Enzyme Loading Amount Research Articles

Saccharification of the Pretreated Corn Stover by Microwave Assisted DMSO/AmimCl Co-solvents

In order to enhance the enzymatic saccharification efficiency of corn stover, microwave assisted treatment with dimethyl sulfoxide (DMSO) and 1-ally-3-methylimidazolium (AmimCl) co-solvents was designed to break up the complicated chemical structure of corn stover. The pretreatment conditions were studied to increase the dissolution ratio, extraction ratio and cellulose I crystallinity index (Crl) variation. With the increase of AmimCl concentration, time and temperature of the pretreatment, the dissolution ratio, extraction ratio increased and Crl decreased. Considering the costs of high AmimCl concentration, degradation of biomass at high temperature and long time, the optimum conditions were DMSO/AmimCl co-solvents with a DMSO concentration of 15% (w) and 4 g corn stover per 100 g co-solvents. The optimum temperature and time were 110 degrees C and 60 min, respectively. Under the optimal conditions, the ratios of corn stover dissolution and extraction were as high as 46.6% and 22.9%, respectively. The cellulose saccharification ratio had a great increase within 14 h of enzymatic hydrolysis with an enzyme loading amount of 14 FPU/g solids. The saccharification ratio for the extracts was 71.4% in 14 h, while the ratio was only 12.5% for the raw corn stover in 20 h. Powder X-ray diffraction was used to determine the cellulose crystal structure. The cellulose I crystallinity index of corn stover residues decreased obviously and the crystal form of AmimCl extracts were transformed from I to II. From the SEM pictures of the materials, it could be seen that the outer surface of corn stover residues became rough, which was good for enzyme accessibility and saccharification. The AmimCl extracts had lost their natural status completely. By simple vacuum distillation, AmimCl could be recycled and its solubility kept almost constant. According to H-1 NMR spectroscopy analysis, the structures of fresh and recycled AmimCl were almost the same. From the results mentioned above, it can be concluded that microwave assisted treatment with DMSO AmimCl co-solvents was a feasible method for corn stover pretreatment.

The Effect of <i>Gleditsia</i> Saponin on Simultaneous Saccharification and Fermentation of Furfural Residue for Ethanol Production

Simultaneous saccharification and fermentation is an attractive process configuration for bioethanol. However, the process cost in SSF is still high. Further reductions in cost are expected with reduction of enzyme addition and the use of waste agricultural or industrial materials as feedstock. Surfactant addition is a simple method to reduce the amount of enzyme loading. In the present work we explore the effect of Gleditsia Saponin, an inexpensive and natural surfactant, on SSF of furfural residue, a main waste of furfural industry in China, for ethanol production. Gleditsia Saponin addition even at low concentration, 0.04 g/l, increased the ethanol yield by 9.9%. Meanwhile, when Gleditsia Saponin addition was 0.16 g/l, the yield was increased by 22.8% and the enzyme activity significantly increased in the liquid fraction at the end of SSF, which would contribute to recover and recycle enzyme. Gleditsia Saponin addition at 1.25 g/l had a better performance in improving SSF efficiency than that of 2.5 g/l Tween-20 addition. The positive effects of Gleditsia Saponin addition on SSF, such as less enzyme loading and shorter residence time could also been observed.

Different behaviors of single and multi wall carbon nanotubes for studying electrochemistry and electrocatalysis of choline oxidase

This work presents a detailed comparison between single and multi wall carbon nanotubes (SWCNTs & MWCNTs) in an effort to understand which could be a better supporting material for studying the electrochemistry and electrocatalysis of enzymes. Choline oxidase (ChOx) was chosen as a model enzyme for evaluation of the electrodes’ performance. The enzyme was adsorbed on either SWCNT or MWCNT modified electrode, in the presence of a typical room temperature ionic liquid (RTIL), and its electron transfer and electroanalytical response toward choline was investigated. RTIL/MWCNTs/GC electrode was uniformly covered by ChOx. Besides, higher electrical conductivity, better reversibility of the ChOx redox reaction and higher electron transfer rate of the enzyme indicated more facile and rapid rate of electron transfer. On the other hand, RTIL/SWCNTs/GC electrodes showed higher amount of enzyme loading, higher enzyme–substrate affinity, lower detection limit, better sensitivity and wider linear range. Consequently, MWCNTs are preferable for kinetic study of ChOx, while SWCNTs are more convenient for biosensing applications.

