Concentration,Partial Characterization,and Immobilization of Lipase Extract from <Emphasis Type="Italic">P. brevicompactum</Emphasis> by Solid-State Fermentation of Babassu Cake and Castor Bean Cake

One relevant limitation hindering the industrial application of microbial lipases has been attributed to their production cost, which is determined by the production yield, enzyme stability among other. The objective of this work was to evaluate the concentration and immobilization of lipase extracts from Penicillium brevicompactum obtained by solid-state fermentation of babassu cake and castor bean cake. The precipitation with ammonium sulfate 60% of saturation of crude extract obtained with babassu cake as raw material showed an enhancement in hydrolytic and esterification activities from 31.82 to 227.57 U/g and from 170.92 to 207.40 U/g, respectively. Concentrated lipase extracts showed preference to medium-chain triglycerides and fatty acids. It is shown that the enzyme activity is maintained during storage at low temperatures (4 and −10°C) for up to 30 days. Higher esterification activities were achieved when the lipase extract was immobilized in sodium alginate and activated coal.     

Enzymatic Hydrolysis of Polylactic Acid Fiber

This study investigated the optimization of the enzymatic processing conditions for polylactic acid (PLA) fibers using enzymes consisting of lipases originating from different sources. The hydrolytic activity was evaluated taking into consideration the pH, temperature, enzyme concentration, and treatment time. The structural change of the PLA fibers was measured in the optimal treatment conditions. PLA fiber hydrolysis by lipases was maximized for lipase from Aspergillus niger at 40 °C for 60 min at pH 7.5 with 60% (owf) concentration, for lipase from Candida cylindracea at 40 °C for 120 min at pH 8.0 with 70% (owf) concentration, and for lipase from Candida rugosa at 45 °C for 120 min at pH 8.0 with 70% (owf) concentration. There was a change in protein absorbance of the treatment solution before and after all lipase treatments. The analyses of the chemical structure change and structural properties of the PLA due to lipase treatment was confirmed by tensile strength, differential scanning calorimetry, wide-angle X-ray scattering diffractometry, Fourier transform infrared spectroscopy, and scanning electron microscopy.     

Catalytic effect of calix[n]arene based sol–gel encapsulated or covalent immobilized lipases on enantioselective hydrolysis of (R/S)-naproxen methyl ester

In this paper the catalytic performance of the immobilized lipases was investigated in the presence of calixarene based polymers using different immobilization techniques. For this reason, Candida rugosa lipase was encapsulated in sol–gel matrices using alkoxysilane precursors and calix[n]arene based silica polymers as additives. The hydrolytic activities of encapsulated lipases were evaluated and compared with the free lipase and covalently immobilized lipases (CnP-L). These encapsulated lipases were also used in the kinetic resolution of the R/S-Naproxen methyl ester. The results indicated that the C6P encapsulated lipase has significantly higher conversion and enantioselectivity as compared to the free lipase; other encapsulated lipases and CnP-L. The optimal pH and temperature region of the C6P encapsulated lipase in the kinetic resolution of the R/S-naproxen methyl ester were 7.0 and 55?°C. Nevertheless, the encapsulated lipases have good stability, adaptability and reusability in comparison with the free enzyme.     

Lipase from a Brazilian strain ofPenicillium citrinum

A lipases (glycerol ester hydrolases E. C. 3.1.1.3) from a brazilian strain ofPenicillium citrinum has been investigated. When the microorganism was cultured in the simple medium (1.0% olive oil and 0.5% yeast extract), using olive oil in as carbon source in the inocula, the enzyme extracted showed maximum activity (409 IU/mL). In addition, decrease of yeast extract concentration also reduces the lipase activity. Nevertheless, when yeast extract was replaced by ammonium sulfate, no activity was detected. Purification by precipitation with ammonium sulfate showed best activity in the 40–60% fraction. The optimum temperature for enzyme activity was found in the range of 34–37°C. However, after 30 min at 60°C, the enzyme was completely inactivated. The enzyme showed optimum at pH 8.0. The dried concentrated fraction (after dialysis and lyophilization) maintained its lipase activity at room temperature (28°C) for 8 mo. This result in lipase stability suggests an application of lipases fromP. citrinum in detergents and other products that require a high stability at room temperature.     

