Improving esterification activity of Burkholderia cepacia lipase encapsulated in silica by bioimprinting with substrate analogues

Bioimprinting is a promising, though relatively unexplored, approach to improving the performance of enzymes. In this study, bioimprinting with substrate analogues of fatty acids was systematically conducted to improve the esterification activity of Burkholderia cepacia lipase that had undergone a sol–gel immobilization procedure with methyltrimethoxysilane (MTMS) and tetramethoxysilane (TMOS) as the precursors. The specific activity of the bioimprinted lipases was 3682.0 μmol h^?1 mg protein, which was a 47.9- and 2.5-fold increase over the free and non-imprinted immobilized lipases, respectively. Compared to the free and non-imprinted immobilized lipases, bioimprinted lipases exhibited better thermal stability, and their activity did not change after being incubated at 60 °C for 12 h. Bioimprinted lipases were more easily affected by alcohol than the non-imprinted ones, whose specific activity could be markedly enhanced by ethanol, isopropanol and n-butanol by factors of 1.23-, 1.28- and 1.12-fold, respectively. The reasons for the improvement of imprinted enzyme activity are also discussed based on the surface structure, specific surface area and average pore diameter of the silane particles.     

An improved method of lipase preparation incorporating both solvent treatment and immobilization onto matrix

A simple and effective preparation of lipases for use in organic solvents is hereby proposed. Lipases in aqueous solution were treated with isopropanol, immediately followed by immobilization onto a commercially available macroporous resin CRBO2 (crosslinked polystyrene with N-methylglucamine as a functional group). The dual modification of lipases by (1) isopropanol treatment and (2) immobilization improved the activity and stability of lipases more significantly than either of the two treatments alone. The degree of lipase activation was dependent on isopropanol–buffer (v/v) ratio and the source of lipase used. Among the lipases tested, Rhizopus oryzae lipase was more significantly activated. The maximum specific activity of R. oryzae lipase after dual modification was 94.9 mmol h⁻¹ g⁻¹, which was, respectively, 3.3-, 2.5- and 1.5-fold of untreated free, untreated immobilized and treated free lipases. The conformations of the treated and untreated free lipases were investigated by circular dichroism (CD) measurement. Changes in the far- and near-UV CD spectra of lipase indicate that lipase activation is accompanied by changes in secondary and tertiary structures of lipases. The increase in negative molar elipticity at 222 nm suggests that the α-helical content of lipase increase after pretreatment.     

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%.     

Purification and properties of enantioselective lipase from a newly isolated Bacillus cereus C71

A lipase from Bacillus cereus C71 was purified to homogeneity by ammonium sulfate precipitation, followed by Phenyl-Sepharose chromatography, DEAE ion exchange chromatography and CIM^® QA chromatography. This purification procedure resulted in a 1092-fold purification of lipase with 18% yield. The molecular mass of the purified enzyme was determined to be approximately 42 kDa by SDS-PAGE and mass spectrometer. The lipase was stable in the pH range of 8.5–10.0, with the optimum pH 9.0. The enzyme exhibited maximum activity at 33 °C and retained 92% of original activity after incubation at 35 °C for 3 h. The protein hydrolyzed p-nitrophenyl esters with acyl chain lengths between C4 and C12. Enzyme activity was strongly inhibited in the presence of Cu²⁺ and Zn²⁺ but promoted by non-ionic surfactants. The lipase demonstrated higher enantioselectivity toward R-isomer of ethyl 2-arylpropanoate than the commercial lipases, and can be used potentially as a catalyst to prepare optically pure pharmaceuticals.     

Thermophilic lipase from Thermomyces lanuginosus: Gene cloning,expression and characterization

