Enzymatic synthesis of a sucrose-containing linear polyester in nearly anhydrous organic media

A variety of enzymes have been found to acylate sucrose in anhydrous pyridine. The enzymic reaction is highly selective; with trifluoroethylbutyrate as ester donor, enzyme-catalyzed transesterification of sucrose yielded sucrose 1'-butyrate and sucrose 6, 1'-dibutyrate. No sucrose-tributyrates were formed. Using a similar technique, a long-chain linear sucrose polyester has been prepared using Proleather, an alkaline protease from a Bacillus sp. This protease catalyzes the esterification of sucrose with bis(2, 2, 2-trifluoroethyladipate) in a 1:1 ratio to yield a sucrose-containing polyester with M(w) = 2100 and M(n) = 1600 for a polydispersity of 1. 31. Polymers with molecular weights in excess of 13, 000 have been prepared by this enzymic approach, indicating that molecules containing over 30 sucrose units have been produced. The polyester is extremely water soluble and soluble in polar organic solvents. As with the sucrose dibutyrate, the polyester has ester linkages at the C6 and C1' positions on the sucrose. The polyester can be depolymerized using Proleather in aqueous buffer, pH7. After 9 days in aqueous buffer, Proleather catalyzed the breakdown of the polyester to an M(w) of ca. 900. This sucrose-containing polyester may have applications as a water-absorbent, biodegradable plastic for use as diapers and hygienic products, water-treatment chemicals, and components of drug delivery systems.     

Influence of different silica derivatives in the immobilization and stabilization of a Bacillus licheniformis protease (Subtilisin Carlsberg)

Alcalase 2T, a commercial preparation of Subtilisin Carlsberg, was covalent immobilized onto physiochemically characterized silica supports. The effect of mean pore diameter and surface chemistry on enzyme activity in the hydrolysis of casein has been examined. Two sets of chemically distinct silica supports were used presenting terminal amino (S_APTES) or hydroxyl groups (S_TESPM-pHEMA). The percentage of immobilized protein was smaller in S_APTES (31–39%) than in S_TESPM-pHEMA (62–71%), but presented higher total and specific activity. Silicas with large pores (S₁₀₀₀, 130/1200 Å) presented higher specific activities relative to those with smaller pore sizes (S₃₀₀, 130/550 Å). The influence of glutaraldehyde concentration and the time of enzyme coupling to the S₁₀₀₀S_APTES supports was examined. The apparent K_m value for the S₁₀₀₀S_APTES immobilized enzyme is lower than the soluble one which may be explained by the partitioning effects of the substrate. No intraparticle diffusion limitations were observed for the immobilized enzyme and therefore the substrate diffusion does not influence the observable kinetics. Finally, the optimum pH, optimum temperature, thermal stability, operational stability, and storage stability of the immobilized and freely soluble enzymes were compared.     

Enzymatic synthesis of inulin-containing hydrogels

The Bacillus subtilis protease Proleather FG-F catalyzed the transesterification of inulin with vinyl acrylate (VA) in dimethylformamide (DMF). The reaction conversion for different VA concentrations was greater than 57% after 96 h at 50 degrees C. The degree of substitution (DS, defined as the amount of acrylate groups per 100 inulin fructofuranoside residues) with acrylate moieties can be controlled by varying the molar ratio of VA to inulin. Reasonable yields were obtained (44-51%, 2 days) using a two-step purification methodology. Inulin derivatized with VA (Inul-VA) was characterized by gel permeation chromatography, and its structure was established by (1)H, (13)C, and (1)H-(1)H correlation spectroscopy and (1)H-(13)C heteronuclear multiple quantum coherence NMR. The main positional isomer was at the 6 position of the fructofuranoside residue and two other minor isomers were observed at the 3 and 4 positions. Thus, the enzymatic reaction was largely regioselective. Furthermore, the inulin fructose residues were monosubstituted. Gels with swelling ratios at equilibrium of up to ca. 20 were prepared by free radical polymerization of aqueous solutions of Inul-VA with different DS and monomer concentrations. Gel pore sizes were calculated from swelling experiments and range from 19 to 57 A. To our knowledge, this work reports the first successful enzymatic modification of a polysaccharide solubilized in 100% DMF solution.     