Partial Characterization of Inulinases Obtained by Submerged and Solid-State Fermentation Using Agroindustrial Residues as Substrates: A Comparative Study

Inulinase belongs to an important class of enzymes as it can be used to produce high-fructose syrups by enzymatic hydrolysis of inulin and fructooligosaccharides, which has been used as functional food. This work aimed to carry out a partial characterization of the crude enzymatic extract of two different inulinases, obtained by solid-state fermentation (SSF) and submerged fermentation (SmF), using agroindustrial residues as substrates. The crude enzymatic extract obtained by SmF showed an optimal pH and temperature for hydrolytic activity of 4.5 and 55?°C, respectively; and that obtained by SSF conducted to optimal pH and temperature of 5.0 and 55?°C, respectively. Both enzymes presented high thermostability, with a D value of 230.4 h and 123.1 h for SmF and SSF, respectively. The inulinase produced by SmF showed highest stability at pH?4.4, while inulinase obtained by SSF was more stable at pH?4.8. The results showed that inulinase obtained by SmF is less susceptible to pH effect and the inulinase obtained by SSF is more resistant to higher temperatures.     

Characterization of Thermo-stable Endoinulinase from a New Strain <Emphasis Type="Italic">Bacillus Smithii</Emphasis> T7

A new thermophilic inulinase-producing strain, which grows optimally at 60 °C, was isolated from soil samples with medium containing inulin as a sole carbon source. It was identified as a Bacillus smithii by analysis of 16s rDNA. Maximum inulinase yield of 135.2 IU/ml was achieved with medium pH7.0, containing inulin 2.0%, (NH₄)H₂PO₄ 0.5%, yeast extract 0.5%, at 50 °C 200 rpm shaker for 72-h incubation. The purified inulinase from the extracellular extract of B. smithii T7 shows endoinulinolytic activity. The optimum pH for this endoinulinase is 4.5 and stable at pH range of 4.0–8.0. The optimum temperature for enzyme activity was 70 °C, the half life of the endoinulinase is 9 h and 2.5 h at 70 °C and 80 °C respectively. Comparatively lower Michaelis–Menten constant (4.17 mM) and higher maximum reaction velocity (833 IU/mg protein) demonstrate the endoinulinase’s greater affinity for inulin substrate. These findings are significant for its potential industrial application.     

Characterization of Cellulolytic Extract from <Emphasis Type="Italic">Pycnoporus sanguineus</Emphasis> PF-2 and Its Application in Biomass Saccharification

The aim of this work was to evaluate the biochemical features of the white-rot fungi Pycnoporus sanguineus cellulolytic complex and its utilization to sugarcane bagasse hydrolysis. When cultivated under submerged fermentation using corn cobs as carbon source, P. sanguineus produced high FPase, endoglucanase, β-glucosidase, xylanase, mannanase, α-galactosidase, α-arabinofuranosidase, and polygalacturonase activities. Cellulase activities were characterized in relation to pH and temperature. β-Glucosidase and FPase activities were higher at 55 °C, pH 4.5, and endoglucanase activity was higher at 60 °C, in a pH range of 3.5–4.0. All cellulase activities were highly stable at 40 and 50 °C through 48 h of pre-incubation. Crude enzymatic extract from P. sanguineus was applied in a saccharification experiment using acid-treated and alkali-treated sugarcane bagasse as substrate, and the hydrolysis yields were compared to that obtained by a commercial cellulase preparation. Reducing sugar yields of 60.4% and 64.0% were reached when alkali-treated bagasse was hydrolyzed by P. sanguineus extract and commercial cellulase, respectively. Considering the glucose production, it was observed that P. sanguineus extract and commercial cellulase ensured yields of 22.6% and 36.5%, respectively. The saccharification of acid-treated bagasse was lower than that of alkali-treated bagasse regardless of the cellulolytic extract. The present work showed that P. sanguineus has a great potential as an enzyme producer for biomass saccharification.     

