Application of immobilized invertase to continuous hydrolysis of concentrated sucrose solutions

Invertase immobilized onto corn grits was utilized in the hydrolysis of highly concentrated sucrose solutions producting liquid sugar solutions containing glucose and fructose. Comparisons of conversion efficiencies of this immobilized invertase in a continuous stirredtank reactor and a plug-flow reactor indicated that the plug-flow reactor has an higher efficiency. Continuous sucrose hydrolysis was then performed in 0.1- and 1-L tubular reactors. This tenforld scaling-up was achieved without any noticeable loss in efficiency. This process thus was scaled-up to a 17.6-L pilot reactor set in a cane sugar refinery. This reactor was fed with highly concentrated sucrose solutions [71% (w/w)] to produce invert sugar syrup with the desired inversion degree. It allows a productivity equal to 9.1 kg sucrose hydrolyzed/h in the case of a 69% (w/w) sucrose initial concentration with a 72% conversion rate.     

Dextran synthesis by immobilized dextran sucrase

Dextran sucrase has been produced by fermentation of Leuconostoc mesenteroides NRRL B-512, with and without continuous sucrose addition to improve enzyme production. The enzyme preparation has been concentrated from the fermentation broth by ultrafiltration and purified by gel permeation chromatography on Ultrogel. The specific activity of the dextran sucrase was greatly enhanced by calcium chloride addition to the purified enzyme. This enzyme preparation has been immobilized by covalent coupling onto an amino porous silica support (Spherosil) activated with glutaraldehyde. Immobilized dextran sucrase derivatives with an activity up to 830 dextran sucrase units per g. support could thus be obtained. The effect of the support specific area on coupling efficiency and reaction kinetics has been investigated, and the effect of intraparticular diffusion underlined. The molecular weight distribution of the dextran has been determined when varying several parameters.     

Recovery of yeast invertase from ethanol fermentation broth

Summary Ethanol fermentation broth produced by an aggregated form ofSaccharomyces uvarum strain contained invertase when sucrose-based raw materials were used. The amount of invertase in the borth was in the range of 1.4 to 4.8 units/ml, which was affected by the dilution rate, the concentration of corn steep liquor, and the type of sugar used. The activity of invertase in the broth could be maintained at 0.8 units/ml over two months. When the broth was passed through DEAE-cellulose beads and eluted with a NaCl-Tris-HCl buffer solution, a 75% recovery yield of invertase with 9-fold purification and 30-fold concentration could be achieved.     

Chemistry and properties of starch based desiccants

Desiccants currently used in industry include molecular sieves, lithium chloride, silica gel, and corn grits. Of these, only corn grits (a form of ground corn) are biodegradable and derived from a renewable resource. A major component of the corn grits, starch, is the primary adsorptive material in the corn grits. Other polysaccharides, including cellulose and hemicellulose also have adsorptive properties. The use of alpha-amylase (EC 3.2.1.1) to modify porosity and surface properties of starch resulted in materials with enhanced water sorption properties compared to the native material. This paper reviews the chemical and structural properties of starch, corn grits, and cellulose-based scaffolds on which starch can be affixed, in order to attain structures that might someday find uses in a range of desiccant applications for industrial, commercial, and residential processes.     

蔗糖脂肪酸脂对离体和活体大豆蔗糖酶活力和构象的影响

研究了蔗糖脂肪酸脂(SFE)对离体和活体大豆蔗糖酶活力和构象的影响。当SFE的浓度大于1.0mmol/L后,开始激活离体蔗糖酶的活力。在大豆的开花期和结荚期,SFE可以增加蔗糖酶的活力。荧光实验结果表明,在小于2mmol/L时,SFE不改变蔗糖酶的荧光最大发射峰峰位和峰高。施用SFE后,大豆叶片中果糖、葡萄糖和蔗糖酶的含量,分别是对照的170%±10%,190%±10%,和260%±20%;而蔗糖的含量几乎不变。对蔗糖酶 的SDS-凝胶电泳图进行扫描分析的结果表明,经SFE处理后的蔗糖酶含量比对照高两倍左右。这些结果说明SFE可以显著增加活体蔗糖酶的活力,但活力的增加既不是因为蔗糖酶构象的改变,也不是蔗糖(作为底物)诱导所致,而是SFE增加了大豆叶片中蔗糖酶的含量引起的。     

Expression of Pichia anomala INV1 gene in Saccharomyces cerevisiae results in two different active forms of hypoglycosylated invertase

