Specific activities of the resin after immobilization of enzymes (5 mg enzyme/gresin) and percentage of residual activity after immobilization (2 h, room temperature, in 50 mM phosphate buffer pH 8) relative to the free protein. The specific activity of the resin after immobilization of the T4L-HLADHs (1 mg enzyme/gresin) was generally very low compared to the native imm-HLADH (Table 3).
At the determined optimum temperature (65◦C), the immobilized enzyme retained 63% of the activity compared to the free enzyme. At the optimum pH for the free (pH 5) and immobilized enzyme (pH 7), the immobilized enzyme retained approximately 62% of the activity of the free enzyme after 20 min of reaction.
Materials and Methods 1. Enzyme Production
Free and immobilized Endo BI-1 were reacted with RNase B at 45 °C at each pH for 20 min, and the relative activity, measured as the ability of the enzyme to deglycosylate RNase B, was measured by MALDI-ToF MS. Release of N-glycans from bovine colostrum whey proteins by free and immobilized Endo BI-1 Bovine colostrum whey proteins were isolated from bovine colostrum whey kindly provided by La Belle (Bellingham, WA, USA).
Comparison of anti-pathogenic activities of the human and bovine milk N-glycome: Fucosylation is a key factor. Comparison of the human and bovine milk N-glycomes via high-performance microfluidic slide liquid chromatography and tandem mass spectrometry.J.
Results and Discussion
HPAEC-PAD chromatogram of the reaction of 600 g/L sucrose at 50◦C with pXd-INV entrapped in PVA. As shown, the activity of the PVA lens-shaped particles was maintained up to approximately 40◦C.
Materials and Methods 1. Materials
The apparent activity of the immobilized biocatalysts was determined using a methodology developed in our group . The thermostability of lens-shaped PVA particles was analyzed by incubating the immobilized biocatalyst at different temperatures (4–60◦C) for 24 h in 100 mM sodium acetate buffer (pH 5.0).
In the case of the reference, substrate was added after the enzyme was first inactivated by incubation in TCA. This suggested that the first immobilization step in both cases is the covalent attachment of the enzyme.
Techniques for Preparation of Cross-Linked Enzyme Aggregates and Their Applications in Bioconversions
Cross-Linking Enzyme Immobilization
They showed that coimmobilization with PEI increased the crosslinking efficiency even at low concentrations of the enzyme. It is assumed that the surfactants can be washed out of the active sites after the cross-linking reaction . As expected, the operational stability of the spherical CLEAs was higher than that of the free enzyme.
The optimum operating temperature of the magnetic CLEAs was better than that of the free enzyme and nonmagnetic CLEAs. The thermal and storage stabilities of the CLEAs were better than those of the free enzymes. The thermal and pH stabilities of the multi-CLEAs were clearly better than those of the free enzymes.
The activities of multi-CLEA in bean protein and zein hydrolysis were superior to those of the free enzymes.
Application for Processing by CLEAs
Due to the high melting temperatures and relatively low decomposition temperatures of polyamides with aliphatic-aromatic structures, the synthesis of polyamides by melting processes is usually difficult. The synthesis of silver nanoparticles by conventional methods is energy and capital intensive and environmentally unfriendly due to the use of toxic solvents or additives that are difficult to remove and degrade on an industrial scale. One of the investigated enzymes, laccase, is widely used for catalytic degradation in industrial wastewater treatment .
Control of the solution flow rates in laminar flow enabled obtaining h-CLEA laccase particles with a size of 220±10 nm. The activity of h-CLEA laccase was comparable to the activity of the free enzyme at neutral pH, indicating that the laccase in the hollow structures had the same three-dimensional structure as free laccase. Endocrine Disrupting Chemicals (EDCs) are defined as "exogenous chemical substances or mixtures that alter the structure or functions of the endocrine system and cause adverse effects at the level of the organism, its progeny, populations or subpopulations of organisms, based on scientific principles, data, weight of evidence and the precautionary principle” .
The activity and stability of magnetite CLEAs and the reusability of CLEAs were reported to be enhanced; a higher yield of hydrolysis was obtained with SCB.
Preparation of cross-linked enzyme aggregates using bovine serum albumin as a protein feed.Anal. Poly-lysine supports cross-linked enzyme aggregates with efficient enzymatic activity and high operational stability. Cellulase cross-linked enzyme aggregates (CLEA) activities can be modulated and enhanced by precipitant selection.J.
Preparation of cross-linked enzyme aggregates in water-in-oil emulsion: application to trehalose synthase.J. Combined cross-linked enzyme aggregates (combi-CLEA) for efficient integration of ketoreductase and cofactor regeneration system. J. Magnetic combined cross-linked enzyme aggregates (combi-CLEA) for regeneration of cofactors in chiral alcohol synthesis.
A novel cross-linked enzyme aggregates (CLEAs) of papain and neutrase production, partial characterization and application.Int.
Rapid Immobilization of Cellulase onto Graphene Oxide with a Hydrophobic Spacer
The relative activity of free and immobilized cellulase was evaluated in the pH range from 3.0 to 8.0 and from 30 to 80 °C (Figure 6). The highest activities of free and immobilized cellulase were observed at pH 5.0, as shown in Figure 6a. Immobilized cellulase showed significantly higher relative activity than free cellulase at pH 3.0 to 6.0.
It is noted that the relative activity of immobilized cellulase was about two times higher than that of free cellulase at pH 3.0-4.0. The immobilized cellulase showed relatively higher activities at a temperature range of 30 to 80◦C compared to the free cellulase. As shown in Figure 8, the relative activity of free and immobilized decreased with the increase of incubation time, the activity of free cellulase decreased sharply than that of the immobilized cellulase.