Screening and Identification of a Fungal β-Glucosidase and the Enzymatic Synthesis of Gentiooligosaccharide

After screening with 0.1% esculoside and 0.03% FeCl₃, we identified from rotten wood a fungal isolate HML0366 that produces high amount of β-glucosidase. Phenotypic and rDNA internal transcribed spacer sequence analyses indicated that the isolate belongs to Aspergillus oryzae. The β-glucosidase produced by HML0366 had an activity of 128 U/g. high performance liquid chromatography analysis also demonstrated a high transglycosylation activity of the crude enzyme. The β-glucosidase was stable between pH 4–10 at 60 °C. A gentiobiose yield of 30.86 g/L was achieved within 72 h of the enzymatic reaction at pH 5 and 55 °C using 50% glucose as the substrate. For the first time, we report here the isolation of an A. oryzae strain producing β-glucosidase with high hydrolytic activities. The crude enzyme has a high transglycosylation activity, which enables the enzymatic synthesis of gentiooligosaccharides.     

Constitutive Expression of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> Lipase LIP2 in <Emphasis Type="Italic">Pichia pastoris</Emphasis> Using GAP as Promoter

A gene encoding Yarrowia lipolytica lipase LIP2 (YlLIP2) was cloned into a constitutive expression vector pGAPZαA and electrotransformed into the Pichia pastoris X-33 strain. The high-yield clones obtained by high copy and enzyme activity screening were chosen as the host strains for shaking flask and fermentor culture. The results showed that glucose was the optimum carbon source for YlLIP2 production, and the maximum hydrolytic activity of recombinant YlLIP2 reached 1,315 U/ml under the flask culture at 28 °C, pH 7.0, for 48 h. The fed-batch fermentation was carried out in 3- and 10-l bioreactors by continuously feeding glucose into the growing medium for achieving high cell density and YlLIP2 yields. The maximum hydrolytic activity of YlLIP2 and cell density obtained in the 3-l bioreactor were 10,300 U/ml and 116 g dry cell weight (DCW)/l, respectively. The peak hydrolytic activity of YlLIP2 and cell density were further improved in the 10-l fermentor where the values respectively attained were 13,500 U/ml and 120 g DCW/l. The total protein concentration in the supernatant reached 3.3 g/l and the cell viability remained approximately 99% after 80 h of culture. Furthermore, the recombinant YlLIP2 produced in P. pastoris pGAP and pAOX1 systems have similar content of sugar (about 12%) and biochemical characteristics. The above results suggest that the GAP promoter-derived expression system of P. pastoris is effective for the expression of YlLIP2 by high cell density culture and is probably an alternative to the conventional AOX1 promoter expression system in large-scale production of industrial lipases.     

Operational and Thermal Stability Analysis of <Emphasis Type="Italic">Thermomyces lanuginosus</Emphasis> Lipase Covalently Immobilized onto Modified Chitosan Supports

The aim of this paper was to evaluate different strategies of chitosan activation using cross-linking reagent like glycidol, epichlorohydrin, and glutaraldehyde for Thermomyces lanuginosus lipase (TLL) immobilization. Operational activity and stability by esterification of oleic acid with ethanol and thermal inactivation using these derivatives were investigated. Derivative obtained by sequentially activation with glycidol, ethylenediamine, and glutaraldehyde and subsequent TLL immobilization showed the best performance, with high hydrolytic activity value. Its stability was 15-fold higher than solubilized TLL in the evaluated inactivation conditions (60 °C, 25 mM sodium phosphate buffer pH 7). After 5 cycles of oleic acid esterification, only a few percentage of its conversion has reduced. On the other hand, glycidol-activated chitosan derivative showed very low hydrolytic activity value. Epichlorohydrin-activated chitosan derivative showed regular hydrolytic activity value. Both derivatives showed low immobilization yields. Operational stability of this last derivative was very low, where after the first cycle of oleic acid esterification, only 56% of its initial conversion was obtained.

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Graphical Abstract ?