The Pichia anomala invertase gene (INV1) was introduced at different copy numbers into a sucrose-nonfermenting mutant of Saccharomyces cerevisiae and expressed from its own promoter sequences. The level reached in the production of invertase by the transformants (up to 540 units/10(10) cells) was in agreement with the INV1 gene dosage. Two forms of multimeric active and glycosylated invertase displaying different subcellular locations and molecular masses could be detected in the transformants. One was found to be present in the culture medium and in the periplasm, and the other could only be detected inside the cell. Each of the two heterologous forms of invertase was shown to be an oligomer composed of identical subunits. The difference found in the apparent molecular masses of their monomers (81.5 and 78.3 kDa, respectively) seems to be due to the size of their N-linked oligosaccharide chains (on average 2.4 and 1.9 kDa, respectively), since the number of sugar chains (9) and the molecular mass of the protein moiety (60.5 kDa) are identical in both forms. The shorter size of their oligosaccharides must also be the reason for the lower apparent molecular masses of the heterologous invertases when compared with the enzyme purified from P. anomala. The hypoglycosylated invertase accumulated within the cells of the transformants to an unusual level (up to 130 units/10(10) cells). Such accumulation of active enzyme inside the cells, as well as its underglycosylation, could be due to intrinsic properties of the P. anomala invertase that are determined by the particular primary structure of its protein moiety.     

Production,separation and purification of yeast invertase as a by-product of continuous ethanol fermentation

Summary A strain of Saccharomyces uvarum produced extracellular invertase in a chemostat reactor using a medium containing corn steep liquor and sugars. The production of yeast invertase increased with increase in corn steep liquor concentration. The production rate of invertase was maximal at a dilution rate of 0.75 h^–1. The production rate of invertase was found to be affected by the type of sugar substrate and fermentation temperature. The invertase in the crude broth could be purified by one-step DEAE chromatography. An 84% enzyme recovery with ninefold purification and 30-fold concentration could be achieved using this simple isolation procedure. Offsprint requests to: L. F. Chen     

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.     

Maltodextrin hydrolysis in a fluidized-bed immobilized enzyme reactor

The present work deals with maltodextrin hydrolysis by glucoamylase immobilized onto corn stover in a fluidized bed reactor. An industrial enzyme preparation was covalently grafted onto corn stover, yielding an activity of up to 372 U/g and 1700 U/g for support particle sizes of 0.8 and 0.2 mm, respectively. A detailed kinetic study, using a differential reactor, allowed the characterization of the influence of mass transfer resistance on the reaction catalyzed by immobilized glucoamylase. A simple and general mathematical model was then developed to describe the experimental conversion data and found to be valid.     

Formulation of filamentous fungi for bioremediation

Mycelia of Marasmiellus troyanus embedded in calcium alginate granules with corn cob grits as a nutritive amendment were viable after one year with refrigeration but inviable when stored at room temperature. With refrigeration, Phanerochaete chrysosporium mycelia and spores embedded in alginate were both viable after one year. At room temperature, spores encapsulated in alginate granules gave good viability while mycelial formulations did not. In all trials, corn cob grits was superior to saw dust for extending shelf life. Corn cob grits-amended granules of both species were able to germinate and grow in both uncontaminated soil and chemical waste-contaminated soil. © Rapid Science Ltd. 1998     

Invertase immobilization via its carbohydrate moiety

After periodate oxidation of its glycosidic component, invertase was covalently bound onto three types of modified solid supports: glycidyl methacrylate, styrene-divinylbenzene copolymers, and bead cellulose. Direct reaction of the invertase aldehyde groups that were formed with amino groups of the support and use of the modified Ugi reaction have been employed as immobilization procedures. Apart from binding methods, the important effects of the buffer, support, conditions of periodate oxidation, and the length of the spacer on the activity of the enzyme conjugate have been investigated. Superior conjugate activity was obtained, via modified Ugi reaction, by the immobilization of a suitably oxidized invertase to a styrene-divinylbenzene copolymer having free amino groups.     

Osmotic regulation of invertase formation and secretion by protoplasts of Saccharomyces

Osmotic regulation of invertase formation and secretion by protoplasts of Saccharomyces was examined using sorbitol, KCl, NaCl, or magnesium sulfate as the osmotic support. The synthesis and secretion of the enzyme is remarkably sensitive to the osmolarity of the supporting medium irrespective of the particular support employed. Invertase formation was inhibited at high osmolarity and was maximal at 0.65 to 0.75 osmolal, even though some leakage of the intracellular enzyme alpha-glucosidase and of ultraviolet (UV)-absorbing materials occurred under these conditions. The reduction of invertase formation and secretion due to high osmolarity was eliminated promptly when protoplasts were transferred into a medium of lower osmolarity. The rate of fructose uptake and of threonine incorporation into protein was decreased by high osmolarity; also reduction of invertase formation could be partially reversed by increasing the level of sugar supplied as energy source. Thus changes in the permeability of the plasma membrane (and presumably also in its structure) are important factors in the response of protoplasts to high osmolarity, though certainly not the complete explanation. Protoplasts suspended in 0.8 m sorbitol, with 10mm fructose as the energy source, increased their invertase level 5- to 10-fold during a 2-hr incubation without substantial release of alpha-glucosidase or UV-absorbing materials. Both the large and small forms of invertase were present in the protoplasts, but only the large form was released into the medium when enzyme was being actively synthesized. Formation and secretion of newly formed invertase and the release of enzyme initially present were inhibited by cycloheximide.     

