Enhancing oxidation activity and stability of iso-1-cytochrome c and chloroperoxidase by immobilization in nanostructured supports

The immobilization of enzymes in inorganic materials has been widely used because it can produce an enhancement of the catalytic stability and enzymatic activity. In this article, the effect of the immobilization of iso-1-cytochrome c (CYC-Sc) from Saccharomyces cerevisiae and chloroperoxidase (CPO) from Caldariomyces fumago on the enzyme stability and catalytic oxidation of styrene was studied. The immobilization was carried out in three silica nanostructured supports with different pore size MCM-41 (3.3 nm), SBA-15 (6.4 nm) and MCF (12.1 nm). The adsorption parameters and leaching degree of immobilized enzymes were determined. Catalytic parameters of immobilized and free enzymes were determined at different temperatures (20–60 °C) and in different acetonitrile/water mixtures (15–85% of acetonitrile). The results show that there is low leaching of the enzymes in the three supports assayed and the adsorption capacity (q_max) was higher as the pore size of the support increased. The pore size also produces the enhancement of peroxidase activities on the styrene oxidation. Thus, CPO adsorption into SBA-15 and MCF showed remarkable thermal and solvent stabilities at 40 °C showing a total turnover numbers of 48,000 and 54,000 times higher than free CPO, respectively. The enhancement of activity and stability doubtless is interesting for the potential industrial use of peroxidases.     

Production of biofuel from waste cooking palm oil using nanocrystalline zeolite as catalyst: process optimization studies

The catalytic cracking of waste cooking palm oil to biofuel was studied over different types of nano-crystalline zeolite catalysts in a fixed bed reactor. The effect of reaction temperature (400-500 °C), catalyst-to-oil ratio (6-14) and catalyst pore size of different nanocrystalline zeolites (0.54-0.80 nm) were studied over the conversion of waste cooking palm oil, yields of Organic Liquid Product (OLP) and gasoline fraction in the OLP following central composite design (CCD). The response surface methodology was used to determine the optimum value of the operating variables for maximum conversion as well as maximum yield of OLP and gasoline fraction, respectively. The optimum reaction temperature of 458 °C with oil/catalyst ratio=6 over the nanocrystalline zeolite Y with pore size of 0.67 nm gave 86.4 wt% oil conversion, 46.5 wt% OLP yield and 33.5 wt% gasoline fraction yield, respectively. The experimental results were in agreement with the simulated values within an experimental error of less than 5%.     

Optimization and kinetic study on the synthesis of palm oil ester using Lipozyme TL IM

Enzymatic synthesis of palm oil esters (POE) was carried out via alcoholysis of palm oil (PO) and oleyl alcohol (OA) catalyzed by Lipozyme TL IM. The optimum reaction conditions were: temperature: 60 °C; enzyme load: 24.7 wt%; substrate ratio: 1:3 (PO/OA), impeller speed: 275 rpm and reaction time: 3 h. At the optimum condition, the conversion of POE was 79.54%. Reusability study showed that Lipozyme TL IM could be used for 5 cycles with conversion above 50%. The alcoholysis reaction kinetic follows the Ping-Pong Bi-Bi mechanism characterized by the V_max, K_m(PO), and K_m(OA) values of 32.7 mmol/min, 0.3147 mmol/ml and 0.9483 mmol/ml, respectively. The relationship between initial reaction rate and temperature was also established based on the Arrhenius law.     

Isolation and immobilization of alkaline protease on mesoporous silica and mesoporous ZSM-5 zeolite materials for improved catalytic properties

Alkaline protease from brinjal leaf (Solanum melongena) having milk clotting activity has been purified to 9.44 fold to a final specific activity of 45.71 U/mg. SDS-PAGE of the final preparation revealed a single protein band of approx 14 kDa. Purified enzyme was characterized and was successfully immobilized into the amorphous mesoporous silica (SBA-15) and crystalline mesoporous zeolite (Nano-ZSM-5) using entrapment method. Maximum immobilization of 63.5% and 79.77% was obtained with SBA-15 and Nano-ZSM-5, respectively. This protocol serves as a novel approach for bioprocesses, mainly as milk coagulant for local dairy products and particularly, cheese making, and opens the new dimension of further research and other innovation.     