An extracellular lipase gene ln1 from thermophilic fungus Thermomyces lanuginosus HSAUP₀₃80006 was cloned through RT-PCR and RACE amplification. Its coding sequence predicted a 292 residues protein with a 17 amino acids signal peptide. The deduced amino acids showed 78.4% similarity to another lipase lgy from T. lanuginosus while shared low similarity with other fungi lipases. Higher frequencies hydrophobic amino acids related to lipase thermal stability, such as Ala, Val, Leu and Gly were observed in this lipase (named LN). The sequence, -Gly-His-Ser-Leu-Gly-, known as a lipase-specific consensus sequence of mould, was also found in LN. High level expression for recombinant lipase was achieved in Pichia pastoris GS115 under the control of strong AOX1 promoter. It was purified to homogeneity through only one step DEAE-Sepharose anion exchange chromatography and got activity of 1328 U/ml. The molecular mass of one single band of this lipase was estimated to be 33 kDa by SDS-PAGE. The enzyme was stable at 60 °C and kept 65% enzyme activity after 30 min incubation at 70 °C. It kept half-activity after incubated for 40 min at 80 °C. The optimum pH for enzyme activity was 9.0 and the lipase was stable from pH 8.0 to 12.0. Lipase activity was enhanced by Ca²⁺ and inhibited by Fe²⁺, Zn²⁺, K⁺, and Ag⁺. The cell-free enzyme hydrolyzed and synthesized esters efficiently, and the synthetic efficiency even reached 81.5%. The physicochemical and catalytic properties of the lipase are extensively investigated for its potential industrial applications.     

Development of a monolith based immobilized lipase micro-reactor for biocatalytic reactions in a biphasic mobile system

This paper reports a simple method for producing macroporous silica-monoliths with controllable porosity that can be used for the immobilization of lipases to generate an active and stable micro-reactor for biocatalysis. A range of commercially available lipases has been examined using the hydrolysis reactions of 4-nitrophenyl butyrate in water–decane media. The kinetic studies performed have identified that a similar value for k_cat is obtained for the immobilized Candida antarctica lipase A (0.13 min⁻¹) and the free lipase in solution (0.12 min⁻¹) whilst the immobilized apparent Michaelis constant K_m (3.1 mM) is 12 times lower than the free lipase in solution (38 mM). A 96% conversion was obtained for the immobilized C. antarctica lipase A compared to only 23% conversion for the free lipase. The significant higher conversions obtained with the immobilized lipases were mainly attributed to the formation of a favourable biphasic system in the continuous flowing micro-reactor system, where a significant increase in the interfacial activation occurred. The immobilized C. antarctica lipase A on the monolith also exhibited improved stability, showing 64% conversion at 80 °C and 70% conversion after continuous running for 480 h, compared to 40 and 20% conversions under the same temperature and reaction time for the free lipase.     

Purification,characterization and application of acidic lipase from Pseudomonas gessardii using beef tallow as a substrate for fats and oil hydrolysis

Beef tallow, a slaughter house waste was used as a substrate for lipase production, employing Pseudomonas gessardii. The strain, P. gessardii was isolated from the beef tallow acclimatized soil. The crude lipase activity at 139 U/ml by volume was obtained at optimized conditions of pH 5.0 and temperature of 37 °C. After purification, a 7.59-fold purity of lipase with specific activity of 1120 U/mg protein and molecular mass of 92 kDa was obtained. The purified lipase showed maximum activity and stability at pH 5.0 and 30 °C. Ca²⁺ had a stimulatory effect on the lipase activity compared to the other metal ions studied. The relative activity was enhanced with the addition of Triton X-100 with lower hydrophilic–lipophilic balance (HLB) value as 13.0 and DMSO with the lowest partition coefficient (log P) value, as 1.378. The amino acid composition and the functional groups of lipase were confirmed by HPLC and FT-IR spectroscopy. The purified lipase had the highest hydrolytic activity towards slaughterhouse wastes and vegetable oils. This work provides a potential biocatalyst for the wide applications in oleochemical and biotechnological industries.     

Characterization of a spore surface lipase from the biocontrol agent Metarhizium anisopliae

A Metarhizium anisopliae spore surface lipase (MASSL) strongly bound to the fungal spore surface has been purified by ion exchange chromatography on DEAE sepharose followed by ultrafiltration and hydrophobic interaction chromatography on phenyl sepharose. Electrophoretic analyses showed that the molecular weight of this lipase is ～66 kDa and pI is 5.6. Protein sequencing revealed that identified peptides in MASSL shared identity with several lipases or lipase-related sequences. The enzyme was able to hydrolyze triolein, the animal lipid cholesteryl stearate and all ρNP ester substrates tested with some preference for esters with a short acyl chain. The values of K_m and V_max for the substrates ρNP palmitate and ρNP laurate were respectively 0.474 mM and 1.093 mMol min^?1 mg^?1 and 0.712 mM and 5.696 mMol min^?1 mg^?1. The optimum temperature of the purified lipase was 30 °C and the enzyme was most stable within the most acid pH range (pH 3–6). Triton X-100 increased and SDS reduced enzyme lipolytic activity. MASSL activity was stimulated by Ca²⁺, Mg²⁺ and Co²⁺ and inhibited by Mn²⁺. The inhibitory effect on activity exerted by EDTA and EGTA was limited, while the lipase inhibitor Ebelactone B completely inhibited MASSL activity as well as PMSF. Methanol 0.5% apparently did not affect MASSL activity while β-mercaptoethanol activated the enzyme.     