     

Investigation of Yeast Invertase Immobilization onto Cupric Ion-Chelated,Porous, and Biocompatible Poly(Hydroxyethyl Methacrylate-<Emphasis Type="Italic">n</Emphasis>-Vinyl Imidazole) Microspheres

Cupric ion-chelated poly(hydroxyethyl methacrylate-n-vinyl imidazole) (poly(HEMA-VIM)) microspheres prepared by suspension polymerization were investigated as a specific adsorbent for immobilization of yeast invertase in a batch system. They were characterized by scanning electron microscopy, surface area, and pore size measurements. They have spherical shape and porous structure. The specific surface area of the p(HEMA-VIM) spheres was found to be 81.2 m²/g with a size range of 70–120 μm in diameter, and the swelling ratio was 86.9%. Then, Cu(II) ion chelated on the microspheres (546 μmol Cu(II)/g), and they were used in the invertase adsorption. Maximum invertase adsorption was 51.2 mg/g at pH 4.5. Cu(II) chelation increases the tendency from Freundlich-type to Langmuir-type adsorption model. The optimum activity for both free and adsorbed invertase was observed at pH 4.5. The optimum temperature for the poly(HEMA-VIM)/Cu(II)-invertase system was found to be at 55 °C, 10 °C higher than that of the free enzyme at 45 °C. V _max values were determined as 342 and 304 U/mg enzyme, for free and adsorbed invertase, respectively. K _m values were found to be same for free and adsorbed invertase (20 mM). Thermal and pH stability and reusability of invertase increased with immobilization.     

Physical and Covalent Immobilization of <Emphasis Type="Italic">Lipase</Emphasis> onto Amine Groups Bearing Thiol-Ene Photocured Coatings

In this study, amine groups containing thiol-ene photocurable coating material for lipase immobilization were prepared. Lipase (EC 3.1.1.3) from Candida rugosa was immobilized onto the photocured coatings by physical adsorption and glutaraldehyde-activated covalent bonding methods, respectively. The catalytic efficiency of the immobilized and free enzymes was determined for the hydrolysis of p-nitrophenyl palmitate and also for the synthesis of p-nitrophenyl linoleate. The storage stability and the reusability of the immobilized enzyme and the effect of temperature and pH on the catalytic activities were also investigated. The optimum pH for free lipase and physically immobilized lipase was determined as 7.0, while it was found as 7.5 for the covalent immobilization. After immobilization, the optimum temperature increased from 37 °C (free lipase) to 50–55 °C. In the end of 15 repeated cycles, covalently bounded enzyme retained 60 and 70 % of its initial activities for hydrolytic and synthetic assays, respectively. While the physically bounded enzyme retained only 56 % of its hydrolytic activity and 67 % of its synthetic activity in the same cycle period. In the case of hydrolysis V _max values slightly decreased after immobilization. For synthetic assay, the V _max value for the covalently immobilized lipase was found as same as free lipase while it decreased dramatically for the physically immobilized lipase. Physically immobilized enzyme was found to be superior over covalent bonding in terms of enzyme loading capacity and optimum temperature and exhibited comparable re-use values and storage stability. Thus, a fast, easy, and less laborious method for lipase immobilization was developed.     

Purification and Partial Characterization of an Exo-polygalacturonase from Paecilomyces variotii Liquid Cultures

An extracellular polygalacturonase (PG) produced from Paecilomyces variotii was purified to homogeneity through two chromatography steps using DEAE-Fractogel and Sephadex G-100. The molecular weight of P. variotii PG was 77,300 Da by gel filtration and SDS-PAGE. PG had isoelectric point of 4.37 and optimum pH 4.0. PG was very stable from pH 3.0 to 6.0. The extent of hydrolysis of different pectins by the purified enzyme was decreased with an increase in the degree of esterification. PG had no activity toward non-pectic polysaccharides. The apparent K _m and V _max values for hydrolyzing sodium polypectate were 1.84 mg/mL and 432 μmol/min/mg, respectively. PG was found to have temperature optimum at 65 °C and was totally stable at 45 °C for 90 min. Half-life at 55 °C was 50.6 min. Almost all the examined metal cations showed partial inhibitory effects under enzymatic activity, except for Na⁺¹, K⁺¹, and Co⁺² (1 mM) and Cu⁺² (1 and 10 mM).     

