Comparison of catalytic properties of free and immobilized cellobiase novozym 188.

The enzyme cellobiase from Novo was immobilized in controlled pore silica particles by covalent binding with the silane-glutaraldehyde method with protein and activity yields of 67 and 13.7%, respectively. The activity of the free enzyme (FE) and immobilized enzyme (IE) was determined with 2 g/L of cellobiose, from 40 to 75 degrees C at pH 3.0-7.0 for FE and from 40 to 70 degrees C at pH 2.2-7.0 for IE. At pH 4.8 the maximum specific activity for the FE and IE occurred at 65 degrees C: 17.8 and 2.2 micromol of glucose/(min x mg of protein), respectively. For all temperatures the optimum pH observed for FE was 4.5 whereas for IE it was shifted to 3.5. The energy of activation was 11 kcal/mol for FE and 5 kcal/mol for IE at pH 4.5-5, showing apparent diffusional limitation for the latter. Thermal stability of the FE and IE was determined with 2 g/L of cellobiose (pH 4.8) at temperatures from 40 to 70 degrees C for FE and 40 to 75 degrees C for IE. Free cellobiase maintained its activity practically constant for 240 min at temperatures up to 55 degrees C. The IE has shown higher stability, retaining its activity in the same test up to 60 degrees C. Half-life experimental results for FE were 14.1, 2.1, and 0.17 h at 60, 65, and 70 degrees C, respectively, whereas IE at the same temperatures had half-lives of 245, 21.3, and 2.9 h. The energy of thermal deactivation was 80.6 kcal/mol for the free enzyme and 85.2 kcal/mol for the IE, suggesting stabilization by immobilization.

Characterization of cyclodextrin glycosyltransferase from Bacillus firmus strain no. 37.

The enzyme cyclodextrin glycosyltransferase (CGTase), EC 2.4.1.19, which produces cyclodextrins (CDs) from starch, was obtained from Bacillusfirmus strain no. 37 isolated from Brazilian soil and characterized in the soluble form using as substrate 100 g/L of maltodextrin in 0.05 M Tris-HCl buffer, 5 mM CaCl2, and appropriate buffers. Enzymatic activity and its activation energy were determined as a function of temperature and pH. The activation energy for the production of beta- and gamma-CD was 7.5 and 9.9 kcal/mol, respectively. The energy of deactivation was 39 kcal/mol. The enzyme showed little thermal deactivation in the temperature range of 35-60 degrees C, and Arrhenius-type equations were obtained for calculating the activity, deactivation, and half-life as a function of temperature. The molecular weight of the enzyme was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, giving 77.6 kDa. Results for CGTase activity as a function of temperature gave maximal activity for the production of beta-CD at 65 degrees C, pH 6.0, and 71.5 mmol of beta-CD/(min x mg of protein), whereas for gamma-CD it was 9.1 mmol of gamma-CD/(min mg of protein) at 70 degrees C and pH 8.0. For long contact times, the best use of the enzymatic activity occurs at 60 degrees C or at a lower temperature, and the reaction pH may be selected to increase the yield of a desired CD.

Selection of stabilizing additive for lipase immobilization on controlled pore silica by factorial design.

Candida rugosa lipase was covalently immobilized on silanized controlled pore silica (CPS) previously activated with glutaraldehyde in the presence of several additives to improve the performance of the immobilized form in long-term operation. Proteins (albumin and lecithin) and organic molecules (beta-cyclodextrin and polyethylene glycol [PEG]-1500) were added during the immobilization procedure, and their effects are reported and compared to the behavior of the immobilized biocatalyst in the absence (lacking) of additive. The selection of the most efficient additive at different lipase loadings (150-450 U/g of dry support) was performed by experimental design. Two 22 full factorial designs with two repetitions at the center point were employed to evaluate the immobilization yield. A better stabilizing effect was found when small amounts of albumin or PEG-1500 were added simultaneously to the lipase onto the support. The catalytic activity had a maximum (193 U/mg) for lipase loading of 150 U/g of dry support using PEG-1500 as the stabilizing additive. This immobilized system was used to perform esterification reactions under repeated batch cycles (for the synthesis of butyl butyrate as a model). The half-life of the lipase immobilized on CPS in the presence of PEG-1500 was found to increase fivefold compared with the control (immobilized lipase on CPS without additive).

Kinetic studies of lipase from Candida rugosa: a comparative study between free and immobilized enzyme onto porous chitosan beads.

