Surface immobilization and entrapping of enzymes on glutaraldehyde crosslinked gelatin particles

A mild and reproducible method has been developed for the surface-immobilization of enzymes on glutaraldehyde crosslinked gelatin beads. In this method glutaraldehyde is used in a dual capacity, as crosslinking agent and as the enzyme coupling agent. Glucoamylase (exo-α-1,4-d-glucosidase, EC 3.2.1.3), β-d-fructofuranosidase (invertase, EC 3.2.1.26) and β-d-glucoside (cellobiase, β-d-glucoside glucohydrolase, EC 3.2.1.21) have been successfully immobilized by this method, on the surface of the crosslinked gelatin particles. The method can be combined with the existing technology for the production of gelatin-entrapped enzymes. Thus, dual immobilized enzyme conjugates of glucoamylase and invertase have been prepared using this method, by entrapment of one enzyme in, and surface-binding of the other to, the gelatin matrix. The coupling of glucoamylase onto cross-linked gelatin particles by precipitation with poly(hexamethylenebiguanide hydrochloride) was also tested.     

A unique and highly efficient non-viral DNA/siRNA delivery system based on PEI-bisepoxide nanoparticles

Delivery of DNA and siRNA into mammalian cells is a powerful technique in treating various diseases caused by single gene defects. Herein, we report a highly efficient delivery system using 1,4-butanediol diglycidyl ether (bisepoxide) crosslinked polyethylenimine (PEI) nanoparticles (PN). The nanoparticle/DNA complexes (nanoplexes) exibited approximately 2.5- to 5.0-fold gene transfer efficacy and decreased cytotoxicity in cultured cell lines, compared to the native PEI (25 kDa) (gold standard) and commercially available transfection agents such as Lipofectamine 2000 and Fugene. The bisepoxide crosslinking results in change in amine ratio in PEI; however, it retains the net charge on PN unaltered. A series of nanoparticles obtained by varying the degree of crosslinking was found to be in the size range of 69-77 nm and the zeta potential varying from +35 to 40 mV. The proposed system was also found to deliver siRNA efficiently into HEK cells, resulting in approximately 70% suppression of the targetted gene (GFP).     

Studies on extracellular and intracellular purified amylases from a thermophilic Bacillus stearothermophilus

Extracellular and intracellular amylases have been purified from a thermophilic Bacillus stearothermophilus and further studies have been made with the purified enzyme. The molecular weights for extra- and intracellular α- and β-amylases were found to be 47 000, 58 000, 39 000 and 67 000, respectively. α-Amylase (1,4-α-d-glucan glucanohydrolase, EC 3.2.1.1) and glucoamylase (1,4-α-d-glucan glucohydrolase, EC 3.2.1.3) were glycoproteins, whereas β-amylase (1,4-α-d-glucan maltohydrolase, EC 3.2.1.2) had little or no carbohydrate moiety. Extracellular FI (α-amylase), FIII (glucoamylase), FIV and FV (α-amylase) had carbohydrate moieties of 14.4, 27.0, 11.0 and 12.5%, respectively, whereas intracellular amylases FI (α-amylase), FII (β-amylase) and FIII (α-amylase) contained 15.2, 0.8 and 13.4% carbohydrate, respectively. The amino acid profile of the amylase protein digest showed a total number of 16 amino acids with aspartic acid showing the highest value followed by glutamic acid and leucine plus isoleucine. Compared to other thermostable amylases, proline and histidine contents were low. Both α- and β- amylase had the - SH group at their active site, which was essential for enzyme activity. EDTA and parachloromercuribenzoate exhibited dose dependent non-competitive inhibition of enzyme activity indicating the involvement of a divalent cation and the - SH group for activity.     

Crosslinking of enzyme coaggregate with polyethyleneimine: A simple and promising method for preparing stable biocatalyst of Serratia marcescens lipase

Crosslinking of enzyme aggregates is a promising method for enzyme immobilization. In this work, crosslinked enzyme coaggregates of Serratia marcescens lipase with polyethyleneimine (CLECAs-SML-PEI) were prepared using polyethyleneimine (PEI) as coprecipitant and glutaraldehyde as crosslinking reagent. The crude lipase solution at a low protein concentration (0.1 mg/ml), with PEI at a mass ratio of 3:1 (PEI/protein, w/w), was found to be most adequate for the coprecipitation of SML. After crosslinking of the coaggregate of SML-PEI with 0.2% (w/v) glutaraldehyde under ambient temperature, over 70% of the total lipase activity was recovered. Compared with the free SML, the optimum temperature of the CLECAs-SML-PEI was enhanced from 50 °C to 60 °C and its thermal stability was also significantly improved. CLECAs-SML-PEI showed excellent operational stability in repeated use in aqueous–toluene biphasic system for asymmetric hydrolysis of trans-3-(4′-methoxyphenyl)glycidic acid methyl ester (MPGM), without significant inactivation after 10 rounds of repeated use.     

