Crude Lipase Research Articles

Agroindustrial Wastes as Alternative for Lipase Production by Candida viswanathii under Solid-State Cultivation: Purification, Biochemical Properties, and Its Potential for Poultry Fat Hydrolysis.

The aims of this work were to establish improved conditions for lipase production by Candida viswanathii using agroindustrial wastes in solid-state cultivation and to purify and evaluate the application of this enzyme for poultry fat hydrolysis. Mixed wheat bran plus spent barley grain (1 : 1, w/w) supplemented with 25.0% (w/w) olive oil increased the lipase production to 322.4%, compared to the initial conditions. When olive oil was replaced by poultry fat, the highest lipase production found at 40% (w/w) was 31.43 U/gds. By selecting, yeast extract supplementation (3.5%, w/w), cultivation temperature (30°C), and substrate moisture (40%, w/v), lipase production reached 157.33 U/gds. Lipase was purified by hydrophobic interaction chromatography, presenting a molecular weight of 18.5 kDa as determined by SDS-PAGE. The crude and purified enzyme showed optimum activity at pH 5.0 and 50°C and at pH 5.5 and 45°C, respectively. The estimated half-life at 50°C was of 23.5 h for crude lipase and 6.7 h at 40°C for purified lipase. Lipase presented high activity and stability in many organic solvents. Poultry fat hydrolysis was maximum at pH 4.0, reaching initial hydrolysis rate of 33.17 mmol/L/min. Thus, C. viswanathii lipase can be successfully produced by an economic and sustainable process and advantageously applied for poultry fat hydrolysis without an additional acidification step to recover the released fatty acids.

Characterization of Crude and Partially Purified Lipase from Geotrichum candidum Obtained with Different Nitrogen Sources

AbstractLipases from Geotrichum candidum were produced in two different medium: A = 12 % (w/v) clarified corn steep liquor (CCSL) + 0.6 % (w/v) soybean oil (SO) and B = 3.5 % (w/v) yeast hydrolysate (YH) + 0.7 % (w/v) SO. Lipases were partially purified from both media by hydrophobic interaction chromatography using 3.0 mol L−1 of NaCl as mobile phase, and they were characterized in the crude and partially purified forms. The recovery of lipase activity from CCSL and YH via HIC were 96 and 94.3 %, and the purification factors were 44.3 and 86.7‐fold, respectively. All evaluated lipases had similar optimum pH (7.0–7.7), but, for the CCSL crude lipase, optimum temperature (47 °C) was 10 °C higher than others lipases evaluated. CCSL crude lipase possessed a higher thermo stability than YH crude lipase, e.g., at 37 °C (pH 7.0) the half‐life of CCSL crude lipase was 19.25 h and at pH 8.0 (30 °C) the half‐life was 48 h, which are five and ten times higher than with YH crude lipase, respectively. On the other hand, the YH crude lipase possessed a higher catalytic constant (kcat = 2.3 min−1) but with almost the same catalytic efficiency (Km/kcat = 32.12 mg mL min−1) in relation to CCSL crude lipase. The lipases differ in biocatalytic properties between substrates, suggesting that the two lipases can be employed for different applications.

Pilot-scale aqueous two-phase floatation for direct recovery of lipase derived from Burkholderia cepacia strain ST8

Abstract The efficient and economical pilot-scale production of lipase is necessary for meeting the growing demand of lipase especially in bioprocessing industry. Aqueous two-phase flotation (ATPF) is a purification technique that can be applied in the recovery and purification of biomolecules. ATPF is based on a combination of aqueous two-phase systems (ATPS) and solvent sublation (SS). In this report lipase was recovered and purified from the fermentation broth of Burkholderia cepacia (B. cepacia) using an alcohol/salt ATPF system on a pilot scale. The working parameters of ATPF, including concentration of crude lipase feedstock, types of alcohol and salt, concentrations of alcohol and salt, volumes of buffer solution and alcohol, were investigated for their effects on the partitioning behavior of lipase in ATPF. ATPF comprised of 1-propanol and ammonium sulphate was successfully established for feasible and cost effective separation of B. cepacia ST8 lipase from liquid fermentation broth. The alcohol/salt ATPF system showed a purification factor of 12.2, a separation efficiency of 93% and a selectivity of 40. Furthermore, a comparison has been made between small-scale and large-scale ATPF production. Our results showed that this novel pilot-scale ATPF has the potential to be applied at industrial scale.