Influence of biodegradation on crystallization of poly (butylene adipate‐co‐terephthalate)

The biodegradation of aromatic‐aliphatic biodegradable polyester poly (butylene adipate‐co‐terephthalate) (PBAT) was studied under mesophilic (37°C) and thermophilic (55°C) anaerobic conditions. Anaerobic sludge from municipal wastewater treatment plant was utilized as an inoculum. Non‐isothermal crystallization kinetics of PBAT before and after biodegradation was explored by differential scanning calorimetry. Under mesophilic anaerobic conditions (37°C), the biodegradation after 126 days was only 2.2%, molecular weight changed from 93 000 to 25 500 g/mol, and the crystallization behavior was changed only slightly. However, biodegradation under thermophilic anaerobic conditions (55°C) caused much bigger changes: biodegradation according to biogas production reached after 126 days 8.3%, molecular weight changed from 93 000 to 9430 g/mol, and the crystallization behavior was changed significantly. While T_m increased only slightly, T_c on the other hand increased significantly for the sample after biodegradation at 55°C. Also, the crystallization rate was slower (particularly at lower cooling rates), but crystallinity was slightly higher. The diffraction pattern was observed by X‐ray diffraction.     

Use of an (Hemi) Cellulolytic Enzymatic Extract Produced by Aspergilli Species Consortium in the Saccharification of Biomass Sorghum

This study evaluated the production of lignocellulose-degrading enzymes by solid-state fermentation (SSF) using a microbial consortium of Aspergillus fumigatus SCBM6 and A. niger SCBM1 (AFN extract). The fungal strains were cultivated in sugarcane bagasse (SCB) and wheat bran (WB) as lignocellulosic substrates for 7 days at 30 °C. After SSF, the highest peaks of enzyme production were 150 and 80 U g⁻¹ for β-xylosidase and β-glucosidase at 48 h, 375 U g⁻¹ for xylanase at 96 h, and 80 U g⁻¹ for endoglucanase and 4 U g⁻¹ for cellulase activity on filter paper (FPase) at 144 h. The efficiency of the produced AFN extract was investigated in the enzymatic hydrolysis of crude biomass sorghum (BS) and after the removal of extractives (ES). After saccharification, the glucose and xylose concentrations were 10× superior in ES than in BS hydrolysate (2.5 g L⁻¹ after 12 h). The presence of inhibitors of alcoholic fermentation, such as formic acid, was also reduced in ES hydrolysates, indicating that the removal of extractives positively contributed to the effectiveness of enzymatic hydrolysis of biomass sorghum using AFN extract.

     

Synthesis and properties of polyimides bearing acid groups on long pendant aliphatic chains

A series of novel polyimide electrolytes having long pendant sulfo‐ or phosphoalkoxy groups were synthesized for fuel‐cell applications. Sulfodecyloxy‐, phosphodecyloxy‐, and sulfophenoxydodecyloxy‐substituted benzidine monomers were synthesized from dihydroxybenzidine. These monomers were copolymerized with naphthalene tetracarboxylic dianhydride and fluorenylidene dianiline to give the corresponding polyimides. A flexible, ductile, and self‐standing membrane was obtained via casting from the polyimide solution. Because the acid groups were on long pendant side chains and away from the main chains, the polyimide membrane showed improved oxidative and hydrolytic stability in comparison with the polyimides with sulfonic acid groups on the main chains or on the short side chains. High thermal stability (no glass‐transition temperature and a decomposition temperature > 200 °C) was also obtained. The polyimide membrane displayed high proton conductivity of 10^?1 S cm^?1 at 120 °C. © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 3995–4005, 2006     

Lactose Hydrolysis by β-Galactosidase Covalently Immobilized to Thermally Stable Biopolymers

Lactose has been hydrolyzed using covalently immobilized β-galactosidase on thermally stable carrageenan coated with chitosan (hydrogel). The hydrogel’s mode of interaction was proven by Fourier transform infrared spectroscopy, differential scanning calorimetry (DSC), and Schiff’s base formation. The DSC thermogram proved the formation of a strong polyelectrolyte complex between carrageenan and chitosan followed by glutaraldehyde as they formed one single peak. The modification of carrageenan improved the gel’s thermal stability in solutions from 35 °C to 95 °C. The hydrogel has been proven to be efficient for β-galactosidase immobilization where 11 U/g wet gel was immobilized with 50% enzyme loading capacity. Activity and stability of free and immobilized β-galactosidase towards pH and temperature showed marked shifts in their optimum pH from 4.5–5 to 5–5.5 and temperature from 50 °C to 45–55 °C after immobilization, which reveals higher catalytic activity and reasonable stability at wider pHs and temperatures. The apparent K _m of the immobilized enzyme increased from 13.2 to 125 mM, whereas the V _max increased from 3.2 to 6.6 μmol/min compared to the free enzyme, respectively. The free and immobilized enzymes showed lactose conversion of 87% and 70% at 7 h, respectively. The operational stability showed 97% retention of the enzyme activity after 15 uses, which demonstrates that the covalently immobilized enzyme is unlikely to leach. The new carrier could be suitable for immobilization of other industrial enzymes.     