Preparation and properties of an immobilized Barley β-amylase

Barely β-amylase (α-1,4-glucan maltohydrolase, EC 3.2.1.2) has been immobilized by covalent fixation to amino derivatives of epichlorohydrin crosslinked Sepharose mediated by cyclohexyl isocyanide and acetaldehyde. The enzyme conjugates contain up to 35% of the total activity of the β-amylase added to the coupling mixture. The profiles of activity versus pH and ionic strength are essentially the same for free and immobilized β-amylase, whereas the resistance to inactivation during storage and use is considerably enhanced by immobilization. Columns with immobilized β-amylase have been used for continuous degradation of starch. At 45°C, half of the initial activity remains after seven weeks, and the corresponding figure at 23°C is 85 percent.     

Immobilization of enzymes on crosslinked gelatin particles activated with various forms and complexes of titanium(IV) species

A number of methods of activating the surface of glutaraldehyde crosslinked gelatin beads with titanium(IV) compounds, for subsequent enzyme coupling, have been investigated. Glucoamylase (exo-1,4-α-d-glucosidase, EC 3.2.1.3) was so immobilized using titanium(IV)-urea, -acrylamide, -citric acid and -lactose complexes; however, immobilized enzyme preparations with low activities were obtained (0.36–1.28 U g^?1). Activation with uncomplexed titanium(IV) chloride, however, of both moist and freeze-dried crosslinked gelatin particles resulted in highly active immobilized glucoamylase preparations (1.74–26.6 U g^?1). Dual immobilized enzyme conjugates of glucoamylase and invertase (β-d-fructofuranosidase, EC 3.2.1.26) were also prepared using this method. Invertase was served on the entrapped enzyme while glucoamylase was coupled on the surface of titanium(IV)-activated gelatin pre-entrapped invertase particles. A dual gelatin coupled glucoamylase/gelatin entrapped glucoamylase was prepared (3.8 U g^?1) and ～72.5% of the total combined activity was due to the surface bound enzyme.     

Application of cellulase and pectinase from fungal origin for the liquefaction and saccharification of biomass

Commercial cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] from Trichoderma viride and pectinase [poly(1,4-α-d-galacturonide) glycanohydrolase, EC 3.2.1.15] from Aspergillus niger have been applied to produce fermentation syrups from sugar-beet pulp and potato fibre. Cellulosic, hemicellulosic and pectic polysaccharides of these substrates were hydrolysed extensively. Recovery of enzymes has been investigated in a packed-column reactor, connected with a hollow-fibre ultrafiltration unit. Enzymes appeared to be stable in this type of reactor, although part of the enzyme activity was lost, especially by adsorption onto the substrate residue.     

Action of antioxidants on membrane-toxic effects of anticholinesterase agents

It was found therapeutic-preventive effectiveness of antioxidants (1,4-dihydropyridine derivatives) at poisoning with malathion insecticide. The effect of 1,4-dihydropyridine derivatives can be attributed to a prevention of lipid peroxidation. Antioxidants do not affect the toxicity of 0,0-dimethyl-0-2,2-dichlorvinylphosphate. Thus, antioxidants are pathogenetic drugs for treatment of poisonings with cholinesterase inhibitors.     

Immobilization of enzymes on spheron: 1. Trypsin and glucoamylase by the titanium-chelation method

In a preliminary study, trypsin (EC 3.4.21.4) and glucoamylase (exo-1,4-α-d-glucosidase, 1,4-α-d-glucan glucohydrolase, EC 3.2.1.3) were immobilized on Spheron by the titanium-chelation method. The activity of trypsin immobilized on Spheron P100 000 was higher against tosyl-l-arginine 4-nitroanilide than against casein. The variation in the specific activities of glucoamylase immobilized on Spherons of different porosities to wards substrates of different molecular weights was examined.     