Purification and characterization of a surfactant-compatible lipase from Aspergillus tamarii JGIF06 exhibiting energy-efficient removal of oil stains from polycotton fabric.

An extracellular lipase with 23,666.66 U/ml/min activity was produced by Aspergillus tamarii JGIF06 under submerged fermentation in mineral salt medium containing coconut oil (2.5 % v/v), tryptone (2 % w/v) and ammonium chloride (2 % w/v), with initial pH of 5 ± 0.2, incubated at 25 °C for 7 days on a rotary shaker at 120 rpm. A 7.9-fold increase in lipase-specific activity was recorded after purification by DEAE Sepharose ion exchange and Sephadex G200 column chromatography. The apparent molecular mass of this enzyme was revealed as 50 kDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The optimal lipase activity was recorded at pH 4 and 37 °C. The enzyme revealed broad specificity towards different vegetable oils. The K m and V max of the lipase on olive oil was found to be 330.4 mg and 53,690 U/ml/min, respectively. The lipase activity was stable in the presence of surfactants such as cetrimonium bromide, sodium dodecyl sulphate and Tween 80, and metal ions and reagents such as Ca2+, Ba2+ and 2-mercaptoethanol. However, the activity was greatly reduced in the presence of organic solvents such as chloroform. The stain removal potential of the crude lipase was determined on polycotton fabric pieces stained with peanut oil. Lipase added to cold water alone significantly enhanced the removal of stain by 152 %. The addition of lipase also improved the stain removal efficiency of a commercially available detergent in the presence of either cold (25 ± 2 °C) or hot (65 ± 2 °C) water. The current findings suggest the potentiality of this enzyme for energy-efficient biocatalytic application.

Pseudomonas sp. BCNU 106이 생산하는 유기용매 내성 리파아제의 특성

유기용매 내성 세균 Pseudomonas sp. BCNU 106으로부터 생산된 리파아제 조효소액은 pH 4-10의 넓은 범위의 pH와 37℃에서 매우 안정적이었다. BCNU 106의 리파아제 안정성은 25% xylene, hexane, octane, toluene, chloroform 및 dodecane에서 증가하였으며, 상업적인 고정화 효소와 비교해도 우수한 안정성을 보이고 있다. 그리고 Cu²⁺, Hg²⁺, Zn²⁺ 및 Mn²⁺ 존재 하에서 110% 이상의 상대활성을 나타낸 반면에, Fe²⁺에서는 효소활성이 억제되었다. 게다가 계면활성제인 tween 80과 triton X-100 및 SDS에서도 높은 안정성을 유지됨이 확인되었다. 본 연구에서 유기용매 내성 Pseudomonas sp. BCNU 106의 리파아제는 고정화 효소에 못지않은 효소 활성 및 안정성을 유지함이 밝혀져 다양한 산업공정에서 잠재적인 생물촉매로 적용될 수 있는 가능성을 확인할 수 있었다. A crude extracellular lipase from solvent-tolerant bacterium Pseudomonas sp. BCNU 106 was highly stable in the broad pH range of 4-10 and at temperature of 37℃. Crude lipase of BCNU 106 exhibited enhanced stability in 25% organic solvents such as xylene (121.85%), hexane (120.35%), octane (120.41 %), toluene (118.14%), chloroform (103.66%) and dodecane (102.94%) and showed excellent stability comparable with the commercial immobilized enzyme. In addition, the stability of BCNU 106 lipase retained above 110% of its enzyme activity in the presence of Cu²⁺, Hg²⁺, Zn²⁺ and Mn²⁺, whereas Fe²⁺ strongly inhibited its stability. The detergents including tween 80, triton X-100 and SDS were positive signals for lipase stability. Because of its stability in multiple organic solvents, cations and surfactants, the Pseudomonas sp. BCNU 106 lipase could be considered as a potential biocatalyst in the industrial chemical processes without using immobilization.