Lipase-Catalyzed Synthesis of 6-O-D-Glucosyldecanoate in Tert-Butanol: Reaction Optimization and Effect of Mixing Power Input

ABSTRACTEnzymatic synthesis of 6-O-D-glucosyldecanoate in tert-butanol was performed using substrates D-glucose as acyl acceptor and decanoic acid as acyl donor Candida antarctica lipase B (Novozyme 435) was used as the catalyst in the esterification process. Box Behnken of response surface methodology (RSM) was used in the optimization of selected parameters viz. reaction temperature, reaction time, substrate ratio and amount of enzyme loading. Substrate ratio was found to be the sole significant main effect while the second-order interaction between reaction time and immobilized enzyme loading was found to be a significant interaction factor. Optimized conditions for the process were: substrate molar ratio 1 (mole acyl donor-to-mole acyl acceptor), 7.5 g-L−1 immobilized enzyme loading, reaction temperature 55°C and time of 14.2 hours. When a mixed-flow impeller with downward pumping mode was used in a stirred tank reactor, minimum power input at 6.8 kW-kg−1 solvent was sufficient for an improved yield of 0.77 g glucosyl decanoate per g glucose. The reaction product composed solely of monoester with sugar acylation occurring at 6-OH primary group of D-glucose where 60% (mole) of the product was 6-O-β-D-glucosyldecanoate and 40% (mole) 6-O-α-D-glucosyldecanoate.

Immobilization of penicillin G acylase on macro-mesoporous silica spheres

In this study, macro-mesoporous silica spheres were prepared with a micro-device and used as the support for the immobilization of penicillin G acylase (PGA). To measure the enzymatic activity, the silica spheres with immobilized PGA were placed into a packed-bed reactor, in which the hydrolysis of penicillin G was carried out. The influences of the residence time, the initial concentration of the substrate, the accumulation of the target product 6-aminopenicillanic acid, and the enzyme loading amount on the performance of the immobilized PGA were investigated. The introduction of macropores increased the enzyme loading amount and decreased the internal mass transfer resistance, and the results showed that the enzyme loading amount reached 895 mg/g (dry support), and the apparent enzymatic activity achieved up to 1033 U/g (dry support). In addition, the immobilized PGA was found to have great stability.

Electrochemical characterization of a single-walled carbon nanotube electrode for detection of glucose

We developed glucose biosensing electrodes using single-walled carbon nanotube (SWCNT) films on flexible, transparent poly(ethylene terephthalate). The homogeneous SWCNT films were fabricated by a vacuum filtration method, and the averaged resistivity and transparency of the fabricated flexible SWCNT films were 400 Ω sq −1 and 80%, respectively. The glucose sensing electrodes were constructed by encapsulating glucose oxidase (GOx) by Nafion binder into the SWCNT film, and the variation in current response as a function of enzyme loading amount, Nafion thickness were investigated. 30 mg mL −1 GOx and 2% Nafion was optimal for the detection of glucose. When ferrocene monocarboxylic acid (FMCA) was introduced as diffusional electron mediator, the current responses toward glucose of the Nafion/GOx/SWCNT electrodes in glucose solution containing FMCA were dramatically improved, and the developed sensor was independent of oxygen. In the application of GOx immobilized SWCNT films for glucose detection, a linear electrical response was observed for concentrations ranging from 0.25 to 3.0 mM, and the detection limit and the sensitivity were assessed to be 97 μM and 9.32 μA mM −1 cm −2, respectively. Moreover, according to the Lineweaver–Burk plot, the apparent Michaelis–Menten constant was calculated to be 23.8 mM, and the current responses did not interfere with coexisting electroactive species, indicating that Nafion is an effective permselective polymer barrier.

In situ synthesis of biomolecule encapsulated gold-cross-linked poly(ethylene glycol) nanocomposite as biosensing platform: A model study

In situ synthesis of poly(ethylene glycol) (PEG) hydrogels containing gold nanoparticles (AuNPs) and glucose oxidase (GOx) enzyme by photo-induced electron transfer process was reported here and applied in electrochemical glucose biosensing as the model system. Newly designed bionanocomposite matrix by simple one-step fabrication offered a good contact between the active site of the enzyme and AuNPs inside the network that caused the promotion in the electron transfer properties that was evidenced by cyclic voltammetry as well as higher amperometric biosensing responses in comparing with response signals obtained from the matrix without AuNPs. As well as some parameters important in the optimization studies such as optimum pH, enzyme loading and AuNP amount, the analytical characteristics of the biosensor (AuNP/GOx) were examined by the monitoring of chronoamperometric response due to the oxygen consumption through the enzymatic reaction at − 0.7 V under optimized conditions at sodium acetate buffer (50 mM, pH 4.0) and the linear graph was obtained in the range of 0.1–1.0 mM glucose. The detection limit (LOD) of the biosensor was calculated as 0.06 mM by using the signal to noise ratio of 3. Moreover, the presence of AuNPs was visualized by TEM. Finally, the biosensor was applied for glucose analysis for some beverages and obtained data were compared with HPLC as the reference method to test the possible matrix effect due to the nature of the samples.