Proteolytic inactivation of 1, 4-α-d-glucan 6-α-d-glucosyltransferase purified from Aspergillus niger R-27

An extracellular 1,4-α-d-glucan 6-α-d-glucosyltransferase [d-glucosyltransferase, 1,4-α-d-glucan:1,4-α-d-glucan(d-gluco 6-α-d-glucosyltransferase, EC 2.4.1.24] from Aspergillus niger R-27 has been purified and the kinetics of its proteolytic inactivation with subtilisin studied. The purified enzyme was shown to be homogeneous using disc polyacrylamide gel electrophoresis. It contained 16.0% mannose, 0.19% glucose and 2.95% 2-acetamido-2-deoxy-d-glucose. The characteristic feature of the proteolytic degradation of glucosyltransferase is rapid hydrolysis of ～12 peptide bonds per mol and the formation of an active intermediate product which is more resistant to further proteolysis, but is easily heat-inactivated. The isolation and some properties of glucosyltransferase are also described.     

Coriolus versicolor Laccase Catalyzes the Decarboxylation of 2-(4-Hydroxyphenyl)-glycine and 4-Hydroxymandelic Acid

Cyclodextrin glucanotransferase [1,4-α-D-glucan 4-α-D-(l,4-α-D-glucano)-transferase (cyclizing), EC 2.4.1.19] from an alkalophilic Bacillus species A2–5a had a wider acceptor specificity than that from B. macerans, which was similar to those from B. stearo-thermophilus and B. circulans.

Glucosyl rhamnose produced by the CGTase was identified as glucopyranosyl-α-l,4-rhamnopyranose by α- and β-glucosidase treatments, and ¹H- and ^{1 3}C-NMR analyses.     

An investigation into the pectolytic activity of the yeast Saccharomycopsis fibuliger

Saccharomycopsis fibuliger cells produce an inducible hydrolase, tentatively characterized as a polygalacturonase [poly(1,4-α-d-galacturonide) glycanohydrolase, EC 3.2.1.15], which is associated with the yeast cells and which causes the partial hydrolysis of pectin or poly-d-galacturonic acid. No evidence of pectinesterase (pectin pectyl hydrolyase, EC 3.1.1.11) or pectate lyase [poly(1,4-α-d-galacturonide) lyase, EC 4.1.1.1] activity has been found. Enzyme production took place at an optimum temperature of 28°C, whereas optimum activity was at ～45°C. The optimum pH for pectolytic activity was similar to the optimum pH for cell growth. A reduction in the concentration of dissolved oxygen in the culture medium and an increase in cell age caused an increase in the rate of pectin decomposition within the limits employed. Products of pectin decomposition consisted of a mixture of uronides including d-galacturonic acid.     

Crosslinking of DNA by busulfan. Formation of diguanyl derivatives

High-pressure liquid chromatography has been used to separate the derivatives of guanosine which are formed when this nucleoside is reacted with busulfan. Derivatives which have been identified by a combination of ultraviolet and mass spectrometry include: 7-(delta-hydroxybutyl)guanosine, 1,4-di(7-guanosyl)butane, and 1-(7-guanyl)-4-(7-guanosinyl)butane. The latter two derivatives can be converted to 1,4-di(7-guanyl)butane by mild acid hydrolysis. Using 1,4-di(7-guanyl)butane as a marker for high-pressure liquid chromatography, we have identified this compound as a product of the reaction between DNA and busulfan. These findings verify the previously unconfirmed report that busulfan is a crosslinking agent for DNA and strengthen the hypothesis that differences in the biological properties of alkylating agents result from differences in their reactions with DNA.     

Immobilization of glucoamylase on porous silicas

The influence of the pore structure of silica carriers (macroporous silica gels, silochromes and porous glasses) on the catalytic activity of immobilized glucoamylase (exo 1,4-α-d-glucosidase, 1,4-α-d-glucan glucohydrolase EC 3.2.1.3) has been studied. The dependence of the immobilized glucoamylase activity, in units g^?1, on the carrier pore diameter was found to pass through a maximum within a range 70–100 nm. Macroporous silica gels can be used with success as carriers for glucoamylase immobilization instead of porous glasses and silochromes.     