Immobilization of alkaline serine endopeptidase from Bacillus licheniformis on SBA-15 and MCF by surface covalent binding

An industrial enzyme, alkaline serine endopeptidase, was immobilized on surface modified SBA-15 and MCF materials by amide bond formation using carbodiimide as a coupling agent. The specific activities of free enzyme and enzyme immobilized on SBA-15 and MCF were studied using casein (soluble milk protein) as a substrate. The highest activity of free enzyme was obtained at pH 9.5 while this value shifted to pH 10 for SBA-15 and MCF immobilized enzyme. The highest activity of immobilized enzymes was obtained at higher temperature (60 °C) than that of the free enzyme (55 °C). Kinetic parameters, Michaelis–Menten constant (K_m) and maximum reaction velocity (V_max), were calculated as K_m = 13.375, 11.956, and 8.698 × 10^?4 mg/ml and V_max = 0.156, 0.163 and 0.17 × 10^?3 U/mg for the free enzyme and enzyme immobilized on SBA-15 and MCF, respectively. The reusability of immobilized enzyme showed 80% of the activity retained even after 15 cycles. Large pore sized MCF immobilized enzyme was found to be more promising than the SBA-15 immobilized enzyme due to the availability of larger pores of MCF, which offer facile diffusion of substrate and product molecules.     

Bioconversion of lignocellulosic palm byproducts into enzymes and lipid by newly isolated oleaginous fungi

Direct conversion of palm pressed fiber (PPF) and palm empty fruit bunches (EFB) into enzymes and lipid by oleaginous fungi were performed through solid-state fermentation (SSF). Among the strains tested, TSIP9 converted PPF and EFB into lipid with the highest yield of 31.1 ± 1.7 mg/gram dry substrate (gds) and 37.5 ± 2.2 mg/gds, respectively. It also produced high activity of cellulolytic enzymes. It was identified as Aspergillus tubingensis. The similar fatty acids of its lipid to those of plant oil indicate its suitable use as biodiesel feedstock. The cellulase and xylanase production by this strain was improved when EFB was pretreated with alkaline. When alkaline-pretreated EFB was added with palm kernel cake (PK) as an alternative nitrogen source and the culture conditions were optimized through response surface methodology (RSM), the production of lipid, cellulase and xylanase were increased up to 88.5 ± 4.9 mg/gds, 26.1 ± 0.1 U/gds and 59.3 ± 0.3 U/gds, respectively. This study reveals the potential use of in situ cellulolytic enzymes producing fungi and the optimal conditions for direct conversion of lignocellulosic biomass into lipid.     

Catalytic cracking of palm oil for the production of biofuels: optimization studies

Oil palm is widely grown in Malaysia. Palm oil has attracted the attention of researchers to develop an 'environmentally friendly' and high quality fuel, free of nitrogen and sulfur. In the present study, the catalytic cracking of palm oil to biofuel was studied over REY catalyst in a transport riser reactor at atmospheric pressure. The effect of reaction temperature (400-500 degrees C), catalyst/palm oil ratio (5-10) and residence time (10-30s) was studied over the yield of bio-gasoline and gas as fuel. Design of experiments was used to study the effect of operating variables over conversion of palm oil and yield of hydrocarbon fuel. The response surface methodology was used to determine the optimum value of the operating variables for maximum yield of bio-gasoline fraction in the liquid product obtained.     

Investigations to optimize the catalytic performance of CPO encapsulated in PEG 200-doped silica matrices

A systematic investigation of the experimental conditions that affect the performance of chloroperoxidase (CPO) when encapsulated in organic/inorganic hybrid materials was carried out, aimed at optimizing the enzymatic catalytic efficiency. Sol–gel process was used to synthesize silica matrices and the incorporation of polyethylene glycols (PEGs) has allowed us to modify the properties of the matrices and the interactions between the silica network and the enzyme. Sol–gel process conditions, i.e. PEG/TMOS (tetramethylorthosilicate) molar ratio, aging and drying time, along with the H₂O₂ concentration in the reaction mixture were optimized to obtain a more efficient and reusable catalyst. A stable and easily recycled biocatalyst was obtained, even in the presence of high amounts of oxidizing agent. A stability of up to 3 complete cycles of reaction was obtained. CPO also exhibited an excellent thermostability even at 70 °C, being residual activity after 2 h of incubation greater than 90%, and it was a very favorable result, especially in view of synthetic applications of CPO.Moreover, it was found that the immobilized catalyst performance can be maintained unchanged over at least a month simply by storing the washed matrices at 4 °C. Further optimization of the experimental conditions will lead in the future to a larger-scale synthetic use of CPO.     