The search for an inexpensive support has motivated our group to undertake this work dealing with the use of chitosan as matrix for immobilizing lipase. In addition to its low cost, chitosan has several advantages for use as a support, including its lack of toxicity and chemical reactivity, allowing easy fixation of enzymes. In this article, we describe the immobilization of Candida rugosa lipase onto porous chitosan beads for the enzymatic hydrolysis of olive oil. The binding of the lipase onto the support was performed by physical adsorption using hexane as the dispersion medium. A comparative study between free and immobilized lipase was conducted in terms of pH, temperature, and thermal stability. A slightly lower value for optimum pH (6.0) was found for the immobilized form in comparison with that attained for the soluble lipase (7.0). The optimum reaction temperature shifted from 37 degrees C for the free lipase to 50 degrees C for the chitosan lipase. The patterns of heat stability indicated that the immobilization process tends to stabilize the enzyme. The half-life of the soluble free lipase at 55 degrees C was equal to 0.71 h (Kd = 0.98 h(-1)), whereas for the immobilized lipase it was 1.10 h (Kd = 0.63 h(-1)). Kinetics was tested at 37 degrees C following the hydrolysis of olive oil and obeys the Michaelis-Menten type of rate equation. The Km was 0.15 mM and the Vmax was 51 micromol/(min x mg), which were lower than for free lipase, suggesting that the apparent affinity toward the substrate changes and that the activity of the immobilized lipase decreases during the course of immobilization.

Enhancement of selectivity for producing gamma-cyclodextrin.

The production of cyclodextrins (CDs) by cyclodextrin-glycosyl-transferase (CGTase) from Bacillus firmus was studied, with respect to the effect of the source of starch upon CD yield and on the selectivity for producing gamma-CD. Cyclodextrin production tests were run for 24 h at 50 degrees C, pH 8.0, and 1 mg/L of CGTase, and substrates were maltodextrin or the starches of rice, potato, cassava, and corn hydrolyzed up to D.E. 10. Cornstarch was the best substrate for producing gamma-CD. Later, glycyrrhizin (2.5% [w/v]), which forms a stable complex with gamma-CD, was added to the cornstarch reaction medium and increased the yield of gamma-CD to about four times that produced with only maltodextrin, but the total yield of CDs remained practically unchanged. Therefore, the results showed that the studied CGTase is capable of giving relatively high yield of gamma-CD in the presence of glycyrrhizin as complexant and cornstarch as substrate.

Production of cyclodextrins in a fluidized-bed reactor using cyclodextrin-glycosyl-transferase.

Cyclodextrin-glycosyl-transferase (EC2.4.1.19), produced by Wacker (Munich, Germany), was purified by biospecific affinity chromatography with beta-cyclodextrin (beta-CD) as ligand, and immobilized into controlled pore silica particles (0.42 mm). This immobilized enzyme (IE) had 4.7 mg of protein/g of support and a specific activity of 8.6 mumol of beta-CD/(min.gIE) at 50 degrees C, pH 8.0. It was used in a fluidized-bed reactor (FBR) at the same conditions for producing cyclodextrins (CDs) with 10% (w/v) maltodextrin solution as substrate. Bed expansion was modeled by the Richardson and Zaki equation, giving a good fit in two distinct ranges of bed porosities. The minimum fluidization velocity was 0.045 cm/s, the bed expansion coefficient was 3.98, and the particle terminal velocity was 2.4 cm/s. The FBR achieved high productivity, reaching in only 4 min of residence time the same amount of CDs normally achieved in a batch reactor with free enzyme after 24 h of reaction, namely, 10.4 mM beta-CD and 2.3 mM gamma-CD.

Brazilian bioethanol program.

Brazil is the largest producer of bioethanol, and sugarcane is the main raw material. Bioethanol is produced by both batch and continuous processes, and in some cases, flocculating yeast is used. This article analyzes the Brazilian Ethanol Program. For the 1996-1997 harvest, Brazil produced 14.16 billion L of ethanol and 13.8 million metric t of sugar, from 286 million metric t of sugarcane. These products were produced by 328 industries in activity, with 101 autonomous ethanol plants producing only ethanol, and 227 sugar mills producing sugar and ethanol. The sugar-ethanol market reaches about 7.5 billion US$/yr, accounting for direct and indirect revenues.

Bleaching of kraft pulp with commercial xylanases.

The performance of commercial xylanases in totally chlorine-free bleaching of kraft pulp from conifer was tested with Pulpzyme HC (Novo Nordisk) and Cartazyme NS-10 (Sandoz/Clariant), at 500 U/kg of dry pulp, respectively. The treatment with Pulpzyme (XP) or Cartazyme (XC) has been combined with stages of bleaching using: oxygen (O), sulfuric acid (A), and extraction with hydrogen peroxide (EOP). The following sequences have been tested: OXpAEOP, OXcAEOP, XpOAEOP, XcOAEOP, and OAEOP. Kraft pulp bleached at the Klabin industrial plant using the sequence, CEH (chlorine, alkaline extraction, and hypochlorination) was used for comparison. The following average values were obtained: 1. Kappa number: OXPAEOP, 4.8; OXCAEOP, 4.9; XPOAEOP, 5.0; XCOAEOP, 4.9; OAEOP, 5.6, and CEH, 1.9; 2. Brightness (% ISO values): OXPAEOP, 68.4; OXCAEOP, 70.1; XPOAEOP, 67.9; XcOAEOP, 68.8; OAEOP, 63.8, and CEH, 63.6; and 3. Viscosity (cP): OXPAEOP, 27.6; OXCAEOP, 26.9; XPOAEOP, 23.4; XCOAEOP, 23.1; OAEOP, 25.4, and CEH, 25.2. Pulps that were treated with xylanases, before or after the delignification with oxygen, have shown reduced kappa number and higher brightness than the pulp OAEOP. Enzyme treatment before delignification with oxygen reduces pulp viscosity. Brightness obtained for pulp produced with bleaching sequences containing the enzymatic treatment, when compared with the control, CEH, shows that the xylanases enhance the action of the bleaching agents.