Chemical crosslinking of alpha subunits in the F1 adenosine triphosphatase of Escherichia coli

The arrangement of the subunits in the F1 adenosine triphosphatase of Escherichia coli has been investigated using bifunctional chemical crosslinking agents to covalently link adjacent subunits in the enzyme molecule. The synthesis of the new cleavable crosslinking agent 2,2'-dithiobis(succinimidyl propionate) is described. The crosslinked products resulting from the reaction of the enzyme with 2,2'- and 3,3'-dithiobis(succinimidyl propionate), 3,3'-dithiobis(sulfosuccinimidyl propionate), disuccinimidyl tartrate, dimethyl adipimidate, 1-ethyl-3[3-(dimethylamino)propyl]carbodiimide, and 1,2:3,4-diepoxybutane were analyzed by "three-dimensional" polyacrylamide gel electrophoresis in which they were resolved first in a two-dimensional system. Following cleavage of the crosslinking bridge in the separated products, the constituent subunits were identified by a further one-dimensional gel electrophoresis step. This procedure greatly improved the precision with which crosslinked subunits could be identified. It largely overcame problems due to abnormal migration of crosslinked species on gel electrophoresis and to the formation of multiple species of the same crosslinked subunit dimers. The following crosslinked subunit dimers were identified: alpha alpha, alpha beta, beta gamma, alpha delta, beta epsilon, and gamma epsilon. The trimer alpha alpha delta was recognized. The formation of alpha alpha over alpha beta dimers was favored when more polar crosslinking agents were used. The constraints placed by the finding of adjacent alpha subunits upon current models for the arrangement of the subunits in the F1 ATPase are discussed.     

Jack bean α-mannosidase digestion profile of hybrid-type N-glycans: effect of reaction pH on substrate preference

Jack bean α-mannosidase (JBM) is a well-studied plant vacuolar α-mannosidase, and is widely used as a tool for the enzymatic analysis of sugar chains of glycoproteins. In this study, the JBM digestion profile of hybrid-type N-glycans was examined using pyridylamino (PA-) sugar chains. The digestion efficiencies of the PA-labeled hybrid-type N-glycans Manα1,6(Manα1,3)Manα1,6(GlcNAcβ1,2Manα1,3)Manβ1,4GlcNAcβ1,4GlcNAc-PA (GNM5-PA) and Manα1,6(Manα1,3)Manα1,6(Galβ1,4GlcNAcβ1,2Manα1,3)Manβ1,4GlcNAcβ1,4GlcNAc-PA (GalGNM5-PA) were significantly lower than that of the oligomannose-type N-glycan Manα1,6(Manα1,3)Manα1,6Manβ1,4GlcNAcβ1,4GlcNAc-PA (M4-PA), and the trimming pathways of GNM5-PA and GalGNM5-PA were different from that of M4-PA, suggesting a steric hindrance to the JBM activity caused by GlcNAcβ1-2Man(α) residues of the hybrid-type N-glycans. We also found that the substrate preference of JBM for the terminal Manα1-6Man(α) and Manα1-3Man(α) linkages in the hybrid-type N-glycans was altered by the change in reaction pH, suggesting a pH-dependent change in the enzyme-substrate interaction.     

Structure of Dextran Synthesized by Dextrin Dextranase from Acetobacter capsulatus ATCC 11894

The structure of dextran synthesized from maltotetraose by dextrin dextranase (EC 2.4.1.2) from Acetobacter capsulatus ATCC 11894 was analyzed. When the Acetobacter dextran (AD) was acetolyzed, glucose and maltose were produced. AD was allowed to react with α-amylases. AD was digested by bacterial saccharifying α-amylase and bacterial liquefying α-amylase, and glucose, maltose, and maltotriose were produced. The structure of the fraction obtained from dextranase-digested AD by activated charcoal chromatography, which did not contain glucose, isomaltose, and isomaltotriose, was investigated by methylation analysis, and the ratio of 2,3,4,6-tetra-O-methyl-: 2,3,4-tri-O-methyl-: 2,3,6-tri-O-methyl-: 2,3-di-O-methyl-alditol acetate was estimated as 22.9:46.8:15.5:14.8. This result indicated the existence of α-1,4 branches and that of α-1,4 linkages in α-1,6 glucosyl linear chains. Native AD was calculated to be constructed with 6.23 branching points and 6.53 α-1,4 linked glucosyl residues per 100 glucosyl units. Though AD was digested slightly by rat intestinal acetone powder, high molecular weight polymers remained. Therefore AD could be used as a dietary fiber.     