The thermal deactivation constant (kd) of the immobilized cellulase (0.013) was six times lower than that of the free enzyme (0.078).
In addition, the good reusability and high thermal stability of immobilized cellulases make them quite attractive for industrial applications.
Materials and Methods 1. Materials
Cellulase activity was determined based on the amount of glucose released from the CMC solution under cellulase catalysis. One unit (U) of cellulase is defined as the amount of enzyme that hydrolyzes CMC to produce 1 mg of glucose equivalent per minute under assay conditions. The thermal stability of cellulase was investigated by incubating free and immobilized enzyme from 30 to 180 minutes at optimal pH and at 50 °C.
Co-immobilization of three cellulases on Au-doped magnetic silica nanoparticles for cellulose degradation. Immobilization of glycerol dehydrogenase and NADH oxidase for enzymatic synthesis of 1,3-dihydroxyacetone with in situ cofactor regeneration.J. Immobilization of glucose oxidase on graphene and cobalt phthalocyanine composite and its application for the determination of glucose.
Covalent immobilization of glucose oxidase on Poly(St-GMA-NaSS) monodisperse microspheres via BSA as spacer arm.Appl.
Immobilization of Cellulase on a Functional
Inorganic–Organic Hybrid Support: Stability and Kinetic Study
Materials and Methods
Physico-chemical pretreatment of the renewable biomass was carried out in the presence of 1.5% H2SO4 at 150◦C during 1 hour. Production of bacterial cellulose by Acetobacter xylinum using acetic acid hydrolyzate of bagasse as a low-cost carbon source. BioResources. Green synthesis of bacterial cellulose via acetic acid prehydrolysis liquor of agricultural maize stalk used as carbon source.Bioresour.
Optimizing bacterial cellulose production in surface culture: Evaluation of the effects of substrate mass transfer on bioreaction (Part 1).Eng. Production of bacterial cellulose by static cultivation using beer culture broth waste. Korejski J. Statistical optimization of culture conditions for bacterial cellulose production by Acetobacter xylinumBPR 2001 from maple syrup. Carbohydr.
The bio-based economy or bioeconomy is defined by the European Commission as "sustainable production.
The Development of Yeast Cell Surface Display 1. Development of the Surface Display Technology
Successful expression of heterologous proteins on the surface of yeast requires fusion with a good yeast anchor protein. Therefore, the choice of anchor protein thus depends on the characteristics of the POI (e.g. active site location, application, complex formation, etc.). Considering the inherent properties of POI, the anchor protein can be fused to either the N or C terminus of the POI, and Figure 2 shows how the POI-anchor fusion is localized on the yeast surface based on the commonly used anchor protein systems and Table 2 shows the features of the yeast anchor protein systems, presented in the following sections.
Covalently bound cell wall mannoprotein; a major component of the cell wall; plays a role in stabilizing the cell wall; Juxtaposed Assembly of Non-Native Adapters and Secreted Enzymes on Yeast (JANNASEY)—this strategy is developed based on biomimicry of the "cellulosome". Complex adapter assembly (CAA)—this is a direct biomimicry of the natural bacterial cellulose assembly, where one or more adapter subunits are assembled on the bacterial surface.
Complex assembly is regulated by the valence (monovalent if it contains a single cohesin and polyvalent if it contains more than one) of the adapter subunit.
Yeast Surface Display Studies for Bioethanol Production from Lignocellulosic Biomass The selected studies presented in this section are those that have applied the yeast surface display
Furthermore, for the direct use of pretreated lignocellulosic substrates, the studies presented in this subsection so far have reported the application of the direct yeast surface display (DYSD) strategy. Looking at the ability to convert half of the expected ethanol from the substrate (ESCY of >50%), it can be generalized that the direct co-expression (DSYD-CD) of cellulases performed well considering that the substrates are acidic (PASC) and alkali (BBglucan) pretreated and soluble (CMC). Substrate transport in the yeast cell - this area involves the efficient transport of fermentable sugars into the cell.
Enzyme functionality refers to the retention or enhancement of the enzyme activity once it is heterologously expressed in yeast. The details about the identity and the protein engineering methods for the transporters of the mentioned sugars are as provided in the reference material . In the studies presented in Section 3, so far only the combination of the cellobiose transport and utilization pathway with yeast surface display has been investigated for the direct co-display (DYSD) , and complex adapter assembly strategies (JANNASEY) .
This may cause an improved accessibility of the enzyme in the center of the initially densely packed pocket. Method A (Figure 6a): a tea bag for trapping the enzyme and magnetic stirring bar were used. The enzyme on the carrier was tightly packed so that no movements of the particles were possible.
The column oven of the HPLC (CTO-20AC) (Shimadzu, Kyoto, Japan) was set at 40◦C and the UV detection was set at 216 nm and the flow rate was 1 mL/min with the heptane:2-propanol as the mobile phase. Kinetic controlled synthesis of ampicillin and cephalexin in highly condensed systems in the absence of a liquid aqueous phase.J. Enzyme-catalyzed addition of hydrocyanic acid to substituted pivalaldehydes—A new synthesis of (R)-pantolactone.Tetrahedral asymmetry.
Hydroxynitrile lyase from Arabidopsis thaliana: Identification of reaction parameters for the synthesis of enantiopure cyanohydrin by pure and immobilized catalyst.Adv. Hydroxynitrile Lyase Catalyzed Synthesis of Cyanohydrins in Organic Solvents Parameters Affecting Activity and Enantiospecificity.Enzyme. Parameters influencing the stability and activity of αS-hydroxynitrile lyase from Hevea brasiliensis in two-phase systems.Enzyme.
This article is an open access article distributed under the terms of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).