Adsorptive Immobilization of Four Proteases on Different Molecular Sieves

The immobilization of four different proteases on a series of molecular sieves, including microporous hy, nay, nh4y, mcm-22, h beta zeolites, modified y zeolites hday, hnh(4)day, and mesoporous mcm-41, by adsorption was systematically investigated the four proteases were alpha-chymotrypsin, papain, subtilisin, and thermoase (or its pure form thermolysin) the results showed that the enzyme loading amount and the activity of immobilized enzyme were significantly affected not only by the structures and textures of molecular sieves and the enzyme properties. but also by the adsorptive conditions such as buffer ph values and enzyme concentration in most cases, the amount of protease loading on molecular sieves was relatively higher at ph 6 0, but declined with further increasing of ph values the nature of the interaction between protease and molecular sieves is discussed as for alpha-chymotrypsin, its loading amount on h beta zeolites was found to be the highest, whereas the activity of a-chymotrypsin immobilized on mcm-22 was the highest, which is probably due to different adsorption states.

Rheological property of extruded and enzyme treated flaxseed mucilage

Rheological properties of flaxseed mucilage under different extrusion conditions with or without enzyme treatment were studied. The steady-shear tests indicated that the apparent viscosity of flaxseed mucilage fitted well to Power law model and all samples showed a shear-thinning behavior. The consistency index decreased and the flow behavior index increased with an increasing temperature and a decreasing screw speed. The addition of initial moisture content improved the degradation of flaxseed mucilage while further increase affected the action negatively. The empirical equations describing relationships between parameters of rheological models and extrusion factors were obtained. The results showed that extrusion can damage the compact fiber structure which improved the effect of enzyme treatment. The apparent viscosity decreased with the increase in hydrolyzing time and enzyme loading amount.

Fabrication of High-Capacity Biomolecular Carriers from Dispersible Single-Walled Carbon Nanotube−Polymer Composites

One of the most interesting applications for carbon nanotubes is as a support material for bioanalytical devices. In this work, we successfully used an ultraviolet light initiated "graft from" polymerization method to fabricate polymer functionalized carbon nanotubes (PFCNTs) with pendant chains of various functionalities, including poly(ethylene glycol) chains to boost dispersibility and pendant epoxy groups for protein conjugate sites. A model enzyme, alkaline phosphatase, was used to study biomolecule loading efficiency as well as the retention of enzyme activity. Samples with various ratios of the two monomers were fabricated to optimize their use in aqueous environments, and an optimal composition was determined. This method allows the enhancement of enzyme loading amount while retaining high enzyme activity. The morphology of the carbon nanotubes were characterized by STEM and AFM before and after functionalization. In addition, the resulting PFCNTs were analyzed by FT-IR, TGA, and XPS.

Comparison of Different Functionalization Routes for the Fabrication of Enzyme-Single Wall Carbon Nanotube Conjugates

In this work, various methods to functionalize single wall carbon nanotubes with immobilized enzyme for bioanalytical application were investigated. Several different conjugation methods, including both covalent and noncovalent methodologies, were compared and the resulting enzyme loading and retained activity were evaluated. After enzymatic modification, the addition of amphiphilic molecules to aid in nanotube dispersability was also studies and its effect on enzyme activity determined. Different characterization tools were employed to observe the morphological changes before and after the functionalization, including FTIR, TEM, and AFM. Both the enzyme loading amount and percent retained activity was greatest for modification when the linker molecule 1-pyrenebutanoic acid succinimidyl ester was used to form the enzyme-carbon nanotube conjugate. In addition, several possible mechanisms were proposed to explain the loss of enzymatic activity following immobilization. While the addition of amphiphilic surfactant molecules aided in the ultimate dispersability of the carbon nanotubes in aqueous media, enzymatic activity was further decreased.