Factors affecting the oligomeric structure of yeast external invertase

It has been assumed that yeast external invertase is a dimer, with each subunit composed of a 60-kDa polypeptide chain. We now present evidence that at its optimal pH of 5.0, the predominant form of external invertase is an octamer with an average size of 8 X 10(5) Da. During ultracentrifugation the octamer dissociated to lower molecular weight forms, including a hexamer, tetramer, and dimer. All forms of the enzyme were shown to possess identical specific activities and to contain a similar carbohydrate to protein ratio. Although the monomer subunits (1 X 10(5) Da) were heterogenous in carbohydrate content, each subunit possessed nine oligosaccharide chains. When stained for protein and enzyme activity following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, only the oligomeric form of the enzyme appeared to be active. Thus, on partially inactivating invertase with 4 M guanidine hydrochloride both octamer and monomer were evident on the gels but only the former was active. Similarly, incubating at pH 2.5 in the presence of sodium dodecyl sulfate yielded only inactive monomer. The monomer, unlike the active oligomeric aggregate, was unable to hydrolyze sucrose after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Consistent with the in vitro studies, freshly prepared yeast lysate was shown to contain the octameric species of external invertase as the major active form of this enzyme. From these studies and others which employed deglycosylated invertase, it is concluded that the carbohydrate component of external invertase contributes not only to stabilizing enzyme activity, but also to maintaining its oligomeric structure.     

Partial purification of potato tuber invertase and its proteinaceous inhibitor

Improved purification of potato tuber invertase was achieved by utilizing a form of affinity chromatography between the enzyme and Concanavalin A (Con A) bound to Sepharose. Twenty-fold increases in specific activity were routinely obtained with this step and the enzyme was purified 190-fold over that found in the crude homogenate. The Con A-Sepharose chromatography step gave a greater purification than any other step in the invertase isolation procedure. There was up to 170% recovery of the activity loaded onto the column. α-Methyl-d-mannoside, sucrose, d-glucose and d-fructose eluted the enzyme from the Con A-Sepharose column with similar recoveries, although the volume of eluent required varied with the sugar. This unusually high recovery of invertase activity was obtained with some batches of tubers but not with others. There was evidence to suggest that the high recovery, or activation, may be due to the release of an inhibitor from the enzyme in the presence of Con A-Sepharose. Adsorption of invertase to Con A-Sepharose could be eliminated by incubation of the enzyme with α-mannosidase and β-glucosidase, indicating that potato tuber invertase is a glycoprotein. Proteinaceous inhibitor purification was improved by treatment of the tuber extract at low pH.     

T-2 Toxin Production by Fusarium tricinctum on Solid Substrate

A method has been developed to produce and purify gram quantities of T-2 toxin [4beta, 15-diacetoxy-8alpha-(3-methylbutyryloxy)-12, 13-epoxytrichothec-9-en-3alpha-ol], a mycotoxin elaborated by a strain of Fusarium tricinctum isolated from toxic corn. After growing for 3 weeks at 15 C on 1,200 g of white corn grits, F. tricinctum NRRL 3299 elaborated at least 9.0 g of T-2 toxin, and 2.3 g of crystalline product was recovered. A lesser amount of toxin was produced on rice, but none was detected in wheat incubated at 20 C. The amount of toxin measured in white corn grits declined as the incubation temperature was raised to 20, 25, and 32 C.     

