Initially, a new moderate halophilic strain was locally isolated from seawater. The partial 16S rRNA sequence analysis positioned the organism in Marinobacter genus and was named 'Marinobacter litoralis SW-45'. This study further demonstrates successful utilization of the halophilic M. litoralis SW-45 lipase (MLL) for butyl ester synthesis from crude palm fruit oil (CPO) and kernel oil (CPKO) in heptane and solvent-free system, respectively, using hydroesterification. Hydrolysis and esterification of enzymatic [Thermomyces lanuginosus lipase (TLL)] hydrolysis of CPO and CPKO to free fatty acids (FFA) followed by MLL-catalytic esterification of the concentrated FFAs with butanol (acyl acceptor) to synthesize butyl esters were performed. A one-factor-at-a-time technique (OFAT) was used to study the influence of physicochemical factors on the esterification reaction. Under optimal esterification conditions of 40 and 45 °C, 150 and 230 rpm, 50% (v/v) biocatalyst concentration, 1:1 and 5:1 butanol:FFA, 9% and 15% (w/v) NaCl, 60 and 15 min reaction time for CPO- and CPKO-derived FFA esterification system, maximum ester conversion of 62.2% and 69.1%, respectively, was attained. Gas chromatography (GC) analysis confirmed the products formed as butyl esters. These results showed halophilic lipase has promising potential to be used for biosynthesis of butyl esters in oleochemical industry.
Enzymatic catalysis is considered to be among the most environmental friendly processes for the synthesis of fine chemicals. In this study, lipase from Thermomyces lanuginosus (Lecitase Ultra™) was used to catalyze the synthesis of flavor esters, i.e., methyl butanoate and methyl benzoate by esterification of the acids with methanol in a microfluidic system. Maximum reaction rates of 195 and 115 mM min(-1) corresponding to catalytic efficiencies (k cat/K M) of 0.30 and 0.24 min(-1) mM(-1) as well as yield conversion of 54 and 41 % were observed in methyl butanoate and methyl benzoate synthesis, respectively. Catalytic turnover (k cat) was higher for methyl butanoate synthesis. Rate of synthesis and yield decreased with increasing flow rates. For both esters, increase in microfluidic flow rate resulted in increased advective transport over molecular diffusion and reaction rate, thus lower conversion. In microfluidic synthesis using T. lanuginosus lipase, the following reaction conditions were 40 °C, flow rate 0.1 mL min(-1), and 123 U g(-1) enzyme loading found to be the optimum operating limits. The work demonstrated the application of enzyme(s) in a microreactor system for the synthesis of industrially important esters.
This study enhanced the production of thermostable organic solvent-tolerant (TS-OST) lipase by locally isolated thermotolerant Rhizopus sp. strain using solid-state fermentation (SSF) of palm kernel cake (PKC). The optimum conditions were achieved using a series of statistical approaches. The cultivation parameters, which include fermentation time, moisture content, temperature, pH, inoculum size, various carbon and nitrogen sources, as well as other supplements, were initially screened by the definitive screening design, and one-factor-at-a-time using PKC as the basal medium. Three significant factors (olive oil concentration, pH, and inoculum size) were further optimized using face-centred central composite design. The results indicated a successful and significant improvement of lipase activity by almost two-fold compared to the initial screening production. The findings showed that the optimal conditions were 2% (v/w) inoculum size, 2% (v/w) olive oil, 0.6% (w/w) peptone, 2% (v/w) ethanol, 70% moisture content at initial pH 10.0 and 45 °C within 72 h of fermentation. Process optimization resulted in maximum lipase activity of 58.63 U/gram dry solids (gds). The analysis of variance showed that the statistical model was significant (p value <0.0001) and reliable with a high value of R2 (0.98) and adjusted R2 (0.96). This indicates a better correlation between the actual and predicted responses of lipase production. By considering this study, the low-cost PKC through SSF appears to be promising in the utilization of agro-industrial waste for TS-OST lipase production. This is because satisfactory enzyme activity could be attained that promises industrial applications.