Immobilization of acidic lipase derived from Pseudomonas gessardii onto mesoporous activated carbon for the hydrolysis of olive oil

Mesoporous activated carbon (MAC) derived from rice husk is used for the immobilization of acidic lipase (ALIP) produced from Pseudomonas gessardii. The purified acidic lipase had the specific activity and molecular weight of 1473 U/mg and 94 kDa respectively. To determine the optimum conditions for the immobilization of lipase onto MAC, the experiments were carried out by varying the time (10–180 min), pH (2–8), temperature (10–50 °C) and the initial lipase activity (49 × 10³, 98 × 10³, 147 × 10³ and 196 × 10³ U/l in acetate buffer). The optimum conditions for immobilization of acidic lipase were found to be: time—120 min; pH 3.5; temperature—30 °C, which resulted in achieving a maximum immobilization of 1834 U/g. The thermal stability of the immobilized lipase was comparatively higher than that in its free form. The free and immobilized enzyme kinetic parameters (K_m and V_max) were found using Michaelis–Menten enzyme kinetics. The K_m values for free enzyme and immobilized one were 0.655 and 0.243 mM respectively. The immobilization of acidic lipase onto MAC was confirmed using Fourier Transform-Infrared Spectroscopy, X-ray diffraction analysis and scanning electron microscopy.     

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.     

Improving the activity and stability of Yarrowia lipolytica lipase Lip2 by immobilization on polyethyleneimine-coated polyurethane foam

In this study, polyurethane foam (PUF) was used for immobilization of Yarrowia lipolytica lipase Lip2 via polyethyleneimine (PEI) coating and glutaraldehyde (GA) coupling. The activity of immobilized lipases was found to depend upon the size of the PEI polymers and the way of GA treatment, with best results obtained for covalent-bind enzyme on glutaraldehyde activated PEI-PUF (MW 70,000 Da), which was 1.7 time greater activity compared to the same enzyme immobilized without PEI and GA. Kinetic analysis shows the hydrolytic activity of both free and immobilized lipases on triolein substrate can be described by Michaelis–Menten model. The K_m for the immobilized and free lipases on PEI-coated PUF was 58.9 and 9.73 mM, respectively. The V_max values of free and immobilized enzymes on PEI-coated PUF were calculated as 102 and 48.6 U/mg enzyme, respectively. Thermal stability for the immobilization preparations was enhanced compared with that for free preparations. At 50 °C, the free enzyme lost most of its initial activity after a 30 min of heat treatment, while the immobilized enzymes showed significant resistance to thermal inactivation (retaining about 70% of its initial activity). Finally, the immobilized lipase was used for the production of lauryl laurate in hexane medium. Lipase immobilization on the PEI support exhibited a significantly improved operational stability in esterification system. After re-use in 30 successive batches, a high ester yield (88%) was maintained. These results indicate that PEI, a polymeric bed, could not only bridge support and immobilized enzymes but also create a favorable micro-environment for lipase. This study provides a simple, efficient protocol for the immobilization of Y. lipolytica lipase Lip2 using PUF as a cheap and effective material.     