Immobilization and stabilization of invertase on Cajanus cajan lectin support

Use of lectins as ligands for the immobilization and stabilization of glycoenzymes has immense application in enzyme research and industry. But their widespread use could be limited by the high cost of their production. In the present study preparation of a novel and inexpensive lectin support for use in the immobilization of glycoenzymes containing mannose or glucose residues in their carbohydrate moiety has been described. Cajanus cajan lectin (CCL) coupled covalently to cyanogen bromide activated Seralose 4B could readily bind enzymes such as invertase, glucoamylase and glucose oxidase. The immobilized and glutaraldehyde crosslinked preparations of invertase exhibited high resistance to inactivation upon exposure to enhanced temperature, pH, denaturants and proteolysis. Binding of invertase to CCL-Seralose was however found to be readily reversible in the presence of 1.0 M methyl alpha-D mannopyranoside. In a laboratory scale column reactor the CCL-Seralose bound invertase was stable for a month and retained more than 80% of its initial activity even after 60 days of storage at 4 degrees C. CCL-Seralose bound invertase exhibited marked stability towards temperature, pH changes and denaturants suggesting its potential to be used as an excellent support for the immobilization of other glycoenzymes as well.     

Reversible immobilization of glutaryl acylase on sepabeads coated with polyethyleneimine

The immobilizaton of the enzyme glutaryl-7-aminocephalosporanic acid acylase (GA) was performed via ionic adsorption onto several supports: a new anionic exchange resin, based on the coating of Sepabeads internal surfaces with polyethyleneimine (PEI) of different molecular weights, and conventional EC-Q1A-Sepabeads and DEAE-agarose. Immobilization occurred very rapidly in all cases, but the adsorption strength was much higher in the case of PEI-Sepabeads than in the other supports at pH 7 (e.g., at 150 mM NaCl, 90% of the enzyme was eluted from the DEAE agarose and 15% was eluted from the EC-Q1A-Sepabeads, whereas no desorption was detected with the best PEI-Sepabeads). Interestingly, the adsorption strength of the GA was increased when it was immobilized on PEI-Sepabeads with higher molecular weights. For instance, enzyme desorption was detected from 75 mM NaCl for the derivative prepared onto Sepabeads coated with PEI 700 Da, whereas in the derivative prepared with the highest molecular weight PEI (600 000 Da) no enzyme desorption was detected below 150 mM NaCl. Optimal PEI-Sepabeads (prepared with PEI of 600 000 Da) was even much more thermostable than the covalent derivative prepared onto cyanogen bromide agarose. Moreover, this derivative presented a half-life 26-fold higher than that of the soluble enzyme at 45 degrees C, and the support could be reused 10 times after the full desorption of the enzyme without decreasing loading capacity.     

Influence of the conditions of trypsin immobilization onto Spherosil on coupling efficiency

Summary The effect of several parameters (pH, time of reaction, temperature, enzyme concentration) on trypsin immobilization onto glutaraldehyde-activated amine-Spherosil was investigated. This activated support could be stored over long periods of time without any important loss of capacity for trypsin coupling. When increasing the amount of trypsin bound to the carrier, enzymatic activity shows an optimal value, beyond which an augmentation of Spherosil enzyme content results in a lowered activity. The influence of the number of available reactive aldehyde groups on silica was investigated by coupling L-lysine to activated support either prior to or simulataneously with trypsin immobilization. In both cases, the activity of trypsin derivatives is decreased when L-lysine concentration is increased, yet the activity of trypsin derivatives is never equal to zero, even in presence of a large excess of L-lysine. This suggests the presence of two types of reactive groups on the activated support.     

Immobilization of invertase on modified nylon tubes

Invertase was immobilized on O-alkylated nylon tubes modified with amine arms and activated with glutaraldehyde. The performance of the immobilized invertase derivative was dependent on the nylon tube modification that preceded the enzyme coupling procedure. Optimal experimental conditions for the tube modification and the enzyme coupling step are described and discussed.     

Poly(glycidylmethacrylate) brushes generated on poly(VBC) beads by SI-ATRP technique: Hydrazine and amino groups functionalized for invertase adsorption and purification

Crosslinked-poly(vinylbenzylchloride), poly(VBC), beads were prepared by suspension polymerization and poly(glycidylmethacrylate) was grafted by surface-initiated-atom radical polymerization (SI-ATRP) technique. Epoxy groups of the grafted poly(GMA) were reacted with hydrazine and ammonia to create an affinity binding sites. The hydrazine and amine functionalized poly(VBC-g-GMA) beads were used as an affinity support for adsorption of invertase from solution and yeast crude extract. The influence of pH, equilibrium time, ionic strength and initial invertase concentration on the adsorption capacities of both hydrazine and amine functionalized beads has been investigated. Maximum invertase adsorptions onto hydrazine and amine functionalized beads, were 86.7 and 30.4 mg/g at pH 4.0 and 5.5, respectively. The experimental equilibrium data fitted well to the Temkin isotherm model. Finally, the hydrazine functionalized poly(VBC-g-GMA) beads were used for the purification of invertase from crude yeast extract in a batch system and the purity of the eluted invertase from the hydrazine functionalized beads was determined as 92% by HPLC from single step purification protocol.