was found to be satisfactory to produce a stable and functioning biocatalyst which could maintain high reactivity for repeating 10 batches with ester conversion above 81.3%.     

Gene cloning,overexpression and characterization of a novel organic solvent tolerant and thermostable lipase from Galactomyces geotrichum Y05

Although the lipase of Geotrichum candidum has been extensively reported, little attention has been focused on molecular genetic and biochemical characterizations of Galactomyces geotrichum lipases. A lipase gene from G. geotrichum Y05 was cloned from both genomic DNA and cDNA sources. Nucleotide sequencing revealed that the ggl gene has an ORF of 1692 bp without any introns, encoding a protein of 563 amino acid residues, including a potential signal sequence of 19 amino acid residues. The amino acid sequence of this lipase showed 86% identity to lipase of Trichosporon fermentans WU-C12. The mature lipase gene was subcloned into pPIC9K vector, and overexpressed in methylotrophic Pichia pastoris GS115. Active lipase was accumulated to the level of 100.0 U/ml (0.4 mg/ml) in the shake-flask culture, 10.4-fold higher than the activity of the original strain (9.6 U/ml). This yield dramatically exceeds that previously reported with 23–50 U/ml, 0.06 mg/ml and 0.2 mg/ml. The purified lipase exhibited several properties of significant industrial importance, such as pH and temperature stability, wide organic solvent tolerance and broad hydrolysis on vegetable oils. Such a combination of properties makes it a promising candidate for its application in non-aqueous biocatalysis, such as biodiesel production, selective hydrolysis or esterification for enrichment of PUFAs and oil-contaminated biodegradation, which have been drawn considerable attention currently.     

Synthesis of thermo-sensitive copolymer with affinity butyl ligand and its application in lipase purification

In this study, thermo-sensitive N-alkyl substituted polyacrylamide polymer P_NNB was synthesized by using N-hydroxymethyl acrylamide(NHAM), N-isopropyl acrylamide (NIPA) and butyl acrylate (BA) as monomers, and its low critical solution temperature (LCST) was controlled to be 28 °C. The recovery of the thermo-sensitive polymer was over 98%. Butanol as a hydrophobic ligand was covalently attached onto polymer P_NNB and butyl ligand density was 80 μmol g^?1 polymer. The affinity polymer was used for purification of lipase from crude material. Optimized condition was pH 7.0, 35 °C adsorption temperature, 120 min adsorption time and 0.5 mg ml^?1 initial concentration of lipase. The adsorption isotherm accords with a typical Langmuir isotherm. The maximum adsorption capacity (Q_m) of the affinity polymer for lipase was 24.8 mg g^?1polymer. The affinity copolymer could be recycled by temperature-inducing precipitation and there was only about 6% loss of adsorption capacity after five recyclings. Specific activity of lipase was improved from 14 IU mg^?1 to 506 IU mg^?1 protein, and its recovery achieved 82%. The affinity polymer is suitable for the purification of target proteins from the crude material with large volume and dilute solution.