Immobilization can be used to improve the stability of lipases and enhances lipase recovery and reusability, which increases its commercial value and industrial applications. Nevertheless, immobilization frequently causes conformational changes of the lipases, which decrease lipase catalytic activity. in the present work, we synthesized UIO-66 and grafted UIO-66 crystals with proline for immobilization of Candida rugosa lipase (CRL). As indicated by steady-state fluorescence microscopy, grafting of proline onto UIO-66 crystals induced beneficial conformational change in CRL. CRL immobilized on UIO-66/Pro (CRL@UIO-66/Pro) demonstrated higher enzyme activity and better recyclability than that immobilized on UIO-66 (CRL@UIO-66) in both hydrolysis (CRL@UIO-66/Pro: 0.34 U; CRL@UIO-66: 0.15 U) and transesterification (CRL@UIO-66/Pro: 0.93 U; CRL@UIO-66: 0.25 U) reactions. The higher values of kcat and kcat/Km of CRL@UIO-66/Pro also showed that it had better catalytic efficiency as compared to CRL@UIO-66. It is also worth noting that CRL@UIO-66/Pro (0.93 U) demonstrated a much higher transesterification activity as compared to free CRL (0.11 U), indicating that UIO-66/Pro has increased the solvent stability of CRL. Both CRL@UIO-66 and CRL@UIO-66/Pro were also used for the fabrication of biosensors for nitrofen with a wide linear range (0-100 μM), lower limit of detection, and good recovery rate.
In recent years, many efforts have been directed to explore the methods to reduce the production costs of industrial lipase by improving the yield and the use of low-cost agricultural wastes. Coconut dregs, which is a lignocellulosic by-product from coconut oil and milk processing plants, is rich in cellulose (36%) and crude fat (9%). A newly isolated Bacillus stratosphericus has been demonstrated to perform cellulose hydrolysis on coconut dregs producing fermentable sugars. The highest extracellular lipase activity of 140 U/mL has been achieved in submerged fermentation with acid pre-treated coconut dregs. The lipase was found to be active over a wide range of temperatures and pHs. The activity of lipase can be generally increased by the presence of detergent ingredients such as Tween-80, cetyltrimethylammonium bromide, hydrogen peroxide and phosphate per sulphate. The great compatibility of lipase in commercial detergents has also underlined its potential as an additive ingredient in biodetergent formulations.
A novel method for the assay of polyhydroxyalkanoate (PHA)-degrading ability of triacylglycerol lipases was developed. By applying the natural affinity of lipases towards hydrophobic interfaces, a sensitive and rapid densitometry analysis for the evaluation of hydrolytic activity of lipase droplets towards PHA-coated surface was successfully carried out. We found that 12 out of 14 tested lipases which are of fungal, bacterial and animal origin were able to hydrolyze P(3HB-co-92 mol% 4HB) thin film. The patterns and opacity of the hydrolysis spots of lipases on PHA films allowed easy comparison of PHA-hydrolytic strength of lipases. Lipase from the bacterium Chromobacterium viscosum exhibited the highest PHA-degrading activity. The hydrolytic activity of lipases on water insoluble PHA, emulsified p-nitrophenyl laurate and olive oil were also compared and interestingly some lipases showed better activity when PHA was used as a substrate.
Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] is one of the polyhydroxyalkanoate (PHA) copolymers which can be degraded by lipases. In this study, the depolymerizing activity of different known commercial lipases was investigated via microassay using P(3HB-co-92 mol % 4HB) thin film as substrate. Non-enzymatic hydrolysis occurred under conditions in which buffers with pH 12 and 13 were added or temperature of 50 °C and above. Different concentrations of metal ions or detergents alone did not cause the film hydrolysis. The depolymerizing activity of lipases on P(3HB-co-4HB) was optimum in the pH range of 6-8 and at temperatures between 30 and 50 °C. Addition of metal ions and detergents in different concentrations was also shown to cause variable effects on the depolymerizing activity of commercial lipases. Pancreatic extracts from both mouse and chicken showed similar depolymerizing activity as the commercial lipases on the P(3HB-co-4HB) film. The presence of lipolytic enzymes in the organ extracts was confirmed with another lipase activity assay, p-nitrophenyl laurate assay. For the first time this has produced a direct evidence for the involvement of lipase-like enzymes from animal in the degradation of this PHA. Lipase is most likely the enzyme from pancreas that was involved in the degradation.
