RESUMEN

Two simple and easily reproducible methods for the immobilization of ß-galactosidase (ß-gal) from Aspergillus oryzae on electrospun gelatin nanofiber mats (GFM) were developed. The process was optimized regarding the electrospinning solvent system and the subsequent cross-linking of GFM in order to increase their stability in water. ß-Gal was covalently immobilized on activated gelatin nanofiber mats with hexamethylenediamine (HMDA) as a bifunctional linker and secondly via entrapment into the gelatin nanofibers during the electrospinning process (suspension electrospinning). Optimal immobilization parameters for covalent immobilization were determined to be at pH 7.5, 40 °C, ß-gal concentration of 1 mg/mL and immobilization time of 24.5 h. For suspension electrospinning, the optimal immobilization parameters were identified at pH 4.5 and ß-gal concentration of 0.027 wt.% in the electrospinning solution. The pH and temperature optima of immobilized ß-gal shifted from 30 °C, pH 4.5 (free enzyme) to pH 3.5, 50 °C (covalent immobilization) and pH 3.5, 40 °C (suspension electrospinning). Striking differences in the Michaelis constant (KM) of immobilized ß-gal compared with free enzyme were observed with a reduction of KM up to 50% for immobilized enzyme. The maximum velocity (vmax) of immobilization by suspension electrospinning was almost 20 times higher than that of covalent immobilization. The maximum GOS yield for free ß-gal was found to be 27.7% and 31% for immobilized ß-gal.

Asunto(s)

RESUMEN

Bienzymatic production of laminaribiose from sucrose and glucose was combined with adsorption on zeolite BEA to introduce a first capture and purification step. Downstream processing including washing and desorption steps was characterized and optimized on a milliliter scale in batch mode. Results were then transferred to a packed bed system for enzymatic production and adsorption where the influence of adsorbent particle diameter on purity and productivity was evaluated. Finally, a continuous enzymatic production of laminaribiose was conducted over 10 days. The subsequent downstream processing of the loaded zeolites led to purities of over 0.5 gLaminaribiose gsugar -1 in the desorbate with a total productivity of 5.6 mgLaminaribiose Lenzyme bed -1 h-1 without the use of recycles.

RESUMEN

The first continuous production system of laminaribiose from sucrose and glucose in a bienzymatic reaction is reported in this study. Immobilized laminaribiose phosphorylase and sucrose phosphorylase were used in a packed bed reactor system comprising of a 3-cm glass column at 35 °C with a steady feeding flow rate of 0.1 ml/min. Factors affecting product formation including enzyme ratio, peal concept (both enzymes in one pearl or in separate pearls), and pearl size were studied. An enzyme ratio of 2:1 of laminaribiose phosphorylase (LP) to sucrose phosphorylase (SP) when encapsulated separately in bigger size peals resulted in higher concentration of product. Laminaribiose (0.4 g/(L h)) is produced in the optimized system at steady state. The reaction system proved to be operationally stable throughout 10 days of continuous processing. A half-life time of more than 9 days was observed for both biocatalysts.

Asunto(s)

RESUMEN

Commercial application of biocatalysts depends on the efficiency of the immobilization method and residual enzyme activity. Electrospinning offers a simple and versatile route to immobilize enzymes in submicron-sized fibers and thus improved mass transfer characteristics. Performance of encapsulation of fructosyltransferase from Bacillus subtilis by emulsion, suspension, and coaxial electrospinning was compared. We particularly focused on the effect of hydrophilic properties of a set of biodegradable polymers on support's activity. Bioactivity of electrospun support in aqueous medium increased in order of the matrix hydrophilicity. Additionally, the efficiency of electrospun fibers was compared with Sepabeads®, commercial epoxy-activated resins. In fibers, enzyme loading of 68.1 mg/g and specific enzyme activity of 5.5 U/mg was achieved compared to 49.5 mg/g and 2.2 U/mg on Sepabeads. Fructosyltransferase exhibited high sensitivity towards organic solvents and covalent attachment, respectively. Immobilization of native enzyme in coaxial fibers increased the specific activity to approx. 30 U/mg which corresponds to 24% of that of the free enzyme. Finally, operational stability of fiber supports was examined in a plug-flow reactor and 5% of initial substrate conversion remained after > 2000 cycles. The efficiency of core-shell immobilizates compared to one-dimensional fibers was both in batch and continuous reaction at least 4.4-fold higher.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

