Alcohol fermentation of sweet potato. Membrane reactor in enzymatic hydrolysis

Use of Ultrafiltration membrane systems in stirred cell and in thin-Channel systems for immobilizing enzyme (sweet potato intrinsic and β-amylase) in hydrolysis of sweet potato through a continuous operation mode were studied. Both the filtration rate and reducing sugars, produced as the result of enzymatic hydrolysis, decreased with the filtration time. The immobilized enzymes in the thin-channel system showed a much better performance compared to that in the stirred cell system. Addition of crystalline sweet potato β-amylase to the sweet potato increased both the filtration rate and reducing-sugars content. Alcoholic fermentation of the filtrate resulted in an alcohol content of 4.2%. This represented fermentation of 95% of the sugars with an efficiency of 88%.     

Glucoamylase and α-amylase production by immobilized Aspergillus niger

Summary Aspergillus niger mycelia or spores were immobilized in calcium alginate gel beads and employed for production of glucoamylase and -amylase by repeated batch process. The immobilized mycelium produced lower enzyme activities than immobilized spores germinated in a growth medium and subsequently cultured in an enzyme production medium. In repeated batch experiments, free cells could be used for only 4 4-day batches, whereas with immobilized spores at least 11 4-day batches with a gradual increase in enzyme activities in each successive batch were possible. The activity ratio of glucoamylase and -amylase produced was altered by immobilization.     

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.     

Preparation of an immobilized two-enzyme system, Beta-amylase--pullulanase, to an acrylic copolymer for the conversion of starch to maltose. III. Process kinetic studies on continuous reactors

Kinetic studies on the parameters influencing the potential industrial application of an immobilized two-enzyme system of β-amylase and pullulanase for conversion of starch to a product with high maltose content, have been performed. The apparent Michaelis constant, the apparent product inhibitor constant, and the activation energy have been determined for the immobilized preparation and compared to the values for the corresponding soluble enzyme system. The catalytic activity of the immobilized enzymes was studied in a plug-flow reactor and a continuous feed stirred tank reactor. Mathematical models for these reactors have been formulated and adapted to fit the experimental data. Comparisons of the reactor efficiencies were made and the conditions were found to be such as to favor the plug-flow reactor. Results on operational stability tests at different temperatures and substrate concentrations are given.     

Immobilization of enzymes on Spheron: 2. β-Amylase — The development of a column reactor—separator

The titanium-chelation method has been used to immobilize β-amylase (1,4-α-d-glucan maltohydrolase, EC 3.2.1.2) on to Spheron. On various grades of Spheron, protein coupling yields of 56–76% were obtained with barley and sweet-potato β-amylases. The specific enzymic activities of the immobilized enzymes fell in the range 3.7–7.6% of those of the soluble enzymes. The immobilized enzymes were more stable than the soluble, especially in the presence of l-cysteine and serum albumin. The presence of cysteine and serum albumin brought about increases in activity in the preparations, presumably by regenerating essential thiol groups in the enzyme which had been oxidized during the operations. Maltose could be separated from amylopectin and other large polysaccharides by chromatography on Spheron P100, and a system was developed in which maltose, produced by hydrolysis of amylopectin applied in pulses to a column of immobilized β-amylase, was separated from starting material and by-products on a second column of Spheron P100.     

Effects of poly(ethylene glycol) and salt on the binding of α-amylase from the fermentation broth of Bacillus amyloliquefaciens by Cu2+-β-CD affinity adsorbent

α-Amylase from Bacillus amyloliquefaciens was purified by the immobilized metal ion affinity adsorbent, β-CD_cl-IDA-Cu²⁺. The adsorbent was prepared by reacting the cross-linked β-cyclodextrin (β-CD) with the ligand, iminodiacetic acid (IDA). The copper ion was further linked to the adsorbent. Poly(ethylene glycol) (PEG) was added to the fermentation broth to improve the adsorption efficiency of the adsorbent toward α-amylase. The effort was to provide hydrophobic interactions with the impurities which might interfere with the adsorption of α-amylase. It also provided a polymer shielding effect to prevent non-specific interactions. With the addition of PEG, the adsorption efficiency could be increased to 98%. Imidazole containing a phosphate buffer and NaCl was used to elute the bound α-amylase. By consecutive adsorption/desorption steps, up to 81% of the α-amylase activity could be recovered. Regarding the reutilization of the affinity adsorbents, α-amylase could be adsorbed and desorbed six times consecutively without a significant loss of α-amylase activity.     