Optimization of Candida sp. 99-125 lipase catalyzed esterification for synthesis of monoglyceride and diglyceride in solvent-free system

Esterification of glycerol and oleic acid catalyzed by lipase Candida sp. 99-125 was carried out to synthesize monoglyceride (MAG) and diglyceride (DAG) in solvent-free system. Beta-cyclodextrin as an assistant was mixed with the lipase powder. Six reaction variables, initial water content (0–14 wt% of the substrate mass), the glycerol/oleic acid molar ratio (1:1–6:1), catalyst load (3–15 wt% of the substrate mass), reaction temperature (30–60 °C), agitator speed (130–250 r/min) and beta-cyclodextrin/lipase mass ratio (0–2) were optimized. The optimal conditions to the synthesis of MAG and DAG were different: the optimal glycerol/oleic acid molar ratio, beta-cyclodextrin/lipase mass ratio, catalyst load and reaction temperature were 6:1, 0, 5%, 50 °C for MAG, and 5:1, 1.5, 10%, 40 °C for DAG, respectively. The optimal water content and agitator speed for both MAG and DAG were 10% and 190 r/min, respectively. Under the optimal conditions, 49.6% MAG and 54.3% DAG were obtained after 8 h and 4 h, respectively, and the maximum of 81.4% MAG plus DAG (28.1% MAG and 53.3% DAG) was obtained after 2 h under the DAG optimal condition. Above 90% purity of MAG and DAG can be obtained by silica column separation.     

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

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

Biocatalytic methanolysis activities of cross-linked protein-coated microcrystalline lipase toward esterification/transesterification of relevant palm products

Biocatalysis by immobilized lipase is an efficient alternative process for conversion of crude vegetable oil with high free fatty acid content to biodiesel, which is the limit of the conventional alkaline-catalyzed reaction. In this study, influences of solid-state organic and inorganic buffer core matrices with different pK_a on catalytic performance of cross-linked protein coated microcrystalline biocatalysts prepared from Thermomyces lanuginosus lipase (CL-PCMC-LIP) toward esterification of palmitic acid (PA), transesterification of refined palm oil (RPO), and co-ester/transesterification of crude palm oil (CPO) to fatty acid methyl ester (FAME) was studied. Glycine, CAPSO (3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid), and TAPS ([(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]-1-propanesulfonic acid) were shown to be potent core matrices for these reactions. The optimal reaction contained 4:1 [methanol]/[fatty acid] molar equivalence ratio with 20% (w/w) CL-PCMC-LIP on glycine in the presence of tert-butanol as a co-solvent. Deactivation effect of glycerol on the biocatalyst reactive surface was shown by FTIR, which could be alleviated by increasing co-solvent content. The maximal FAME yields from PA, RPO, and CPO reached 97.6, 94.9, and 95.5%, respectively on a molar basis under the optimum conditions after incubation at 50 °C for 6 h. The biocatalyst retained >80% activity after recycling in five consecutive batches. The work demonstrates the potential of CL-PCMC-LIP on one-step conversion of inexpensive crude fatty acid-rich feedstock to biodiesel.     

Synthesized tyrosyl hydroxyphenylacetate,a novel antioxidant,anti-stress and antibacterial compound

New tyrosyl ester derivative, a naturally occurring phenol with interesting biological properties, has been synthesized in good yield by a direct esterification of tyrosol (Ty) with p-hydroxyphenylacetic acid (p-HPA) using Candida antarctica lipase as a catalyst. The response surface methodology was used to modulate the effects of the enzyme amount (10–50 mg), the tert-butanol/hexane (v/v) ratio (0.16–0.84), the temperature (35–55 °C) and the reaction time (15–45 h) on the tyrosyl hydroxyphenylacetate (Ty-HPA) conversion yield. Under the optimal predicted conditions (enzyme amount: 10 mg, solvents volume ratio 0.16, reaction temperature; 45 °C and 34 h of incubation), a high conversion yield of 79.33 ± 4% was reached. The obtained ester was purified and characterized by NMR, LC/MS and FT-IR methods. ABTS free radical quenching potency demonstrated that the esterified tyrosol (Ty-HPA) was more effective than the natural separated antioxidants: Ty and p-HPA. Furthermore, when used at a non-cytotoxic concentration (100 μM), tyrosyl ester showed significant effectiveness in preventing iron-induced oxidative stress in blood cells compared to the two separated compounds. The antibacterial activity of Ty, p-HPA, mixed solution of Ty + p-HPA and Ty-HPA was performed by determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) using a micro-well dilution method. Compared to the separated substrates, synthesized ester exhibits the most antibacterial effect mainly against Gram+ bacteria.     