Effects of pore characters of mesoporous resorcinol–formaldehyde carbon gels on enzyme immobilization

This paper demonstrates, for the first time, the use of resorcinol–formaldehyde carbon gels (RFCs) as enzyme carriers. The immobilization behavior of Bacillus licheniformis serine protease in RFCs of different pore characters was investigated. RFCs derived with (RF1) and without (RF2) cationic surfactant (trimethylstearylammonium chloride; C18) resulted in predominantly microporous, and mesoporous characters, respectively. It was found that support pore size and volume were key parameters in determining immobilized enzyme loading, specific activity, and stability. RF2, with higher mesopore volume (V_mes: RF1 = 0.21 cm³/g; RF2 = 0.81 cm³/g) and mesopore size radius (RF1 = 1.7–3.8 nm; RF2 = 7.01 nm), accommodated approximately fourfold more enzyme than RF1. Serine protease loading in RF2 could reach as high as 21.05 unit/g support. In addition, RF2 was found to be a better support in terms of serine protease operation and storage stability. Suitable mesopore size likely helped preventing immobilized enzyme from structural denaturation due to external forces and heat. However, immobilized enzyme in RF1 gave 12.8-fold higher specific activity than in RF2, and 2.1-fold higher than soluble enzyme. Enzyme leaching was found to be problematic in both supports, nonetheless, higher desorption was observed in RF2. Enhancement of interaction between serine protease and RFCs as well as pore size adjustment will be necessary for repeated use of the enzyme and further process development.     

Biocatalytic esterification of citronellol with lauric acid by immobilized lipase on aminopropyl-grafted mesoporous SBA-15

Mesoporous SBA-15 was synthesized under acidic condition at 40 °C with a non-ionic triblock copolymer (P123) as the template. The synthesis gel composition used was 1 SiO₂:0.017 P123:2.9 HCl:202.6 H₂O. Functionalization of SBA-15 with 3-aminopropyltriethoxysilane (APTES) by post-synthesis method was performed under reflux for 2 h. The mesoporous samples were characterized using Fourier transform infrared (FT-IR), nitrogen adsorption, transmission electron microscopy (TEM) and scanning electron microscopy (SEM). They were then utilized as supports for the immobilization of lipase to be subsequently used for the esterification of citronellol and lauric acid. Leaching and reusability tests were also conducted on the immobilized enzymes. Functionalization resulted in about 10% improvement in enzyme loading, leading to higher activity. The immobilized enzyme was also more stable to low pH and high temperature while showing better retention (up to 95%) of enzyme molecules. Immobilized lipase maintained 90% of its esterification activity in non-aqueous system even after 4 cycles of use. The improvements were associated with enhanced surface hydrophobicity, changes in pore shapes and stronger enzyme–support interactions with minimal effects to the enzymatic activity.     

Effects of the immobilization supports on the catalytic properties of immobilized mushroom tyrosinase: a comparative study using several substrates

Mushroom tyrosinase was immobilized from an extract onto glass beads covered with one of the following compounds: the crosslinked totally cinnamoylated derivatives of glycerine, D-sorbitol, D-manitol, 1,2-O-isopropylidene-alpha-D-glucofuranose, D-glucuronic acid, D-gulonic acid, sucrose, D-glucosone, D-arabinose, D-fructose, D-glucose, ethyl-D-glucopyranoside, inuline, dextrine, dextrane or starch, or the partially cinnamoylated derivative 3,5,6-tricinnamoyl-D-glucofuranose which was obtained by the acid hydrolysis of 1,2-O-isopropylidene-alpha-d-glucofuranose. The enzyme was immobilized by direct adsorption onto the support and the quantity of tyrosinase immobilized was found to increase with the hydrophobicity of the supports. The kinetic constants of immobilized tyrosinase acting on the substrates, 4-tert-butylcatechol, dopamine and DL-dopa, were studied. When immobilized tyrosinase acted on 4-tert-butylcatechol, the values of K(m)(app) were lower than these obtained for tyrosinase in solution while, when dopamine and DL-dopa were used, the K(m)(app) were higher. The order of the substrates as regards their ionizable groups, DL-dopa (two ionizable groups)>dopamine (one ionizable group)>4-tert-butylcatechol (no ionizable group) coincided with the order of the K(m)(app) values shown by tyrosinase immobilized on the hydrophobic supports, and was the inverse of that observed for tyrosinase in solution. The K(m)(app) values of immobilized tyrosinase were in all cases higher than those of soluble tyrosinase and depended on the nature of the support and the hydrophobicity of the substrate, meaning that it is possible to design supports with different degrees of selectivity towards a mixture of enzyme substrates in the reaction medium.     