Optimization of culture conditions and properties of lipase from Penicillium camembertii Thom PG-3

The culture medium including nitrogen source, carbon source and metal ions, for lipase from Penicillium camembertii Thom PG-3 was optimized and the optimal medium consisted of soybean meal (fat free) 4%, Jojoba oil 0.5%, (NH₄)₂HPO₄, 0.1% Tween 60, initial pH 6.4 and the inoculation was at 28 °C for 96 h. The lipase activity produced was enhanced 3.9-fold and reached 500 U/ml. The lipase was purified 19.8-fold by pH precipitation, ethanol precipitation and ammonium sulphate precipitation as well as DEAE-cellulose chromatography. The purified lipase showed one polypeptide band in SDS-polyacrylamide gel electrophoreses (SDS-PAGE) with molecular weight 28.18 kDa. The optimal pH and temperature for activity of lipase were 6.4 and 48 °C, respectively, which are higher than those lipases from other penicillium sources. The P. camembertii Thom lipase is 1,3-positional specificity for hydrolysis of triglyceride and hydrolyses plant oil preferentially to animal oil. The lipase can be used in short chain ester synthesis with an esterification degree of 95%.     

Enhancement of lipase stability and productivity through chemical modification and its application to latex-based polymer emulsions

The primary focus of this research was to employ amino-group specific chemical modifications for improving the productivity and stability of two commercially produced lipases, Lipase-A from Candida antarctica (CAL_UM) and Greasex from Humicola lanuginosa (HLL_UM), for application in a latex-based paint formulation. The modified lipases showed higher percentage increase (benzoic anhydride-modified, HLL_BA, 150%; PEG-modified, HLL_PEG,162% at 75 °C) as well as higher absolute productivities 41, 50, 52 and 53 μmole substrate mg⁻¹ lipase for unmodified, CAL_PEG, HLL_PEG and HLL_BA, respectively at 37 °C. The half-lives of thermal inactivation for all modified variants were improved from 40 to 166% at 50, 60 and 70 °C relative to unmodified lipases. The higher thermal stability and catalytic efficiency (k_cat/K_m) with concomitant lower activity (k_cat) indicates that enhanced productivity is likely to be due to the modified enzymes being better able to resist thermal denaturation over the time course of the productivity experiments. Importantly, both lipases, CAL_BA (60%) and HLL_BA (55%) retained the highest activity in paint compared with CAL_UM (36%) and HLL_UM (39%) after 20 weeks incubation at 25 °C. The long term stability of the modified lipases illustrates their potential value for commercial paint and other industrial applications.     

Combination of two lipases more efficiently catalyzes methanolysis of soybean oil for biodiesel production in aqueous medium

A combination of two lipases was employed to catalyze methanolysis of soybean oil in aqueous medium for biodiesel production. The two lipase genes were cloned from fungal strains Rhizomucor miehei and Penicillium cyclopium, and each expressed successfully in Pichia pastoris. Activities of the 1,3-specific lipase from R. miehei (termed RML) and the non-specific mono- and diacylglycerol lipase from P. cyclopium (termed MDL) were 550 U and 1545 U per ml respectively, and enzymatic properties of these supernatant of fermentation broth (liquid lipase) were stable at 4 °C for >3 months. Under optimized conditions, the ratio of biodiesel conversion after 12 h at 30 °C, using RML alone, was 68.5%. When RML was assisted by addition of MDL, biodiesel conversion ratio was increased to >95% under the same reaction conditions. The results suggested that combination of lipases with different specificity, for enzymatic conversion of more complex lipid substrates, is a potentially useful strategy for biodiesel production.     

Lipase immobilisation: an equilibrium study of lipases immobilised on hydrophobic and hydrophilic/hydrophobic supports

This work investigates the enzyme-support equilibrium behaviour in immobilised lipase biocatalysts. Equilibrium data determines the maximum enzyme up-take by unit weight of support. Four lipases were immobilised on two polymeric supports, respectively. They were Lipase PS from Pseudomonas, Lipolase 100L from Humicola, SP871 from Rhizomucor miehel and QL from Alcaligenes. The supports were Accurel EP100 (a polypropylene material) and 45SAA (a polypropylene/silica composite). Experimentally, equilibrium was expressed in terms of lipase loading (LU/g support) versus residual lipase concentration (LU/dm³). Activity, efficiency and operational stability of the immobilised lipases were assayed by solvent-free esterification of oleic acid and octanol.Equilibrium data were modelled by the Langmuir, Freundlich and Redlich–Peterson formulae. It was found that Lipolase 100L/Accurel, PS/45SAA and SP871/45SAA systems conformed to the Langmuir behaviour, while Lipase PS/Accurel and SP871/Accurel systems followed the Freundlich behaviour and Lipolase 100L/45SAA, QL/45SAA and QL/Accurel EP100 resembled Redlich–Peterson behaviour. Whereas immobilisation on Accurel EP100 resulted in classical equilibrium isotherms with all four lipases, immobilisation on support 45SAA resulted in two-plateau equilibrium curves which included a step change in the isotherm for all lipases studied, except for SP871. Quantitatively, for 1 g lipase, Accurel and 45SAA had a maximum capacity of 140 and 260 kLU for PS, 112 and 550 kLU for Lipolase 100L, 320 and 800 kLU for SP871 and 18 and 29 kLU for QL, respectively.     