Enzymatically active colloidal crystal arrays

We report the construction of three-dimensional (3D) colloidal crystal arrays (CCA) and hollow colloidal crystal arrays (HCCA) derived from the self-assembly of polyelectrolytes (PE)-coated polystyrene (PS) particles and their use as models of high surface area systems to immobilize peroxidase (POD). POD molecules could infiltrate into the deep layers of CCA and HCCA through their interconnected pores and strongly adsorbed at the PE shell of the colloidal particles. And the total enzyme loading amount and bioactivities increased linearly with the thickness of the CCA till ca. 10 μm. Compared with flat substrates with the same geometrical area, CCA and HCCA exhibit much higher enzyme loading abilities (∼43 and 53 times respectively) and the resulting bioactivities (∼35 and 41 times respectively) due to their inherently higher surface area and 3D interconnected porous structures. In addition, HCCA could load approximately 30% more POD than CCA because some POD molecules could infiltrate into the interior of the hollow capsule under salt condition.

The effect of Tween-20 on simultaneous saccharification and fermentation of softwood to ethanol

Simultaneous saccharification and fermentation (SSF) of steam-pretreated wood constitutes an attractive process configuration for ethanol production from biomass. However, the high enzyme addition in SSF contributes to a high process cost. In this study we explore the effect of the non-ionic surfactant Tween-20 as an additive in SSF. Tween-20 addition at 2.5 g/l had several positive effects on SSF: (i) the ethanol yield was increased by 8%; (ii) the amount of enzyme loading could be reduced by 50%, while maintaining a constant yield; (iii) the enzyme activity increased in the liquid fraction at the end of SSF, probably by preventing unproductive binding of the cellulases to lignin, which could facilitate enzyme recovery; (iv) the time required to attain maximum ethanol concentration was reduced. Surfactants as an additive in SSF can significantly lower the operational cost of the process. However, less expensive surfactants must be investigated.

Covalent immobilization of lipase onto hydrophobic group incorporated poly(2-hydroxyethyl methacrylate) based hydrophilic membrane matrix

In this study, a hydrophobic group containing monomer, 2-methacrylamidophenyalanine (MAPA) was prepared by using methacrylochloride and phenylalanine. Then, poly(2-hydroxyethyl methacrylate-co-methacrylamido-phenlyalanine) (pHEMA-MAPA) membranes were prepared by UV-initiated photopolymerization of HEMA and MAPA in the presence of an initiator α-α ′-azobisisobutyronitrile (AIBN). The lipase was immobilized onto these membranes by covalent bonding through carbodiimide activation. The amount of enzyme loading on the membranes was increased as the MAPA ratio increased in the membrane structure. Immobilization improved the pH stability of the enzyme as well as its temperature stability. Thermal stability was found to increase with immobilization and at 60 ° C the thermal stability constants were 1.1×10 −1min for free enzyme and 1.2×10 −2min for the immobilized enzyme. The immobilized enzyme activity was found to be quite stable in repeated experiments.

Determination of urate in undiluted whole blood by enzyme electrode

An amperometric enzyme electrode is described for the assay of urate in undiluted, unstirred whole blood. The electrode used Aspergillus flavus uricase (EC.1.7.3.3) cross-linked to bovine serum albumin by means of glutaraldehyde, sandwiched between a dimethyldichlorosilane-treated microporous polycarbonate membrane and an inner cellulosic H 2O 2-selective membrane. The resulting device had a low pH dependence, was capable of repeated use in blood, and gave an acceptable correlation with a standard spectrophotometric method. Electrode steady state and dynamic response were found to be dependent upon the amount of enzyme loading, and could be further optimised by the incorporation of catalase in the enzyme layer.

The production of 6‐aminopenicillanic acid by a multistage tubular reactor packed with immobilized penicillin amidase

A theoretical model equation was derived to find the correlation between the conversion and the amount of immobilized penicillin amidase in column. The theoretical values of the conversion were predicted form this correlation and compared with experimental results. It was observed in a column reactor that the pH drop along the column path was linear versus the enzyme loading and that the enzyme activity was also linearly dependent on pH up to 8.0. In order to diminish the effect of pH drop, a continuous two-stage plug-flow reactor (PFR) with pH adjustment between the two columns was used was used in the experiments, and two- and three-stage PFRs were simulated by computer. In the case of the two-stage PFR, the maximum productivity was demonstrated experimentally and theoretically as well. when an equal amount of the immobilized enzyme was packed in both columns. It was also predicted in the tree-stage PFR system that the optimal distributions of enzyme loading in three columns were found to be 1:1:1. It was demonstrated that the increased number of reactors in series could enhance the level of the maximum productivity with a given amount of enzyme loading.