Forced-flow bioreactor for sucrose inversion using ceramic membrane activated by silanization

A forced-flow enzyme membrane reactor system for sucrose inversion was investigated using three ceramic membranes having different pore sizes. Invertase was immobilized chemically to the inner surface of a ceramic membrane activated by a silane-glutaraldehyde technique. With the cross-flow filtration of sucrose solution, the reaction rate was a function of the permeate flux, easily controlled by pressure. Using 0.5 mum support pore size of membrane, the volumetric productivity obtained was 10 times higher than that in a reported immobilized enzyme column reactor, with a short residence time of 5 s and 100% conversion of the sucrose inversion.     

Effects of pore characters of mesoporous resorcinol–formaldehyde carbon gels on enzyme immobilization

This paper demonstrates, for the first time, the use of resorcinol–formaldehyde carbon gels (RFCs) as enzyme carriers. The immobilization behavior of Bacillus licheniformis serine protease in RFCs of different pore characters was investigated. RFCs derived with (RF1) and without (RF2) cationic surfactant (trimethylstearylammonium chloride; C18) resulted in predominantly microporous, and mesoporous characters, respectively. It was found that support pore size and volume were key parameters in determining immobilized enzyme loading, specific activity, and stability. RF2, with higher mesopore volume (V_mes: RF1 = 0.21 cm³/g; RF2 = 0.81 cm³/g) and mesopore size radius (RF1 = 1.7–3.8 nm; RF2 = 7.01 nm), accommodated approximately fourfold more enzyme than RF1. Serine protease loading in RF2 could reach as high as 21.05 unit/g support. In addition, RF2 was found to be a better support in terms of serine protease operation and storage stability. Suitable mesopore size likely helped preventing immobilized enzyme from structural denaturation due to external forces and heat. However, immobilized enzyme in RF1 gave 12.8-fold higher specific activity than in RF2, and 2.1-fold higher than soluble enzyme. Enzyme leaching was found to be problematic in both supports, nonetheless, higher desorption was observed in RF2. Enhancement of interaction between serine protease and RFCs as well as pore size adjustment will be necessary for repeated use of the enzyme and further process development.     

Digestion of protein substrates by subtilisin: immobilization changes the pattern of products

Subtilisin BPN' (Bacillus protease strain N') was immobilized on glass-bead carriers of controlled pore size by the glutaraldehyde method. The Vmax and Km values of the synthetic substrate were similar for immobilized and free enzymes. However, the hydrolytic patterns of immobilized and free enzymes toward casein and carboxymethylated lysozyme were different. The free enzyme rapidly hydrolyzed the substrate in the early stage of the reaction to produce peptides of various sizes. The immobilized enzyme, however, slowly digested the casein and lysozyme during digestion; even in the late stage of digestion the original substrates were present in the reaction mixture. The peptide size produced by immobilized enzyme depended on the pore size of the carrier; enzyme immobilized on glass of smaller pore size produced smaller peptide products. These phenomena found with our system of immobilized protease and a protein substrate can be explained by a multiple attack mechanism, in which the substrate that has been forced to enter the matrix is attacked many times by the protease to be completely hydrolyzed, because the substrate and the intermediate-sized product are trapped inside the matrix under reduced diffusion movement. To explain the effective digestion that forms amino acids, we have proposed that a multiple type of attack is responsible for the intracellular protein degradation that takes place in cellular organelles in which hydrolytic enzymes are entrapped.     