The most significant drawback of bacterial protein production involving inclusion bodies is the subsequent refolding into bioactive form. Implementation of refolding operations in large-scale applications often fails due to low yields and/or low product concentrations. This paper presents a simple method of integrated refolding by dialysis and matrix assisted refolding that combines advantages of both methods, high product concentrations and high refolding yields. Ion exchange resins (IER) and size exclusion media served as refolding additives and were added to solubilized protein prior to refolding by continuous exchange of dialysis buffer. Refolding experiments were performed with fructosyltransferase (FTF, EC 2.4.1.162) from Bacillus subtilis NCIMB 11871 produced as inclusion bodies. Conventional anion exchangers with gel matrix structure enhanced refolding performance by about 43% with final protein concentration of 9 mg/mL and yield improvement is strictly linear dependent on the mass ratio of resins to protein. With the applied setup refolded protein was self-eluted from resin due to pH and salt concentration shift during dialysis. Macroporous resins and gel filtration media showed a negative effect on refolding yields.

Asunto(s)

RESUMEN

For commercial applications refolding process must be fast, inexpensive and highly efficient. In the past many strategies for protein refolding were introduced. Still, simple refolding methods with high product concentrations are still rare. Refolding experiments were performed with fructosyltransferase (FTF, EC 2.4.1.162) from Bacillus subtilis NCIMB 11871 produced as inclusion bodies. Solubilizates were refolded with batch dialysis or by continuous exchange of dialysis buffers with variable ionic strength. By employing dialysis with gentle removal of denaturant the dependence of protein concentration and decreasing refolding yields could be overcome compared to batch dialysis and yields were enhanced by 52% at protein concentrations of approx. 10 mg/mL. The average specific activity of refolded FTF was 123 U/mg, 83% relative to standard FTF. Rising ionic strength of refolding buffers to 600 mM leads to complete renaturation of solubilized protein at equal protein concentration. Buffer composition plays a less significant role on renaturation output. The effect might be correlated with ion charge density of co-solvents.

Asunto(s)

RESUMEN

Immobilization methods and carriers were screened for immobilization of Euglena gracilis extract with laminaribiose phosphorylase activity. The extract was successfully immobilized on three different carriers via covalent linkage. Suitable immobilization carriers were Sepabeads EC-EP/S and ECR 8209M with epoxy groups and ECR 8309M with amino groups as functional units. Immobilization on Sepabeads EC-EP/S resulted in highest retained activity (65%). The immobilizates were characterized for pH, temperature, and buffer molarity preferences. The immobilized enzyme lost 48% of its activity when used seven times. Together with sucrose phosphorylase, laminaribiose phosphorylase was successfully applied for bienzymatic production of laminaribiose from sucrose and glucose with a final laminaribiose concentration of 14.3 ± 2.1 g/L (20% yield).

Asunto(s)

RESUMEN

The integration of all relevant tools for bioreaction engineering has been a recent challenge. This approach should notably favor the production of oligo- and polysaccharides, which is highly complex due to the requirements of regio- and stereoselectivity. Oligosaccharides (OS) and polysaccharides (PS) have found many interests in the fields of food, pharmaceuticals, and cosmetics due to different specific properties. Food, sweeteners, and food ingredients represent important sectors where OS are used in major amounts. Increasing attention has been devoted to the sophisticated roles of OS and glycosylated compounds, at cell or membrane surfaces, and their function, e.g., in infection and cancer proliferation. The challenge for synthesis is obvious, and convenient approaches using cheap and readily available substrates and enzymes will be discussed. We report on new routes for the synthesis of oligosaccharides (OS), with emphasis on enzymatic reactions, since they offer unique properties, proceeding highly regio- and stereoselective in water solution, and providing for high yields in general.