Characterization of transesterification reactions by Mucoromycotina lipases in non-aqueous media

Transesterification properties of Rhizomucor, Rhizopus, Mucor, Umbelopsis and Mortierella crude lipases were investigated in water-free systems using an UV spectrophotometric method. Conversion of p-nitrophenyl palmitate (pNPP) with ethanol was characterized under various reaction conditions. Best conversion yields were generally obtained at 40°C in n-heptane, but temperature of 50°C and the solvents hexane and isooctane proved to be also useful. Then, crude lipases from five strains, namely Rhizomucor miehei NRRL 5282, Rhizopus oryzae NRRL 1526, Rhizopus stolonifer SZMC 13609, Mucor corticolus SZMC 12031 and Mortierella echinosphaera CBS 575.75 were selected for further kinetic studies and to evaluate the effect of the acyl acceptors and donors with various chain lengths on the reaction. In general, ethanol up to 4.25mol/L concentration enhanced the catalytic activities, and a steady conversion with maximum yields ranging from 61.2% to 90.5% was obtained at about 96h. Replacement of ethanol as the acyl acceptor with methanol, isopropanol, n-butanol or n-hexanol resulted in lower conversions when incubated with the same concentration. The selected crude lipases demonstrated high initial affinity toward the medium-chain aryl esters with C8 and C12 acids. Esterification of palmitic acid with ethanol showed much slower catalysis rates resulting in less specific activities than transesterification. The high stability in organic solvents makes the investigated crude enzymes promising candidates for further studies aiming at the application in organic synthesis processes.

Cocoa pod husk, a new source of hydrolase enzymes for preparation of cross-linked enzyme aggregate.

Cocoa pod husk (CPH) is a by-product of cocoa production obtained after removing the beans from the fruit. The analysis of CPH has shown that it contains high amounts of protein. This study is aimed to utilize this protein source in hydrolase enzyme production. In this study, seven hydrolase enzymes (amylase, fructosyltransferase, mannanase, glucosidase, glucanase, lipase and protease) were screened from CPH for the first time for feasible industrial production. Among these hydrolases, lipase was chosen for the next steps of experiments as it has a lot of applications in different industries. The extraction of high active lipase from CPH has been done under optimum conditions. The condition that was optimum for the three major factors was achieved using Face centered central composite design (FCCCD) with response surface methodology (RSM) to obtain the highest enzyme activity of crude lipase from CPH. The optimum condition of extraction is used for preparation of cross-linked enzyme aggregate (CLEA). For the production of immobilized biocatalyst, the technique of CLEA is considered as an effective technique for its industrially attractive advantages. Referring to the results of OFAT, CLEA-lipase was prepared in the best condition at the presence of 30 mM ammonium sulphate, 70 mM glutaraldehyde with 0.23 mM Bovine serum albumin as an additive. Immobilization effectively improved the stability of lipase against various organic solvents.

Zinc Oxide Nanoparticles Supported Lipase Immobilization for Biotransformation in Organic Solvents: A Facile Synthesis of Geranyl Acetate, Effect of Operative Variables and Kinetic Study.

The present study describes grafting of zinc oxide (ZnO) nanoparticles with polyethyleneimine (PEI) followed by modification with glutraldehyde used as the bridge for binding the enzyme to support. The prepared nanocomposites were then characterized using Fourier transform infrared spectroscopy, thermogravimetric analysis, and transmission electron microscopy, utilized for synthesis of geranyl acetate in n-hexane. Among all the three prepared nanocomposites (ZnO + PEI, ZnO + PEI + SAA, ZnO + PEI + GLU), Candida rugosa lipase immobilized on ZnO-PEI-GLU was found to be best for higher ester synthesis. The operating conditions that maximized geranyl acetate resulted in the highest yield of 94% in 6h, molar ratio of 0.1:0.4M (geraniol/vinyl acetate) in the presence of n-hexane as reaction medium. Various kinetic parameters such as V max, K i(G), K m(G), and K m(VA) were determined using nonlinear regression analysis for order bi-bi mechanism. The kinetic study showed that reaction followed order bi-bi mechanism with inhibition by geraniol. Activation energy (E a ) was found to be lower for immobilized lipase (12.31kJmol(-1)) than crude lipase (19.04kJmol(-1)) indicating better catalytic efficiency of immobilized lipase. Immobilized biocatalyst demonstrated 2.23-fold increased catalytic activity than crude lipase and recycled 20 times. The studies revealed in this work showed a promising perspective of using low-cost nanobiocatalysts to overcome the well-known drawbacks of the chemical-catalyzed route.