Selective hydrogenation of 1,10-phenanthrolines by silica-supported palladium nanoparticles

The catalytic hydrogenation of 1,10-phenanthroline (Phen) or 2,9-dimethyl-1,10-phenanthroline (DMPhen) has been achieved using silica-supported palladium nanoparticles (Pd/SiO₂) with metal contents of 1.98 or 9.95 wt%. With either catalyst, the hydrogenation regiochemistry has been effectively controlled by the reaction temperature. The catalyst with the higher metal content was selective for the hydrogenation of one heterocyclic ring of either substrate at 80 °C and for both external rings at 130 °C for Phen and at 160 °C for DMPhen. The catalyst with the lower metal content was more active and exhibited comparable selectivity.     

Purification of l-(+)-lactic acid from pre-treated fermentation broth using vapor permeation-assisted esterification

The vapor permeation-assisted esterification of l-(+)-lactic acid and ethanol was investigated using a zeolite membrane. Pervaporation and vapor permeation were initially investigated for dehydration performances, and the latter showed much better results. The molecular sieve property of the membrane resulted in a high separation factor of over 1000 for all conditions. The maximum flux was 10.24 kg/(m² h) at a feed temperature of 145 °C, a water feed concentration of 10 wt%, and a feed pressure of 4.0 bars, respectively. For vapor permeation-assisted esterification using synthetic solutions, the productivity and ethyl lactate yield strongly depended on the dehydration rate. Realistic purifications were performed with fermentation broths of Pediococcus pentosaceus as the lactic acid producer. Experimental results revealed that most of the lactic acid was converted into ethyl lactate at the final stage of the reaction. After distillation and hydrolysis, high purity l-(+)-lactic acid was obtained with more than 95% recovery yields.     

Decanter cake waste as a renewable substrate for biobutanol production by Clostridium beijerinckii

The aim of this study was to determine if decanter cake waste from a palm oil mill could be used as a renewable substrate for biobutanol production. Decanter cake waste was first hydrolyzed to fermentable sugars by nitric acid and detoxified by activated-charcoal. The detoxified hydrolysate supplemented with whey protein and ammonium sulfate as cheap nitrogen sources, was used for butanol production by growing cells of Clostridium beijerinckii. The detoxified hydrolysate was also used as a co-substrate for direct conversion of butyric acid to butanol in a nitrogen-free medium. By these two steps, C. beijerinckii produced 3.42 g/L of butanol with a yield of 0.28 C-mol butanol/C-mol carbon in the first step and produced 6.94 g/L of butanol with a yield of 0.47 C-mol butanol/C-mol carbon in the second step. This study has showed that decanter cake waste could serve as a low-cost substrate for biobutanol production.     

Real evidence about zeolite as microorganisms immobilizer in anaerobic fluidized bed reactors

Using the scanning electronic microscopy, it was observed that natural zeolite possesses excellent physical characteristics as a support medium in anaerobic fluidized bed reactors (AFBR). Samples for biomass analysis were taken from two identical laboratory-scale AFBR (R-1 and R-2), which were operated with 25% of fluidization. These reactors treated distillery wastewaters (vinasses) at mesophilic temperature (30 ± 2 °C). The experiments were carried out with 0.25–0.50 and 0.50–0.80 mm zeolite particle diameter in reactors R-1 and R-2, respectively. The biomass concentration attached to zeolite in both reactors was found to be in the range of 40–45 g volatile solids/l. COD removal efficiencies as high as 90% were achieved at organic loading rate (OLRs) of up to 20 g COD/l day. The volatile fatty acid (VFA) levels were always lower that the suggested limits for digester failure. The yield coefficient of methane production was 0.29 l CH₄(at STP)/g COD consumed and was virtually independent of the OLR applied. A hybridization technique (fluorescence in situ hybridization, FISH) helped determine the predominant anaerobic microorganisms that colonized the natural zeolite, which were found to be Methanosaeta and Methanosarcinaceae, observing a reduced number of sulphate reducing bacteria. The results obtained for reactors R-1 and R-2 were very similar, showing that the particle size did not significantly influence the microbial community immobilized on zeolite.     