Development of Batch and Continuous Processes on Biodiesel Production in a Packed-Bed Reactor by a Mixture of Immobilized Candida rugosa and Rhizopus oryzae Lipases

In this study, transesterification and esterification were investigated in batch and continuous process using immobilized Candida rugosa and Rhizopus oryzae lipases. In the case of batch process, stepwise reaction method was investigated to prevent the lipase deactivation. Reaction conditions were as follows: temperature, 45 °C; agitation speed, 250 rpm; enzyme concentration, 20%; and water contents 10%. And then, conversion yield was 98.33% at 4 h. In the case of continuous process, circulation and long-term continuous system were investigated for development of efficient mass transfer system. Optimal reaction conditions were as follows: temperature, 45 °C; flow rate, 0.8 mL/min; and water contents, 10%. And then, conversion yield of biodiesel was 97.98% at 3 h. Especially, the maximum conversion yield using a mixture of immobilized lipases exceeded over 90% for 108 h in long-term continuous system under optimal reaction conditions (45 °C; flow rate, 0.8 mL/min; and water contents, 10%). These results should help in determining the best method for the biodiesel production and improving the design and operation of large scale by enzymatic systems.     

Ultrahigh-pressure supercritical fluid extraction and chromatography of Moringa oleifera and Moringa peregrina seed lipids

An ultrahigh-pressure supercritical fluid extraction method was optimized and applied to extract seed oil lipids from two moringa species, namely Moringa oleifera (MO) and Moringa peregrina (MP). A full-factorial design was used to investigate the direct and interaction influence of pressure and temperature in the range of 40 to 80 MPa and 40 to 70 °C, respectively, on the extracted amount of oil from crushed seeds. The results revealed that pressure has a significant positive influence on the extracted amount of oil. The best extraction condition using neat CO₂ was found at 80 MPa and 57 °C, yielding 396 ± 23 and 529 ± 26 mg oil per gram of seeds for MO and MP, respectively. An extraction kinetics study revealed a mainly solubility-controlled extraction of oil, and 28 g of CO₂ was required to extract 400 mg of oil per gram of seeds of MO using the developed method. Addition of ethanol to the sample prior to the extraction increased the proportion of extractable polar lipids as well as the total amount of extracted oil. The developed method increased the extracted amount of oil twofold compared to a reference method based on solvent sonication. The obtained oil consisted mainly of glycerolipids, sterol esters, and phospholipids. Phospholipids, campesterol, and stigmasterol ester concentrations were found to be higher in MO while cholesterol ester was more abundant in MP.

     

Cell Disruption Optimization and Covalent Immobilization of β-D-Galactosidase from <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> YW-1 for Lactose Hydrolysis in Milk

β-D-galactosidase (EC 3.2.1.23) from Kluyveromyces marxianus YW-1, an isolate from whey, has been studied in terms of cell disruption to liberate the useful enzyme. The enzyme produced in a bioreactor on a wheat bran medium has been successfully immobilized with a view to developing a commercially usable technology for lactose hydrolysis in the food industry. Three chemical and three physical methods of cell disruption were tested and a method of grinding with river sand was found to give highest enzyme activity (720 U). The enzyme was covalently immobilized on gelatin. Immobilized enzyme had optimum pH and temperature of 7.0 and 40 °C, respectively and was found to give 49% hydrolysis of lactose in milk after 4 h of incubation. The immobilized enzyme was used for eight hydrolysis batches without appreciable loss in activity. The retention of high catalytic activity compared with the losses experienced with several previously reported immobilized versions of the enzyme is significant. The method of immobilization is simple, effective, and can be used for the immobilization of other enzymes.