Characterization of biotechnologically relevant extracellular lipase produced by Aspergillus terreus NCFT 4269.10.

Enzyme production by Aspergillus terreus NCFT 4269.10 was studied under liquid static surface and solid-state fermentation using mustard oil cake as a substrate. The maximum lipase biosynthesis was observed after incubation at 30 °C for 96 h. Among the domestic oils tested, the maximum lipase biosynthesis was achieved using palm oil. The crude lipase was purified 2.56-fold to electrophoretic homogeneity, with a yield of 8.44%, and the protein had a molecular weight of 46.3 kDa as determined by SDS-PAGE. Enzyme characterization confirmed that the purified lipase was most active at pH 6.0, temperature of 50 °C, and substrate concentration of 1.5%. The enzyme was thermostable at 60 °C for 1 h, and the optimum enzyme–substrate reaction time was 30 min. Sodium dodecyl sulfate and commercial detergents did not significantly affect lipase activity during 30-min incubation at 30 °C. Among the metal ions tested, the maximum lipase activity was attained in the presence of Zn2+, followed by Mg2+ and Fe2+. Lipase activity was not significantly affected in the presence of ethylenediaminetetraacetic acid, sodium lauryl sulfate and Triton X-100. Phenylmethylsulfonyl fluoride (1 mM) and the reducing, β-mercaptoethanol significantly inhibited lipase activity. The remarkable stability in the presence of detergents, additives, inhibitors and metal ions makes this lipase unique and a potential candidate for significant biotechnological exploitation.

Liquid–liquid extraction of lipase produced by psychrotrophic yeast Leucosporidium scottii L117 using aqueous two-phase systems

Aqueous two-phase systems (ATPS) have been used in biomolecules separation and as an efficient alternative to traditional purification systems for lipases extraction. Here, we investigated the partitioning and recovery of lipase derived from Leucosporidium scottii L117 using ATPS and aqueous two-phase micellar systems (ATPMS). Thus, we evaluated three ATPS: (i) polyethylene glycol (PEG)/phosphate salts and (ii) PEG/polyacrylic acid (NaPA) in different molecular weights (1500, 4000 and 8000 g/mol). (iii) Triton X-114 (TX-114)/McIlvaine buffer pH 7.0 in different conditions (2.0% (w/w) of TX-114 at 25.0 and 28.0 °C). The PEG/phosphate and PEG/NaPA systems resulted in a great loss of enzymatic activity; thus these systems do not represent viable alternatives for these lipase extraction. The micellar systems yielded the best results for lipase extraction with enzyme activity balances ranging between 84.7% and 113.05%. After optimizing the micellar system by experimental design of the partition coefficient of lipase increased by 10.3-fold (0.75–7.76). Lipase preferentially partitioned into the micelle-rich phase with KLip = 7.76, %RECBot = 93.85% and PF = 1.2 at 25.03 °C, 5.1 pH and 10.38% TX-114 and KLip = 4.77, %RECBot = 73.53% and PF = 1.97 at 28.00 °C, 4.5 pH and 8.0% TX-114, indicating that the ATPMS represents an alternative to purification/extraction of lipase L. scottii L117. A crude lipase extract was also evaluated to define the optimum pH and temperature. Lipase reached optimal activity at 40 °C, and remained stable in pH values ranging from pH 3.0 to 8.0 and temperatures from 20.0 to 45.0 °C, with relative residual lipase activity above 80% after 30 min of incubation.