Biosynthesis of pullulan using immobilized Aureobasidium pullulans cells

Immobilization of Aureobasidium pullulans by adsorption on solid supports and entrapment in open pore polyurethane foam were attempted. By adsorption, the highest cell loading of 0.012-0.018 g dry wt/cm(2) support was obtained in pH 2.0 medium. Under this acidic condition, the net surface charges (zeta potentials) of both the cells and supports were close to zero and no pullulan was synthesized. Cationic coatings of Cytodex and polyethylenimine were not efficient in enhancing the binding strength between the cells and the supports. Surface immobilized cells and polyurethane foam entrapped cells exhibited a similar fermentation characteristics resulting in ca. 18 g/L pullulan and ca. 5 g/L leaked cells. However, cells entrapped in the polyurethane foam were more shear resistant. The immobilized cells thus could be repeatedly used for pullulan biosynthesis.     

Optimizing Immobilization and Stabilization of Feruloyl Esterase from Humicola Insolens and its Application for the Feruloylation of Oligosaccharides

Feruloyl esterase (FAE)-catalyzed esterification reaction is as a potential route for the biosynthesis of feruloylated oligosaccharides as functional ingredients. Immobilization of FAE from Humicola insolens on metal chelate-epoxy supports was investigated. The study of effects of immobilization parameters using response surface methodology revealed the significance of enzyme/support ratio (3.25-29.25 mg/g support), immobilization time (14-38 h), buffer molarity (0.27-1.25 M) and pH (4.0-8.0). The interactions between enzyme-to-support ratio/buffer molarity and enzyme-to-support ratio/pH were found to be critical for the modulation of the immobilization activity yield and the retention of specific activity, respectively. Optimum conditions for FAE-immobilization on metal chelate Sepabeads® EC-EP R were identified to be 22.75 mg FAE/g support, pH of 5.0, 27.7 h and buffer molarity of 0.86 M. At these conditions, an activity yield of 82.4%, a specific activity retention of 143.4%, and an enzyme activity of 395.4 μmol/min. g support were achieved. Further incubation of the immobilized FAE at pH 10.0 improved its thermostability. Increasing the pore size of the epoxy support improved the retention of FAE hydrolytic activity and the esterifying efficiency of the immobilized biocatalyst. Optimally immobilized and stabilized FAE on metal chelate-epoxy support retained up to 92.9% of the free enzyme feruloylation efficiency to xylooligosaccharides..     

Immobilized Clostridium perfringens neuraminidase. Substrate cleavage and enzyme release during incubation.

Pure Clostridium perfringens neuraminidase was immobilized on Sepharose 4 B, azido-Sepharose 4 B and controlled pore glass (CPG)- glycophase using different coupling procedures. The immobilized enzyme showed increased stability under various conditions relative to the soluble enzyme. The low release of active enzyme from the supports under incubation conditions was quantitated using a highly sensitive radioactive assay. The activity of the immobilized enzyme was dependent on the nature of the support and the substrate. Activity decreased with increasing substrate molecular weight, but the enzyme showed improved cleavage with GD1a micelles and human erythrocytes, substrates having ordered surface properties. Uses of immobilized neuraminidase in biochemistry and cell biology are considered and evaluated relative to the measured release of enzyme from the supports reported and to the molecular size and organization of possible substrates.     

Studies on immobilized Saccharomyces cerevisiae. III. Physiology of growth and metabolism on various supports

In earlier communications general analyses of rapid ethanol fermentation by Saccharomyces cerevisiae immobilized on inert supports were described. In this article physiology of growth and metabolism (parameters like rates of CO(2) evolution and O(2) uptake, respiratory quotient, and generation time) of Saccharomyces cerevisiae immobilized on different supports are reported. Values of the ratio of specific oxygen uptake rate for immobilized cells to free cells have been found to be 0.732, 0.781 and 0.785 for carrier A, carrier B, and covalently crosslinked controlled pore glass (CPG, specific surface area of 439 m(2) g(-1)), respectively. Rates of specific CO(2) evolution for immobilized cells to free cells for these supports are 0.784, 0.822, and 0.783, respectively. Marked reduction in generation time of Saccharomyces cerevisiae on all the supports has been observed. No change in size (4.8-5 mum) and specific growth rate (mu(m) = 0.275 h(-1)) of cells leaving the reactor has been observed.     