Yeasts are a convenient platform for many applications. They have been widely used as the expression hosts. There is a need to have a new yeast expression system to contribute the molecular cloning demands. Eight yeast isolates were screened from various environment sources and identified through ribosomal DNA (rDNA) Internal Transcribed Spacer (ITS). Full sequence of the rDNA ITS region for each isolate was BLASTed and phylogenetic study was constructed by using MEGA4. Among the isolates, isolate WB from 'ragi' (used to ferment carbohydrates) could be identified as a new species in order Saccharomycetales according to rDNA ITS region, morphology and biochemical tests. Isolate SO (from spoiled orange), RT (rotten tomato) and RG (different type of 'ragi') were identified as Pichia sp. Isolates R1 and R2, S4 and S5 (from the surrounding of a guava tree) were identified as Issatchenkia sp. and Hanseniaspora sp., respectively. Geneticin, 50 µg/mL, was determined to be the antibiotic marker for all isolates excepted for isolates RT and SO which used 500 µg/mL and 100 µg/mL Zeocin, respectively. Intra-extracellular proteins were screened for lipolytic activity at 30°C and 70°C. Thermostable lipase activity was detected in isolates RT and R1 with 0.6 U/mg and 0.1 U/mg, respectively. In conclusion, a new yeast-vector system for isolate WB can be developed by using phleomycin or geneticin as the drugs resistance marker. Moreover, strains RT and R1 can be investigated as a novel source of a thermostable lipase.
Purified thermostable recombinant L2 lipase from Bacillus sp. L2 was crystallized by the counter-diffusion method using 20% PEG 6000, 50 mM MES pH 6.5 and 50 mM NaCl as precipitant. X-ray diffraction data were collected to 2.7 A resolution using an in-house Bruker X8 PROTEUM single-crystal diffractometer system. The crystal belonged to the primitive orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 87.44, b = 94.90, c = 126.46 A. The asymmetric unit contained one single molecule of protein, with a Matthews coefficient (V(M)) of 2.85 A(3) Da(-1) and a solvent content of 57%.
An organic solvent-tolerant lipase from Bacillus sp. strain 42 was crystallized using the capillary-tube method. The purpose of studying this enzyme was in order to better understand its folding and to characterize its properties in organic solvents. By initially solving its structure in the native state, further studies on protein-solvent interactions could be performed. X-ray data were collected at 2.0 Å resolution using an in-house diffractometer. The estimated crystal dimensions were 0.09×0.19×0.08 mm. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a=117.41, b=80.85, c=99.44 Å, β=96.40°.
An organic solvent-tolerant S5 lipase was purified by affinity chromatography and anion exchange chromatography. The molecular mass of the lipase was estimated to be 60 kDa with 387 purification fold. The optimal temperature and pH were 45 degrees C and 9.0, respectively. The purified lipase was stable at 45 degrees C and pH 6-9. It exhibited the highest stability in the presence of various organic solvents such as n-dodecane, 1-pentanol, and toluene. Ca2+ and Mg2+ stimulated lipase activity, whereas EDTA had no effect on its activity. The S5 lipase exhibited the highest activity in the presence of palm oil as a natural oil and triolein as a synthetic triglyceride. It showed random positional specificity on the thin-layer chromatography.
In silico and experimental investigations were conducted to explore the effects of substituting hydrophobic residues, Val, Met, Leu, Ile, Trp, and Phe into Gln 114 of T1 lipase. The in silico investigations accurately predicted the enzymatic characteristics of the mutants in the experimental studies and provided rationalization for some of the experimental observations. Substitution with Leu successfully improved the conformational stability and enzymatic characteristics of T1 lipase. However, replacement of Gln114 with Trp negatively affected T1 lipase and resulted in the largest disruption of protein stability, diminished lipase activity and inferior enzymatic characteristics. These results suggested that the substitution of a larger residue in a densely packed area of the protein core can have considerable effects on the structure and function of an enzyme. This is especially true when the residue is next to the catalytic serine as demonstrated with the Phe and Trp mutation.
A thermostable lipase from Geobacillus zalihae strain T1 was chemically modified using propionaldehyde via reductive alkylation. The targeted alkylation sites were lysines, in which T1 lipase possessed 11 residues. Far-UV circular dichroism (CD) spectra of both native and alkylated enzyme showed a similar broad minimum between 208 and 222 nm, thus suggesting a substantial amount of secondary structures in modified enzyme, as compared with the corresponding native enzyme. The hydrolytic activity of the modified enzymes dropped drastically by nearly 15-fold upon chemical modification, despite both the native and modified form showed distinctive α-helical bands at 208 and 222 nm in CD spectra, leading us to the hypothesis of formation of a molten globule (MG)-like structure. As cooperative unfolding transitions were observed, the modified lipase was distinguished from the native state, in which the former possessed a denaturation temperature (T(m)) in lower temperature range at 61 °C while the latter at 68 °C. This was further supported by 8-anilino-1-naphthalenesulfonic acid (ANS) probed fluorescence which indicated higher exposure of hydrophobic residues, consequential of chemical modification. Based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis, a small number of lysine residues were confirmed to be alkylated.