Utilization of agro-industrial wastes for the production of lipase from Stenotrophomonas maltophilia isolated from Arctic and optimization of physical parameters

Abstract In the present work production of lipase by Arctic sediment isolate Stenotrophomonas maltophilia was investigated , and focus was on the lipase production using agro industrial waste. Optimization of physical parameters which enhance the lipase production in agricultural waste such as groundnut cake, rice bran, wheat bran, neem cake, and coconut cake was achieved and significance was validated by ANOVA. The crude lipase was concentrated by precipitation with acetone and further purification was carried out by ion exchange chromatography followed by SDS-PAGE. When screened for extracellular lipase production, 54 isolates showed positive results and significant activity was expressed by sediment isolate KS 46 which was phylogenetically identified to be S. maltophilia . The groundnut cake supported maximum enzyme production (74.117 U/ml) followed by coconut cake (61.911 U/ml), neem cake (58.737 U/ml), wheat bran (49.614 U/ml) and rice bran (28.165 U/ml). Characterization of the enzyme revealed the optimum temperature to be 28 °C and optimum pH to be 6 for groundnut cake, neem cake and coconut cake while pH 7 for rice bran and wheat bran. Maximum lipase production was attained at 60% moisture for all substrates except for neem cake (70%). The enzyme was purified to 2.9 fold and molecular weight was found to be 49.1 KDa. The results suggest that agro wastes such as groundnut cake has the potential to be used as alternative cost effective substrate for lipase production and optimum conditions vary for the same enzyme when different substrates are utilized.

Purification and Characterization of Lipase produced by Pseudomonas aeruginosa isolated from some soils of Basrah Governorate

The present study aimed to Purification and characterization of lipase produced by the isolate Pseudomonas aeruginosa. The results showed that crude lipase was purified by using the precipitation step of several concentrations ratios of cold acetone. The concentration ratio 4:1 gave the best specific activity 6 U/mg of protein. The subsequent purification step using gel filtration was showed that specific activity reached to 40.5U/mg of protein. The gel electrophoresis (SDS-PAGE) of lipase purity were determined. the result showed there is an appearance of a single protein band predominant in all purification steps, While The molecular weight of purified lipase was 68KDa

Lipase catalyzed interesterification of Amazonian patauá oil and palm stearin for preparation of specific-structured oils.

This study showed that enzymatic interesterification of Amazonian oils could be an important tool in order to produce new oils with physicochemical properties that improve the applications of these raw materials. Structured oils of Amazonian patauá oil and palm stearin using two lipases were produced in three different enzymatic systems: first, a crude lipase from the fungus Rhizopus sp (a microorganism isolated in our laboratory); second, a commercial lipase; and third, to check any synergistic effect, a mixture of both lipases (Rhizopus sp and commercial). The lipase from Rhizopus sp was specific in the incorporation of oleic acid at the sn-1,3 positions of the triacylglycerol, resulting in an oil richer in saturated fatty acid in the sn-2 position. This enzyme, produced by solid-state fermentation, even though crude, was fatty acid and positional specific and able to operate at low concentration (2.5%, w/w). In the second enzyme system, the commercial lipase from Thermomyces lanuginosus was not specific in the tested conditions; there was no change in the distribution of saturated and unsaturated fatty acids in the three positions of the triacylglycerol profile, there was only a replacement by the type of fatty acid at the same position. In the third enzyme system, the mixture of both lipases shows no synergic effect. The structured oils retained the concentration of bioactive α- and γ- tocopherol in the three enzyme systems. Triacylglycerol classes and Thermal behavior tests indicated the formation of more homogeneous triacylglycerols, especially the mono and di-unsaturated.

Pretreatment of coconut mill effluent using celite-immobilized hydrolytic enzyme preparation from Staphylococcus pasteuri and its impact on anaerobic digestion.

Biological treatment of oil and grease (O&G)-containing industrial effluents has long been a challenging issue. Practically feasible avenues to bring down their O&G load and enhance treatability are desired. In one such endeavour, the partially purified lipase from Staphylococcus pasteuri COM-4A was immobilized on celite carrier and applied for the enzymatic hydrolysis of unsterilized coconut oil mill effluent. In batch hydrolysis experiments, optimum conditions of 1% (w/v) immobilized lipase beads, one in four effluent dilution, and a contact time of 30 h resulted in 46% and 24% increase in volatile fatty acids and long-chain fatty acids and a concomitant 52% and 32% decrease in O&G and chemical oxygen demand (COD) levels, respectively. Batch anaerobic biodegradation trials with this prehydrolyzed effluent showed 89%, 91%, and 90% decrease in COD, proteins, and reducing sugars, respectively. These results were validated in a hybrid stirred tank--upflow anaerobic sludge blanket reactor. Average COD and O&G reductions effected by the hybrid reactor were found to be 89% and 88%, whereas that by the control reactor without enzymatic hydrolysis were only 60% and 47%, respectively. A maximum of 0.86 L methane gas was generated by the hybrid reactor per gram of VS added. Hence, this celite-immobilized crude lipase, sourced from a native laboratory isolate, seems to be a workable alternative to commercial enzyme preparations for the management of lipid-rich industrial effluents.