Neutron and X-ray crystallographic analysis of Achromobacter protease I at pD 8.0: Protonation states and hydration structure in the free-form

The structure of the free-form of Achromobacter protease I (API) at pD 8.0 was refined by simultaneous use of single crystal X-ray and neutron diffraction data sets to investigate the protonation states of key catalytic residues of the serine protease. Occupancy refinement of the catalytic triad in the active site of API free-form showed that ca. 30% of the imidazole ring of H57 and ca. 70% of the hydroxyl group of S194 were deuterated. This observation indicates that a major fraction of S194 is protonated in the absence of a substrate. The protonation state of the catalytic triad in API was compared with the bovine β-trypsin–BPTI complex. The comparison led to the hypothesis that close contact of a substrate with S194 could lower the acidity of its hydroxyl group, thereby allowing H57 to extract the hydrogen from the hydroxyl group of S194. H210, which is a residue specific to API, does not form a hydrogen bond with the catalytic triad residue D113. Instead, H210 forms a hydrogen bond network with S176, H177 and a water molecule. The close proximity of the bulky, hydrophobic residue W169 may protect this hydrogen bond network, and this protection may stabilize the function of API over a wide pH range.     

Development of sensors for direct detection of organophosphates. Part I: Immobilization, characterization and stabilization of acetylcholinesterase and organophosphate hydrolase on silica supports

Biosensors for organophosphates in solution may be constructed by monitoring the activity of acetylcholinesterase (AChE) or organophosphate hydrolase (OPH) immobilized to a variety of microsensor platforms. The area available for enzyme immobilization is small (< 1 mm2) for microsensors. In order to construct microsensors with increased surface area for enzyme immobilization, we used a sol-gel process to create highly porous and stable silica matrices. Surface porosity of sol-gel coated surfaces was characterized using scanning electron microscopy; pore structure was found to be very similar to that of commercially available porous silica supports. Based upon this analysis, porous and non-porous silica beads were used as model substrates of sol-gel coated and uncoated sensor surfaces. Two different covalent chemistries were used to immobilize AChE and OPH to these porous and non-porous silica beads. The first chemistry used amine-silanization of silica followed by enzyme attachment using the homobifunctional linker glutaraldehyde. The second chemistry used sulfhydryl-silanization followed by enzyme attachment using the heterobifunctional linker N-gamma-maleimidobutyryloxy succinimide ester (GMBS). Surfaces were characterized in terms of total enzyme immobilized, total and specific enzyme activity, and long term stability of enzyme activity. Amine derivitization followed by glutaraldehyde linking yielded supports with greater amounts of immobilized enzyme and activity. Use of porous supports not only yielded greater amounts of immobilized enzyme and activity, but also significantly improved long term stability of enzyme activity. Enzyme was also immobilized to sol-gel coated glass slides. The mass of immobilized enzyme increased linearly with thickness of coating. However, immobilized enzyme activity saturated at a porous silica thickness of approximately 800 nm.     

Continuous production of galacto-oligosaccharides from lactose using immobilized β-galactosidase from Bacillus circulans

Summary -Galactosidase-2 (-d-galactoside galactohydrolase, EC 3.2.1.23) from Bacillus circulans was purified using hydroxyapatite gel chromatography and immobilized onto Duolite ES-762 (phenolformaldehyde resin) and Merckogel (controlled pore silica gel) for continuous production of galacto-oligosaccharides using lactose as the substrate. The maximum amount of ologosaccharides produced by the immobilized enzyme was 35–40% of the total sugar during hydrolysis of 4.56% lactose. Partially purified -galactosidase from B. circulans was also immobilized onto various supports for the same purpose. The stability of the immobilized -galactosidase-2 or partially purified enzyme during a continuous reaction depended on their supports and specific activity. Of the supports tested, Merckogel was best for operational stability. With this support, the enzyme was quite stable with specific activity up to 15 units/g of wet gel; it was reversibly inactivated with more.     

Chitosan-based hybrid immobilization in bienzymatic reactions and its application to the production of laminaribiose

A hybrid-immobilization method was developed to improve the long-term stability of laminaribiose phosphorylase immobilized on epoxy supports Sepabeads EC-EP/S. Entrapment in chitosan retained all of the enzyme activity depending on the amount of entrapped solid materials and increased half-life by a factor of 10–94.4 h. No enzyme activity loss was determined during 12 times reuse. The immobilization method is also applicable to sucrose phosphorylase immobilized on Sepabeads EC-EP/S. Up to 31.9 g/L laminaribiose were produced in bienzymatic batch experiments with reaction-integrated product separation by adsorption on zeolites.