Purification of recombinant α-amylase with immuno-affinity chromatography using monoclonal antibody

A monoclonal antibody against recombinant thermostable α-amylase produced by Escherichia coli was isolated from serum-free medium and immobilized on Sepharose 4B. The adsorption equilibrium between α-amylase and the immobilized immuno-adsorbent showed a Langmuir type isotherm. The breakthrough curve calculated numerically using the averaged volumetric coefficient coincided well with the experimental data. More than 90% of the activity of bound α-amylase could be recovered by eluting with glycine-HCl buffer (pH 2.5). The elution profile at pH 2.5 became sharper with increasing temperature. By using an immuno-affinity column, the recombinant α-amylase produced by E. coli could be purified homogeneously from crude extract enzyme solution with two-step elution.     

Production of thermophilic α-amylase using immobilized transformed Escherichia coli by addition of glycine

Efficient production of thermophilic α-amylase from Bacillus stearothermophilus was investigated using recombinant Escherichia coli HB101/pH1301 immobilized with κ-carrageenan by the addition of glycine. The effects of glycine, the concentrations of κ-carrageenan and KCI on the production of the enzyme as well as the stability of plasmid pHI301 were studied. In the absence of glycine, the enzyme was localized in the periplasmic space of the recombinant E. coli cells and a small amount of the enzyme was liberated in the culture broth. Although the addition of glycine was very effective for release of α-amylase from the periplasm of E. coli entrapped in gel beads, a majority of the enzyme accumulated in the gel matrix. (In this paper, production of the enzyme from recombinant cells to an ambient is expressed by the term “release”, while diffusion-out from gel beads is referred to by the term “liberate”.) Concentrations of KCI and immobilizing support significantly affected on the liberation of α-amylase to the culture broth. Mutants which produced smaller amounts of the enzyme emerged during a successive culture of recombinant E. coli, even under selective pressure, and they predominated in the later period of the passages. The population of plasmid-lost segregants increased with cultivation time. The stability of pHI301 for the free cells was increased by the addition of 2% KCI, which is a hardening agent for carrageenan. Although the viability of cells and α-amylase activity in the beads decreased with cultivation time during the successive culture of the immobilized recombinant E. coli, the plasmid stability was increased successfully by immobilization. Efficient long-term production of α-amylase was attained by an iterative re-activation-liberation procedure using the immobilized recombinant cells. Although the viable cell number, plasmid stability and enzyme activity liberated in the glycine solution decreased at an early period in the cultivation cycles, the process attained steady state regardless of the addition of an antibiotic.     

Starch conversion by soluble and immobilized α-amylase

B. subtilis α-amylase was immobilized on cyanogen bromide activated carboxymethyl cellulose. The conversion of wheat starchwas carried out at 72°C in a stirred tank by soluble and immobilized α-amylase. The initial reaction rate with immobilized α-amylase was lower than with the soluble enzyme, but after 1 hr immobilized α-amylase produced a higher quantity of reducing sugars than the soluble enzyme. The action pattern of immobilized α-amylase was different from that of the soluble enzyme: immobilized α-amylase produced relatively more glucose and maltose, except at the beginning of conversion. Immobilized α- readily hydrolyze G₆. The starch conversion by immobilized α-amylase was not diffusion controlled at a stirring rate of 100-300 rpm.     

Amylase from Lactobacillus cellobiosus D-39 isolated from vegetable wastes: characteristics of immobilized enzyme and whole cell

Cells and partially purified α-amylase (EC 3.2.1.1) of the producer strain, Lactobacillus cellobiosus D-39 were immobilized on acrylamide gel. The enzyme showed marked improvement in operational stability. Both immobilized cells and enzyme were stable for a long period and no appreciable loss activity was detected on keeping at 4°C for 4 months. The amylase activity of immobilized cells and enzyme attained maximum at pH 6.0 and 7.6 respectively and at temperature 60°C for both cases. The effects of various solvents, detergent and metal ions were tested; Triton X-100 gave maximum stimulation of the enzyme activity of immobilized cells whereas metal ions exhibited no such enhancement for either of immobilized cells or enzyme.     