Fast and efficient purification of chloroperoxidase from C. fumago

Chloroperoxidase (CPO) from Caldariomyces fumago is a highly versatile and synthetically important enzyme. Nevertheless, the actual technical application is limited, mainly due to the high costs for purified preparations. Here, a novel fast and very efficient method for the purification of CPO has been developed. It involves separation of CPO from black fungal pigment and most side proteins in aqueous biphasic systems followed by a single chromatographic step. A residual yield of ∼70.4% and a specific activity of 2900 U/mg were obtained. Sufficient purity for technical application was already achieved after biphasic extraction, where CPO recovery from culture broth yielded about 100%. Time required for purification ranges between 30 min and 1 day depending on the desired degree of purity. Thus, a protocol is presented that reduces consumption of time and material and enhances the enzyme quality.     

Enzymatic transformation of 2-O-α-d-glucopyranosyl-l-ascorbic acid (AA-2G) by immobilized α-cyclodextrin glucanotransferase from recombinant Escherichia coli

This work aims to produce 2-O-α-d-glucopyranosyl-l-ascorbic acid (AA-2G) from ascorbic acid and β-cyclodextrin with immobilized α-cyclodextrin glucanotransferase (α-CGTase) from recombinant Escherichia coli. Molecular sieve (SBA-15) was used as an adsorbent, and sodium alginate was used as a carrier, and glutaraldehyde (GA) was used as a cross-linker. The effects of several key variables on α-CGTase immobilization were examined, and optimal immobilization conditions were determined as the following: glutaraldehyde (GA, cross-linker) 0.01% (v/v), SBA-15 (adsorbent) 2 g/L, CaCl₂ 3 g/L, sodium alginate 20 g/L, adsorption time 3 h, and immobilization time 1 h. In comparison with free α-CGTase, immobilized α-CGTase had a similar optimal pH (5.5) and a higher optimal temperature (45 °C). The continuous production of AA-2G from ascorbic acid and β-cyclodextrin in the presence of immobilized α-CGTase was carried out, and the highest AA-2G production reached 21 g/L, which was 2-fold of that with free α-CGTase. The immobilization procedure developed here was efficient for α-CGTase immobilization, which was proved to be a prospective approach for the enzymatic production of AA-2G.     

Preparation and properties of glycerol plasticized-pea starch/zinc oxide-starch bionanocomposites

A bionanocomposite was cast using ZnO nanoparticles stabilized by soluble starch (nano-ZnO) as filler in a glycerol plasticized-pea starch (GPS) matrix. According to the characterization of nano-ZnO particles by Fourier-transform infrared spectroscopy (FT-IR), thermogravimetric analysis (TG) and transmission electron microscopy (TEM), ZnO nanoparitlces (70 wt%) were encapsulated by starch (30 wt%) in nano-ZnO particles of about 10 nm. In GPS/nano-ZnO nanocomposites, loading a low level of nano-ZnO particles improved the pasting viscosity, storage modulus, glass transition temperature and UV absorbance. When the nano-ZnO content varied from 0 to 4 wt%, the tensile yield strength and Young’s modulus increased from 3.94 to 10.80 MPa and from 49.80 to 137.00 MPa, respectively. The water vapor permeability decreased from 4.76 × 10^?10 to 2.18 × 10^?10 g m^?1 s^?1 Pa^?1. The improvement in these properties may be attributed to the interaction between the nano-ZnO filler and GPS matrix.     

Enhanced selective oxidation of trimethylolpropane to 2,2-bis(hydroxymethyl)butyric acid using Corynebacterium sp. ATCC 21245

2,2-Bis(hydroxymethyl)butyric acid (BHMB) is an important multifunctional chemical for the emerging bio-based polymer industry. It can be produced from trimethylolpropane (TMP) by selective oxidation using growing cells of Corynebacterium sp. ATCC 21245. However, this process is limited by the low volumetric productivity and low concentration of the final product. In the present study, we performed sequential batch operation with cell recycling in media containing glycerol, acetic acid, and increasing concentrations of yeast extract. This approach enhanced the conversion of 10 and 15 g/L TMP to 11.0 and 16.3 g/L BHMB at rates of 0.50 and 0.20 g/L^.h, respectively. Applying a cell bleeding strategy resulted in an overall 10-fold improvement in productivity. The consequently prolonged biocatalyst viability resulted in a quantitative conversion of 20 g/L TMP to 22.3 g/L BHMB and a yield of 1.10 g_BHMB/g_TMP (100% molar yield). This work facilitates further studies of the selective oxidation on other industrially important polyols.