     

Selectivity and stability of alkaline protease AL-89 in hydrophilic solvents

The alkaline protease from Bacillus pseudofirmus strain AL-89 used vinyl fatty acid esters of increasing chain length from C10 to C18 equally well as substrates for esterification of sucrose in a reaction mixture of DMF and DMSO (1:1, v/v). The synthesized esters were purified and characterized by NMR and nano-electron spray MS. As evaluated by the initial reaction rates, the primary site of substitution of sucrose was at the C-2 position with the C-3 and C-3′ as secondary substitution sites. The enzyme catalysed the formation of 3-O-acyl sucrose from 2-O-acyl sucrose. The investigation did not reveal if the 3′-O-acyl sucrose was formed the same way. The synthesis of the 2-O-esters showed the characteristics of kinetically controlled reactions, whereas the formation of the 3-O- and 3′-O-esters showed the characteristics of equilibrium controlled reactions. The enzyme catalysed process was effected by initial water content, substrate molar ratio and reaction temperature. Under the reaction conditions of 0% initial water content, a molar ratio of sucrose to vinyl stearate of 1:1.5 and 70 °C an initial formation rate of 13.5, 2.9 and 2.1 μmol min⁻¹ was achieved for 2-O-, 3-O- and 3′-O-stearoyl sucrose respectively with a specific initial synthesis rate of 2-O-stearoyl sucrose of 0.27 μmol min⁻¹ mg⁻¹ biocatalyst. In the absence of substrates the enzyme proved to be more stable in DMF than in water and DMSO at 50 °C. Mixing DMF with DMSO 1:1 (v/v) increased the stability and the half-life was found equal to that in water. In the presence of substrates a residual activity of 40% was observed after 24 h of incubation in the 1:1 (v/v) mixture of DMF and DMSO at 70 °C.     

Immobilization ofβ-fructofuranosidase fromAureobasidium sp. ATCC 20524 on porous silica

Summary -Fructofuranosidase P-1 fromAureobasidium sp. ATCC 20524, which produces a fructo-oligosaccharide (1-kestose) from sucrose, was immobilized covalently onto alkylamine porous silica with glutaraldehyde at high efficiency (44.4%). Optimum pore diameter of porous silica for immobilization of the enzyme was 91.7 nm. The enzymatic profiles of immobilized enzyme were almost identical to the native one except its stabilities to temperature and metal ions were improved. 1-Kestose was produced continuously and selectively from 40% (w/v) sucrose at fast flow rates by a column packed with the immobilized enzyme for up to 26 days, and the effluent concentration of 1-kestose remained in the range 113–135 mg ml^–1.     

Preparation of immuno-affinity membranes for cholesterol removal from human plasma

Anti-low density lipoprotein antibody (anti-LDL) immobilized polyhydroxyethylmethacrylate (pHEMA) based membrane was prepared for selective removal of cholesterol from hypercholesterolemic human plasma. In order to further increase blood-compatibility, a newly synthesized comonomer, methacryloylamidophenylalanine (MAPA) was included in the membrane formulation. p(HEMA-MAPA) membranes were produced by a photopolymerization and then characterized by swelling tests, SEM and contact angle studies. Blood-compatibility tests were also investigated. The water swelling ratio of the p(HEMA-MAPA) membrane increases significantly (133.2.9%) compared with pHEMA (58%). p(HEMA-MAPA) membranes have large pores around in the range of 5-10 microm. All the clotting times increased when compared with pHEMA membranes. Loss of platelets and leukocytes was very low. The maximum anti-LDL antibody immobilization was achieved around pH 7.0. Immobilization of anti-LDL antibody was 12.6 mg/ml. There was a very low non-specific cholesterol adsorption onto the plain p(HEMA-MAPA) membranes, about 0.36 mg/ml. Anti-LDL antibody immobilized membranes adsorbed in the range of 4.5-7.2 mg cholesterol/ml from hypercholesterolemic human plasma. Up to 95% of the adsorbed LDL antibody was desorbed. The adsorption-desorption cycle was repeated 10 times using the same membrane. There was no significant loss in the adsorption capacity.