Branched polyethylenimine derivatives with reductively cleavable periphery for safe and efficient in vitro gene transfer

Twenty-five kDa polyethylenimine (PEI) is one of the most efficient nonviral gene transfer agents currently applied as a golden standard for in vitro transfection. In this study, novel 25 kDa PEI derivatives with reductively cleavable cystamine periphery (PEI-Cys) were designed to reduce carrier-associated cytotoxicity and to enhance further the transfection activity. The Michael-type conjugate addition of 25 kDa PEI with N-tert-butoxycarbonyl-N'-acryloyl-cystamine (Ac-Cys-(t)Boc) and N-tert-butoxycarbonyl-N'-methacryloyl-cystamine (MAc-Cys-(t)Boc) followed by deprotection readily afforded PEI-Cys derivatives, denoted as PEI-(Cys)x(Ac) and PEI-(Cys)x(MAc), with degree of substitution (DS) ranging from 14 to 34 and 13 to 38, respectively. All PEI-Cys derivatives had higher buffer capacity than the parent 25 kDa PEI (21.2 to 23.1% versus 15.1%). Gel retardation and ethidium bromide exclusion assays showed that cystamine modification resulted in largely enhanced interactions with DNA. PEI-(Cys)x(Ac) could condense DNA into small-sized particles of 80-90 nm at and above an N/P ratio of 5/1, which were smaller than polyplexes of 25 kDa PEI (100-130 nm). In comparison, PEI-(Cys)x(MAc) condensed DNA into somewhat larger particles (100-180 nm at N/P ratios from 30/1 to 5/1). Gel retardation and dynamic light scattering (DLS) measurements showed that PEI-Cys polyplexes were quickly unpacked to release DNA in response to 10 mM dithiothreitol (DTT). These PEI-Cys derivatives revealed markedly decreased cytotoxicity as compared with 25 kDa PEI with IC(50) values of >100 mg/L and 50-75 mg/L for HeLa and 293T cells, respectively (corresponding IC(50) data of 25 kDa PEI are ca. 11 and 3 mg/L). The in vitro transfection experiments in HeLa and 293T cells using pGL3 as a reporter gene showed that gene transfection activity of PEI-Cys derivatives decreased with increasing DS and PEI-(Cys)x(MAc) exhibited higher transfection activity than PEI-(Cys)x(Ac) at similar DS. Notably, polyplexes of PEI-(Cys)14(Ac) and PEI-(Cys)13(MAc) showed significantly enhanced gene transfection efficiency (up to 4.1-fold) as compared with 25 kDa PEI formulation at an N/P ratio of 10/1 in both serum-free and 10% serum-containing conditions. The modification of PEI with reductively cleavable periphery appears to be a potential approach to develop safer and more efficient nonviral gene vectors.     

Reversible inhibition of cholinesterases by salts of pyrilium, thiopyrilium and selenopyrilium derivatives

Salts of pyrilium, thiopyrilium and selenopyrilium derivatives at pH 7.5 and temperature of 25 degrees C are studied for their effect on the catalytic activity of acetyl cholinesterase (EC 3.1.1.7) of human blood erythrocytes and butyryl cholinesterase (EC 3.1.1.8) of horse blood serum which is measured by the method of potentiometric titration. All enumerated salts are established to be strong reversible inhibitors of mixed-type cholinesterases, that is testified by small values of the inhibitory constants: competitive Ki, noncompetitive K'i and generalized K epsilon. Pyrilium and selenopyrilium salts inhibit acetyl cholinesterase of human blood erythrocytes to a higher extent than butyryl cholinesterase of horse blood serum, and thiopyrilium salts inhibit the latter to the highest extent. By the value of the inhibitory effect on acetyl cholinesterase of human blood erythrocytes thiopyrilium salts exceed the analogous pyrilium salts, whereas in experiments with butyl cholinesterase of horse blood serum there is an opposite dependence.     

Immobilization of Penicillium funiculosum cellulase on a soluble polymer

The three cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] components of Penicillium funiculosum have been immobilized on a soluble, high molecular weight polymer, poly(vinyl alcohol), using carbodiimide. The immobilized enzyme retained over 90% of cellulase [1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4], and exo-β-d-glucanase [1,4-β-d-glucan cellobiohydrolase, EC 3.2.1.91] and β-d-glucosidase [β-d-glucoside glucohydrolase, EC 3.2.1.21] activities. The bound enzyme catalysed the hydrolysis of alkali-treated bagasse with a greater efficiency than the free cellulase. The potential for reuse of the immobilized system was studied using membrane filters and the system was found to be active for three cycles.