Ala258Phe substitution in Bacillus sp. YX-1 glucose dehydrogenase improves its substrate preference for xylose

Bacillus sp. YX-1 glucose dehydrogenase (BsGDH) with good solvent resistance catalyzes the oxidation of β-d-glucose to d-glucono-1,5-lactone. Xylose is a recyclable resource from hemicellulase hydrolysis. In this work, to improve the preference of BsGDH for xylose, we designed seven mutants inside or adjacent to the substrate binding pocket using site-directed mutagenesis. Among all mutants, Ala258Phe mutant displayed the highest activity of 7.59 U mg⁻¹ and nearly 8-folds higher k_cat/K_m value towards xylose than wild-type BsGDH. The kinetic constants indicated that the A258F mutation effectively altered the transition state. By analysis of modeled protein structure, Ala258Phe created a space to facilitate the reactivity towards xylose. A258F mutant retained good solvent resistance in glycol, ethyl caprylate, octane, decane, cyclohexane, nonane, etc. as with BsGDH. This work provides a protein engineering approach to modify the substrate stereo-preference of alcohol dehydrogenase and a promising enzyme for cofactor regeneration in chiral catalysis.     

Synthesis of structured lipids by two enzymatic steps: Ethanolysis of fish oils and esterification of 2-monoacylglycerols

This paper studies the synthesis of structured triacylglycerols (STAGs), rich in polyunsaturated fatty acids (PUFAs) by a two-step enzymatic process: (i) alcoholysis of fish oils (cod liver and tuna oils) with ethanol to obtain 2-monoacylglycerols (2-MAGs), catalyzed by 1,3 specific lipases and (ii) esterification of these 2-MAGs with caprylic acid (CA, 8:0), also catalyzed by a 1,3 specific lipase, to produce STAGs of structure CA–PUFA–CA. As regards the alcoholysis reaction, three factors have been studied: the influence of the type of lipase used (lipase D from Rhizopus oryzae, immobilized on Accurel MP1000, and Novozym 435 from Candida antarctica), the operational mode of a stirred tank reactor (STR operating in discontinuous and continuous mode) and the intensity of treatment (IOT = lipase amount × reaction time/oil amount). Although higher 2-MAG yields were obtained with lipase D, Novozym 435 was selected due to its greater stability in the operational conditions. The highest 2-MAG yield (63%) was attained in the STR operating in discontinuous mode at an IOT of 1 g lipase × h g oil^?1 (at higher IOT the 2-MAGs were degraded to glycerol). This system was scaled up to 100 times the initial volume, achieving a similar yield (65%) at the same IOT. The 2-MAGs in the final alcoholysis reaction mixture were separated from ethyl esters by solvent extraction using solvents of low toxicity (ethanol and hexane); the 2-MAG recovery yield was over 90% and the purity was approximately 87–90%. Regarding the esterification of the 2-MAGs, the following factors were studied: the influence of the lipase type used, the presence or absence of solvent (hexane) and the reaction time or intensity of treatment (IOT = lipase amount × reaction time/2-MAG amount). Of the five lipases tested, the highest STAG percentages (over 90%) were attained with lipases D and DF, immobilized on Accurel MP1000. These STAGs contain 64% CA, of which 98% is at positions 1 and 3. Position 2 contains 5% CA and 45% PUFAs, which means that all the PUFAs that were located at position 2 in the original oil remain in that position in the final STAGs. The lipase D immobilized on Accurel MP1000 is stable in the operational conditions used in the esterification reaction. Finally the purification of STAGs was carried out by neutralization of free fatty acids with hydroethanolic solution of KOH and extraction of STAGs with hexane. By this method purity was over 95% and separation yields were about 80%.     