양송이 배지에서 유래한 Lipase 생산균을 이용한 바이오디젤 생산

A lipase producing bacterium was isolated from button mushroom bed, which showing high clear zone on agar media containing Tributyrin as the substrate. The strain was identified as Burkholderia cepacia by analysis of 16S rDNA gene sequence. Crude lipase (CL) was partially purified from 70% ammonium sulfate precipitation using the culture filtrate of B. cepacia. Immobilized lipases were prepared by cross-linking method with CL from B. cepacia and Novozyme lipase (NL) onto silanized Silica-gel as support. Residual activitiy of the immobilized CL (ICL) and immobilized NL (INL) was maintained upto 61% and 72%, respectively. Biodiesel (Fatty acid methyl ester, FAME) was recovered by transesterification and methanolysis of Canola oil using NaOH, CL and ICL as the catalysts to compare the composition of fatty acids and the yield of FAME. Total FAME content was NaOH 781 mg L-1, CL 681 mg L-1 and ICL 596 mg L-1, in which the highest levels of FAME was observed to 50% oleic acid (C18:1) and 22% stearic acid (C18:0). In addition, the unsaturated FAME (C18:1, C18:2) decreased, while saturated FAME (C16:0, C18:0) increased according to increasing the reaction times with both CL and ICL, supporting CL possess both transesterification and interesterification activity. When reusability of ICL and INL was estimated by using the continuous reaction of 4 cycles, the activity of ICL and INL was respectively maintained 66% and 79% until the fourth reaction.

Immobilized lipase from Hypocrea pseudokoningii on hydrophobic and ionic supports: Determination of thermal and organic solvent stabilities for applications in the oleochemical industry

Hypocrea pseudokoningii purified lipase was immobilized on hydrophobic supports (phenyl-sepharose, butyl-sepharose, octyl-sepharose, Hexyl Toyopearl, Lewatit, Purolite, Decaoctyl sepabeads) and ionic supports (Duolite, DEAE-agarose, PEI-agarose, MANAE-agarose, and Q-sepharose). The immobilization processes resulted in derivatives with excellent thermal stabilities, increasing the half-life up to 500-fold. The derivatives had excellent stability to organic solvents compared to the crude lipase. In the presence of 50% ethanol, hexyl and Decaoctyl derivatives increased by about 6- and 3.5-fold their stability to organic solvents, respectively. When tested for methanol, phenyl-sepharose derivative also increased their stability to organic solvents in approximately 2-fold. Octyl-sepharose derivative was fully stable for 48h in the presence of propanol, which showed a half-life of about 7.5h. The greater activation of the derivatives occurred using 50% cyclohexane, in which the hexyl derivative obtained an increase in the activity of 9-fold and phenyl and octyl derivatives had their activity increased by 6-fold. The lipase showed activity on different oils. Therefore, the adsorption of lipases in low ionic strength and highly hydrophobic supports is shown to be a simple and rapid tool for the immobilization of H. pseudokoningii lipase. These derivatives strongly increase the chances of this biocatalyst for industrial application.

A new thermostable and organic solvent-tolerant lipase from Staphylococcus warneri; optimization of media and production conditions using statistical methods.

A new thermostable and solvent-tolerant lipase was isolated from newly isolated Staphylococcus warneri from oil-contaminated soil. Optimization of the fermentation media for production of thermostable and organic solvent-tolerant lipase was carried out using two statistical methods, i.e., Plackett-Burman design (PBD) and central composite design (CCD) were used for the optimization of the media components. PBD was used to efficiently select important medium components affecting the lipase production. Out of 15 medium components screened, four components, i.e., olive oil, peptone, maltose, and K2HPO4 were found to contribute positively to lipase production. CCD and response surface methodology (RSM) were used to determine the optimum levels of the selected components using Design-Expert 8.0 software. Production medium with olive oil (1.45 %), peptone (0.28 %), maltose (0.054 %), and K2HPO4 (0.091 %) was optimized with a maximum lipase production of 10.43 IU/ml/min. Similarly, production conditions for the lipase production were optimized by using CCD and RSM. Optimized conditions were found to have an incubation temperature of 55 °C, medium pH of 8.0, agitation of 120 rpm, and inoculum volume of 2 %. RSM revealed the maximum lipase production of 17.21 IU/ml using these optimized production conditions. Crude lipase showed enhanced activity in organic solvents such as diethyl ether, hexane, and cyclohexane.