Asunto(s)

RESUMEN

Co-Immobilization of dextransucrase (DS) and dextranase (DN) into calcium alginate includes the co-entrapment of soluble DS and adsorbed DN. DS converts sucrose into dextran, which is the substrate for DN, so that isomalto-oligosaccharides (IMOs) are follow-up products of dextran hydrolysis. The boundary conditions for the successful preparation are investigated with respect to choice of DN adsorbate, surface modifications using blotting agents and optimal enzyme activity ratios. Further, repetitive batch experiments suggest the selection of medium activity ratios for continuous use (0.3 U(DN)U(-1) (DS), e.g.). Product formation at various cosubstrate:substrate concentrations as well as at different DN:DS ratios are discussed. Moreover, the complexity of the bi-enzymatic system can be reduced considering the molar ratios of cosubstrate:substrate (glucose:sucrose). Based on these factors, a mechanistic kinetic model is developed, which distinguishes the corresponding contributions of the two enzymes upon overall product formation. In general, at low glucose:sucrose ratios isomaltose synthesis is featured primarily by DN action. Yet with increasing amounts of glucose both the quantity and quality of DN substrate changes, so that its contribution to product formation decreases in an exponential manner; still the overall product yield continuously increases due to enhanced DS contribution.

Asunto(s)

RESUMEN

Fermentation kinetics of Penicillium aculeatum ATCC 10409 demonstrated that fungal growth and dextranase release are decoupled. Inoculation by conidia or mycelia resulted in identical kinetics. Two new isoenzymes of the dextranase were characterized regarding their kinetic constants, pI, MW, activation energy and stabilities. The larger enzyme was 3-fold more active (turnover number: 2,230 +/- 97 s(-1)). Pre-treatment of bentonite with H(2)O(2) did not affect adsorption characteristics of dextranase. Enzyme to bentonite ratios above 0.5:1 (w/w) resulted in a high conservation of activity upon adsorption. Furthermore, dextranase could be used in co-immobilizates for the direct conversion of sucrose into isomalto-oligosaccharides (e.g. isomaltose). Yields of co-immobilizates were 2-20 times that of basic immobilizates, which consist of dextransucrase without dextranase.

Asunto(s)

RESUMEN

In order to facilitate the Co-Immobilization of dextransucrase and dextranase, various techniques for the immobilization of industrial endo-dextranase from Chaetomium erraticum (Novozymes A/S) were researched. Adsorption isotherms at various pH-values have been determined for bentonite (Montmorillonite), hydroxyapatite and Streamline DEAE. Using bentonite and hydroxyapatite, highest activity loads (12,000 Ug(-1); 2900 Ug(-1), respectively) can be achieved without a significant change of the apparent Michaelis-Menten constant K(M). For successful adsorption, enzyme to bentonite ratios greater than 0.4 (w/w) have to be used as lower ratios lead to 90% enzyme inactivation due to bentonite contact. In addition, covalent linkage using the activated oxiran carriers Eupergit C and Eupergit C250L as well as linkage with aminopropyl silica via metaperiodate activation of glycosyl moiety of dextranase are discussed. This is also the first report probing the structure of a matrix containing dextranase by use of substrate species with different molecular weights. From this we can observe a relationship between the porosity of Eupergit and dextran dependent activity. For the reactor concept using Co-Immobilisates, hydroxyapatite will be preferred to Eupergit because of its higher specific activity and dispersity.

Asunto(s)

RESUMEN

The acceptor reactions of dextransucrase offer the potential for a targeted synthesis of a wide range of di-, tri- and higher oligosaccharides by the transfer of a glucosyl group from sucrose to the acceptor. We here report on results which show that the synthetic potential of this enzyme is not restricted to 'normal' saccharides. Additionally functionalized saccharides, such as alditols, aldosuloses, sugar acids, alkyl saccharides, and glycals, and rather unconventional saccharides, such as fructose dianhydride, may also act as acceptors. Some of these acceptors even turned out to be relatively efficient: alpha-D-glucopyranosyl-(1-->5)-D-arabinonic acid, alpha-D-glucopyranosyl-(1-->4)-D-glucitol, alpha-D-glucopyranosyl-(1-->6)-D-glucitol, alpha-D-glucopyranosyl-(1-->6)-D-mannitol, alpha-D-fructofuranosyl-beta-D-fructofuranosyl-(1,2':2,3')-dianhydride, 1,5-anhydro-2-deoxy-D-arabino-hex-1-enitol ('D-glucal'), and may therefore be of interest for future applications of the dextransucrase acceptor reaction.

Asunto(s)