Extracellular cold-active lipase of Microbacterium luteolum isolated from Gangotri glacier,western Himalaya: Isolation,partial purification and characterization

A psychrophilic bacterium producing cold-active lipase upon growth at low temperature was isolated from the soil samples of Gangotri glacier and identified as Microbacterium luteolum. The bacterial strain produced maximum lipase at 15 °C, at a pH of 8.0. Beef extract served as the best organic nitrogen source and ammonium nitrate as inorganic for maximum lipase production. Castor oil served as an inducer and glucose served as an additional carbon source for production of cold-active lipase. Ferric chloride as additional mineral salt in the medium, highly influenced the lipase production with an activity of 8.01 U ml^?1. The cold-active lipase was purified to 35.64-fold by DEAE-cellulose column chromatography. It showed maximum activity at 5 °C and thermostability up to 35 °C. The purified lipase was stable between pH 5 and 9 and the optimal pH for enzymatic hydrolysis was 8.0. Lipase activity was stimulated in presence of all the solvents (5%) tested except with acetonitrile. Lipase activity was inhibited in presence of Mn²⁺, Cu²⁺, and Hg²⁺; whereas Fe⁺, Na⁺ did not have any inhibitory effect on the enzyme activity. The purified lipase was stable in the presence of SDS; however, EDTA and dithiothreitol inhibited enzyme activity. Presence of Ca²⁺ along with inhibitors stabilized lipase activity. The cold active lipase thus exhibiting activity and stability at a low temperature and alkaline pH appears to be practically useful in industrial applications especially in detergent formulations.     

Cloning,expression and characterization of an organic solvent tolerant lipase from Pseudomonas fluorescens JCM5963

A novel lipase gene from an organic solvent degradable strain Pseudomonas fluorescens JCM5963 was cloned, sequenced, and overexpressed as an N-terminus His-tag fusion protein in E. coli. The alignment of amino acid sequences revealed that the protein contained a lipase motif and shared a medium or high similarity with lipases from other Pseudomonas strains. It could be defined as a member of subfamily I.1 lipase. Most of the recombinant proteins expressed as enzymatically active aggregates soluble in 20 mM Tris–HCl buffer (pH 8.0) containing sodium deoxycholate are remarkably different from most subfamily I.1 and I.2 members of Pseudomonas lipases expressed as inactive inclusion body formerly described in E. coli. The recombinant lipase (rPFL) was purified to homogeneity by Ni-NTA affinity chromatography and Sephacryl S-200 gel filtration chromatography. The purified lipase was stable in broad ranges of temperatures and pH values, with the optimal temperature and pH value being 55 °C and 9.0, respectively. Its activity was found to increase in the presence of metal ions such as Ca²⁺, Sn²⁺ and some non-ionic surfactants. In addition, rPFL was activated by and remained stable in a series of water-miscible organic solvents solutions and highly tolerant to some water-immiscible organic solvents. These features render this novel lipase attraction for biotechnological applications in the field of organic synthesis and detergent additives.     

Optimization studies to develop a low-cost medium for production of the lipases of Rhizopus microsporus by solid-state fermentation and scale-up of the process to a pilot packed-bed bioreactor

A low-cost lipase preparation is required for enzymatic biodiesel synthesis. One possibility is to produce the lipase in solid-state fermentation (SSF) and then add the fermented solids (FS) directly to the reaction medium for biodiesel synthesis. In the current work, we scaled up the production of FS containing the lipases of Rhizopus microsporus. Initial experiments in flasks led to a low-cost medium containing wheat bran and sugarcane bagasse (50:50 w/w, dry basis), supplemented only with urea. We used this medium to scale-up production of FS, from 10 g in a laboratory column bioreactor to 15 kg in a pilot packed-bed bioreactor. This is the largest scale yet reported for lipase production in SSF. During scale-up, the hydrolytic activity of the FS decreased 57%: from 265 U g⁻¹ at 18 h in the laboratory bioreactor to 113 U g⁻¹ at 20 h in the pilot bioreactor. However, the esterification activity decreased by only 14%: from 12.1 U g⁻¹ to 10.4 U g⁻¹. When the FS produced in the laboratory and pilot bioreactors were dried and added directly to a solvent-free reaction medium to catalyze the esterification of oleic acid with ethanol, both gave the same ester content, 69% in 48 h.