Synthesis of crosslinked lipase aggregates and their use in the synthesis of aspirin

Biocatalytic synthesis of drugs is imperative to avoid the hazardous environmental effects of the pharmaceutical production processes. In view of this, biocatalytic synthesis of aspirin is discussed in the present work. A crude lipase extracted from Geobacillus sp. was crosslinked using N,N-methylenebisacrylamide (N,N-MBAAm) and two different initiators: ammonium persulphate (APS) and γ-irradiation. The crude lipase was crosslinked as such bypassing the separation procedures using a biocompatible crosslinker. The crosslinked lipase was characterized by various techniques and evaluated for its activity profile as a function of time, temperature, pH and substrate concentration and results were compared to its free form. The activity profile of the crosslinked lipase was observed to be only marginally lower than the free lipase. The crosslinked lipases were used as catalysts in the green synthesis of acetylsalicylic acid, aspirin, directly from the acetic acid. The reusability studies of the crosslinked lipases were carried out up to 10 cycles.

Effect of ethanol concentrations on rice bran protease activity and ester synthesis during enzymatic synthesis of oleic acid ethyl ester in a fed-batch system using crude rice bran (Oryza sativa) lipase

Crude rice bran lipase was used for the enzymatic synthesis of Oleic Acid Ethyl Ester (OAEE) in a fed-batch system. It was found that rice bran contains protease (4.19 U/g) and lipase (324.03 U/g). The results show that protease inhibits lipase at a low concentration of ethanol (5 μmol/ml), in which lipase esterification activity was only 45.02 U/g. However, protease activity decreased about 11 times at a higher concentration of ethanol (5–50 μmol/ml). It resulted in an increase in the esterification activity of lipase (324.03 U/g). This finding could be used for explaining the effects of ethanol on the enzymatic synthesis of OAEE using crude rice bran lipase in a fed-batch system. When the ethanol concentration in the reaction system was low (≤ 4 μmol/ml), the final OAEE conversion was only 30%. It was caused both by the inhibition of lipase by rice bran protease and by the effect of low ethanol concentration on the equilibrium position of the lipase-catalysed esterification reaction. However, the final OAEE conversion reached 76–92% when higher ethanol concentrations (12.5–50 μmol/ml) were added at initial reaction. It is suggested that the addition of higher ethanol concentrations (12.5–50 μmol/ml) into the reaction system may reduce protease activity and also increase ethanol concentration in the reaction system to be converted into OAEE.

Biochemical characterisation and application of lipases produced by Aspergillus sp. on solid‐state fermentation using three substrates

SummaryLipase from Aspergillus sp. obtained by solid‐state fermentation (SSF) on wheat bran (LWB), soybean bran (LSB) and soybean bran combined with sugarcane bagasse (LSBBC) were 67.5, 58 and 57.3 U of crude lipase per gram substrate, respectively. The optimum pH of activity and stability of the LWB was between 8 and 9, and the optimum temperature of activity and stability was 50 °C and up to 60 °C, respectively. The LSB and LSBBC showed two peaks of optimum pH (4 and 6) and optimal values of temperature and stability at 50 °C. The LSB was stable in the pH range of 6–7, while LSBBC in the range of pH 4–7. All the enzymes show activities on p‐nitrophenyl esters (butyrate, laurate and palmitate). LWB stood out either on the hydrolysis of sunflower oil, presenting 66.1% of the activity over commercial lipase and on the esterification of oleic acid and ethanol, surpassing the activities of the commercial lipases studied. The thin layer chromatography showed that LWB and LSB have produced ethyl esters from corn oil, while LWB produced it from sunflower oil.