Immobilization of enzymes based on hydrophobic interaction. II. Preparation and properties of an amyloglucosidase adsorbate

Amyloglucosidase from Aspergillus niger (α-1,4 and 1,6 glucan glucohydrolase, EC 3.2.1.3) was immobilized through adsorption onto a hexyl–Sepharose, containing 0.51 mol hexyl-group per mole of galactose. The adsorption limit of the carrier with respect to this enzyme was about 17 mg per gram wet conjugate. The retention of activity upon immobilization was high, varying from essentially full activity at low enzyme content down to 68% at the adsorption limit. The immobilized preparation, as well as the soluble enzyme, showed apparent zero order kinetics within 60% of the substrate's conversion limit. Product inhibition of the soluble enzyme showed a k₁ of 5 · 10^?2M. In the presence of 3M NaCl, adsorbates were formed more rapidly and with a higher yield of immobilized protein, but with lower specific activity. Conjugates resulting from adsorption of amyloglucosidase in identical concentrations, but at different salt contents, showed comparable activities and operational stabilities. Continuous operation for three months reduced conjugate activity to 40%. The thermal stability of the adsorbate was inferior to that of the soluble enzyme, but was noticeably enhanced in the presence of substrate.     

Purification, partial characterization, and covalent immobilization–stabilization of an extracellular α-amylase from Aspergillus niveus

An extracellular amylase secreted by Aspergillus niveus was purified using DEAE fractogel ion exchange chromatography and Sephacryl S-200 gel filtration. The purified protein migrated as a single band in 5 % polyacrylamide gel electrophoresis (PAGE) and 10 % sodium dodecyl sulfate (SDS-PAGE). The enzyme exhibited 4.5 % carbohydrate content, 6.6 isoelectric point, and 60 and 52 kDa molar mass estimated by SDS-PAGE and Bio-Sil-Sec-400 gel filtration column, respectively. The amylase efficiently hydrolyzed glycogen, amylose, and amylopectin. The end-products formed after 24 h of starch hydrolysis, analyzed by thin layer chromatography, were maltose, maltotriose, maltotetraose, and maltopentaose, which classified the studied amylase as an α-amylase. Thermal stability of the α-amylase was improved by covalent immobilization on glyoxyl agarose (half-life of 169 min, at 70 °C). On the other hand, the free α-amylase showed a half-life of 20 min at the same temperature. The optima of pH and temperature were 6.0 and 65 °C for both free and immobilized forms.     

Enzymatic activity of immobilized enzyme determined by isothermal titration calorimetry

The activity of adsorbed β-glucosidase onto spherical polyelectrolyte brushes (SPBs) is investigated by UV-Vis spectroscopy and isothermal titration calorimetry (ITC). By comparing the results of these two methods, we demonstrate that ITC is a precise method for the study of the activity of immobilized enzymes. The carrier particles used for immobilization here consist of a polystyrene core onto which poly(acrylic acid) chains are grafted. High amounts of enzyme can be immobilized in the brush layer at low ionic strength by the polyelectrolyte-mediated protein adsorption (PMPA). Analysis of the activity of β-glucosidase was done in terms of Michaelis-Menten kinetics. Moreover, the enzymatic activity of immobilized enzyme is studied by ITC using cellobiose as substrate. All data show that ITC is a general method for the study of the activity of immobilized enzymes.     

Immobilization of β-amylase on polystyrene cation exchange resin equilibrated with Al3+ions (IR-120 Al3+)

A simple procedure for the immobilization of β-amylase (EC3.2.1.2) on IR-120 Al³⁺ is described. Immobilization brought about a slight shift in the optimum pH towards the alkaline side relative to that of the soluble enzyme. Immobilized β-amylase showed a broader temperature optima (45–55°C) compared with the soluble enzyme (45°C). A six-fold increase in the K_m was also noticed. On storage and repeated use the immobilized enzyme retained approximately 60% of its initial activity up to 120 days at room temperature.     

Properties of immobilized β-d-galactosidase from Bacillus circulans

Partially purified β-d-galactosidase (β-d-galactoside galactohydrolase, EC 3.2.1.23) from Bacillus circulans showed high activity towards both pure lactose and lactose in skim milk, and a better thermal stability than the enzyme from yeast or Escherichia coli. During the course of hydrolysis of lactose catalysed by the enzyme, considerable amounts of oligosaccharides were produced. β-d-Galactosidase from B. circulans was immobilized onto Duolite ES-762, Dowex MWA-1 and sintered alumina by adsorption with glutaraldehyde treatment. The highest activity for hydrolysis of lactose was obtained with immobilization onto Duolite ES-762. During a continuous hydrolysis of lactose, the immobilized enzyme was reversibly inactivated, probably due to oligosaccharides accumulating in the gel. The inactivation was reduced when a continuous reaction was operated at a high percent conversion of lactose in a continuous stirred tank reactor (CSTR). The half-life of the immobilized enzyme was estimated to be 50 and 15 days at 50 and 55°C, respectively, when the reaction was carried out in a CSTR with a percent conversion of lactose >70%.     