In this study, an artificial neural network (ANN) trained by backpropagation algorithm, Levenberg-Marquadart, was applied to predict the yield of enzymatic synthesis of dioctyl adipate. Immobilized Candida antarctica lipase B was used as a biocatalyst for the reaction. Temperature, time, amount of enzyme, and substrate molar ratio were the four input variables. After evaluating various ANN configurations, the best network was composed of seven hidden nodes using a hyperbolic tangent sigmoid transfer function. The correlation coefficient (R2) and mean absolute error (MAE) values between the actual and predicted responses were determined as 0.9998 and 0.0966 for training set and 0.9241 and 1.9439 for validating dataset. A simulation test with a testing dataset showed that the MAE was low and R2 was close to 1. These results imply the good generalization of the developed model and its capability to predict the reaction yield. Comparison of the performance of radial basis network with the developed models showed that radial basis function was more accurate but its performance was poor when tested with unseen data. In further part of the study, the feedforward backpropagation model was used for prediction of the ester yield within the given range of the main parameters.
Synthesis of layered double hydroxides (LDHs) of Zn/Al-NO3- hydrotalcite (HIZAN) and Zn/Al-diocytyl sodium sulfosuccinate (DSS) nanocomposite (NAZAD) with a molar ratio of Zn/Al of 4:1 were carried out by coprecipitation through continuous agitation. Their structures were determined using X-ray diffractometer spectra, which showed that basal spacing for LDH synthesized by both methods was about 8.89 A. An expansion of layered structure of about 27.9 A was observed to accommodate the surfactant anion between the interlayer. This phenomenon showed that the intercalation process took place between the LDH interlayer. Lipase from Candida rugosa was immobilized onto these materials by physical adsorption method. It was found that the protein loading onto NAZAD is higher than HIZAN. The activity of immobilized lipase was investigated through esterification of oleic acid and 1-butanol in hexane. The effects of pore size, surface area, reaction temperature, thermostability of the immobilized lipases, storage stability in organic solvent, and leaching studies were investigated. Stability was found to be the highest in the nanocomposite NAZAD.
Lipase from Candida rugosa was immobilized by entrapment on poly(N-vinyl- 2-pyrrolidone-co-2-hydroxyethyl methacrylate) (poly[VP-co-HEMA]) hydrogel, and divinylbenzene was the crosslinking agent. The immobilized enzymes were used in the esterification reaction of oleic acid and butanol in hexane. The activities of the immobilized enzymes and the leaching ability of the enzyme from the support with respect to the different compositions of the hydrogels were investigated. The thermal, solvent, and storage stability of the immobilized lipases was also determined. Increasing the percentage of composition of VP from 0 to 90, which corresponds to the increase in the hydrophilicity of the hydrogels, increased the activity of the immobilized enzyme. Lipase immobilized on VP(%):HEMA(%) 90:10 exhibited the highest activity. Lipase immobilized on VP(%):HEMA(%) 50:50 showed the highest thermal, solvent, storage, and operational stability compared to lipase immobilized on other compositions of hydrogels as well as the native lipase.
The chemical production of methyl oleate using chemically synthesized fatty acid alcohols and other toxic chemicals may lead to significant environmental hazards to mankind. Being a highly valuable fatty acid replacement raw material in oleochemical industry, the mass production of methyl oleate via environmentally favorable processes is of concern. In this context, an alternative technique utilizing Candida rugosa lipase (CRL) physically adsorbed on multi-walled carbon nanotubes (MWCNTs) has been suggested. In this study, the acid-functionalized MWCNTs prepared using a mixture of HNO3 and H2SO4 (1:3 v/v) was used as support for immobilizing CRL onto MWCNTs (CRL-MWCNTs) as biocatalysts. Enzymatic esterification was performed and the efficiency of CRL-MWCNTs was evaluated against the free CRL under varying conditions, viz. temperature, molar ratio of acid/alcohol, solvent log P, and enzyme loading. The CRL-MWCNTs resulted in 30-110 % improvement in the production of methyl oleate over the free CRL. The CRL-MWCNTs attained its highest yield (84.17 %) at 50 °C, molar ratio of acid/alcohol of 1:3, 3 mg/mL of enzyme loading, and iso-octane (log P 4.5) as solvent. Consequently, physical adsorption of CRL onto acid-functionalized MWCNTs has improved the activity and stability of CRL and hence provides an environmentally friendly means for the production of methyl oleate.