Purification and Some Properties of Active β-Amylase in Rice

An active β-amylase was purified from germinated rice seeds by precipitation with ammonium sulfate, acid treatment, chromatographies on DEAE-cellulose and DEAE-Sephadex A-50, and gel filiations on Sephadex G-75. The purified enzyme was homogeneous in disc electrophoretic analysis.

The molecular weight was estimated to be approximately 53,000 by thin-layer gel filtration and polyacrylamide gel electrophoresis. The isoelectric point was found to be pH 5.0 by disc electrofocusing.

The optimum pH was found to be in the pH range of 5.5 to 6.5. The Km value for soluble starch was 3 mg/ml. The enzyme was inhibited by sulfhydryl reagents or heavy metal ions.

The active β-amylase was oxidatively dimerized by treatment with 0.3 m ferricyanide in 3 m urea. The dimerized enzyme was thought to be one of inert β-amylases in ungerminated rice seeds.     

Free and Protein-bound β-Amylases of Barley Grain. Characterization by Two-dimensional Immunoelectrophoresis

The β-amylases of ungerminated barley (Hordeum distichum L. cv. Emir) were characterized by two-dimensional immunoelectrophoretic techniques in order to elucidate the structure and physiological importance of the latent β-amylase present in cereal grains. Two water-soluble forms with partial immunochemical identity were detected. One of the enzyme forms was found to consist of aggregates between β-amylase and an immunochemically distinct non-active protein. Both β-amylase and the protein could be released from the aggregates with β-mercaptoethanol and, to some extent, with papain. β-Mercaptoethanol increased the extractability of β-amylase and of the non-active protein. The aggregated form of β-amylase was found to predominate in extracts made in the presence of papain. Possible functions of the non-enzymatic protein in formation of latent β-amylase is discussed.     

Kinetics and stability of immobilized chicken liver xanthine dehydrogenase.

Xanthine dehydrogenase (EC 1.2.1.37) was isolated from chicken livers and immobilized by adsorption to a Sepharose derivative, prepared by reaction of n-octylamine with CNBr-activated Sepharose 4B. Using a crude preparation of enzyme for immobilization it was observed that relatively more activity was adsorbed than protein, but the yield of immobilized activity increased as a purer enzyme preparation was used. As more activity and protein were bound, relatively less immobilized activity was recovered. This effect was probably due to blocking of active xanthine dehydrogenase by protein impurities. The kinetics of free and immobilized xanthine dehydrogenase were studied in the pH range 7.5-9.1. The Km and V values estimated for free xanthine dehydrogenase increase as the pH increase; the K'm and V values for the immobilized enzyme go through a minimum at pH 8.1. By varying the amount of enzyme activity bound per unit volume of gel, it was shown that K'm is larger than Km are result of substrate diffusion limitation in the pores of the support material. Both free and immobilized xanthine dehydrogenase showed substrate activation at low concentrations (up to 2 microM xanthine). Immobilized xanthine dehydrogenase was more stable than the free enzyme during storage in the temperature range of 4-50 degrees C. The operational stability of immobilized xanthine dehydrogenase at 30 degrees C was two orders of magnitude smaller than the storage stability, t 1/2 was 9 and 800 hr, respectively. The operational stability was, however, better than than of immobilized milk xanthine oxidase (t 1/2 = 1 hr). In addition, the amount of product formed per unit initial activity in one half-life, was higher for immobilized xanthine dehydrogenase than for immobilized xanthine oxidase. Unless immobilized milk xanthine oxidase can be considerable stabilized, immobilized chicken liver xanthine dehydrogenase is more promising for application in organic synthesis.     

The β-amylase polymorphism of winter common wheat grains

The polymorphism of winter common wheat with respect to β-amylase isoenzymes has been analyzed using electrophoresis in polyacrylamide gel (PAAG) buffered with a Tris-glycine system (pH 8.3). Seven β-amylase isoenzymes have been found in wheat cultivars and the breeding stock. Isoenzymes A, B, and C are the most frequent in Russian and Ukrainian cultivars (51.7 4.7, 30.7 3.8, and 11.9 2.5%, respectively). Two alleles of the β-Amy-D1 locus of the long arm of chromosome 4D have been identified. The substrate-enzyme affine effect can be used to locate the zones of activity of this enzyme by means of staining for proteins. It has been determined that β-amylase zymotypes may play a role in the aggregating capacity of the grain protein complex via the formation of S-S bonds.