Characterization and utilization of Candida rugosa lipase immobilized on controlled pore silica.

Candida rugosa lipase was immobilized by covalent binding on controlled pore silica (CPS) using glutaraldehyde as cross-linking agent under aqueous and nonaqueous conditions. The immobilized C. rugosa was more active when the coupling procedure was performed in the presence of a nonpolar solvent, hexane. Similar optima pH (7.5-8.0) was found for both free and immobilized lipase. The optimum temperature for the immobilized lipase was about 10 degrees C higher than that for the free lipase. The thermal stability of the CPS lipase was also greater than the original lipase preparation. Studies on the operational stability of CPS lipase revealed good potential for recycling under aqueous (olive-oil hydrolysis) and nonaqueous (butyl butyrate synthesis) conditions.

Modeling cellobiose hydrolysis with integrated kinetic models.

The enzyme cellobiase Novozym 188, which is used for improving hydrolysis of bagasse with cellulase, was characterized in its commercial available form and integrated kinetic models were applied to the hydrolysis of cellobiose. The specific activity of this enzyme was determined for pH values from 3.0-7.0, and temperatures from 40-75 degrees C, with cellobiose at 2 g/L. Thermal stability was measured at pH 4.8 and temperatures from 40-70 degrees C. Substrate inhibition was studied at the same pH, 50 degrees C, and cellobiose concentrations from 0.4-20 g/L. Product inhibition was determined at 50 degrees C, pH 4.8, cellobiose concentrations of 2 and 20 g/L, and initial glucose concentration nearly zero or 1.8 g/L. The enzyme has shown the greatest specific activity, 17.8 U/mg, at pH 4.5 and 65 degrees C. Thermal activation of the enzyme followed Arrhenius equation with the Energy of Activation being equal to 11 kcal/mol for pH values 4 and 5. Thermal deactivation was adequately modeled by the exponential decay model with Energy of Deactivation giving 81.6 kcal/mol. Kinetics parameters for substrate uncompetitive inhibition were: Km = 2.42 mM, Vmax = 16.31 U/mg, Ks = 54.2 mM. Substrate inhibition was clearly observed above 10 mM cellobiose. Product inhibition at the concentration studied has usually doubled the time necessary to reach the same conversion at the lower temperature tested.

Thermal stability and energy of deactivation of free and immobilized amyloglucosidase in the saccharification of liquefied cassava starch.

Amyloglucosidase from Novo (Copenhagen, Denmark) was immobilized in controlled pore silica particles with the silane-glutaraldehyde covalent method. Thermal stability of the free and immobilized enzyme (IE) was determined with 30% (w/v) alpha-amylase liquefied cassava starch, pH 4.5, temperatures from 35 to 75 degrees C. Free amyloglucosidase maintained its activity practically constant for 240 min and temperatures up to 50 degree C. The IE has shown higher stability retaining its activity for the same period up to 60 degrees C. Half-life for free enzyme was 20.6, 6.44, 2.07, 0.69, and 0.24 h for 55, 60, 65, 70, and 75 degrees C, respectively, whereas the IE at the same temperatures had half-lives of 116.4, 30.88, 8.52, 2.44, and 0.73 h. The energy of thermal deactivation was thus 50.6 and 57.6 kcal/mol, respectively for the free and IE, confirming stabilization by immobilization.

beta-Cyclodextrin production by simultaneous fermentation and cyclization.

Production of beta-cyclodextrin (CD) with high-dextrose equivalent (DE) starch hydrolysates by simultaneous fermentation and cyclization (SFC) gives higher yields than using only the enzyme CGTase, because fermentation eliminates glucose and maltose that inhibit CD production, while at the same time, produces ethanol that increases yield. A 10% (w/v) solution of cassava starch, liquefied with alpha-amylase, was incubated with CGTase using: only the enzyme, added ethanol (from 1 to 5%), and added yeast S. cerevisiae (12% w/v), plus nutrients, the latter being the SFC process. Reaction conditions were: 38 degrees C, pH 6.0, DE from 2 to 25, and 3.3 mL of CGTase/L. The yield of beta-CD has decreased with an increase in DE, and maximum reaction yields were found for DE equal to 3.54, reaching 5.6, 14.7, and 11.5 mM beta-CD, respectively. For an increase of DE, of approx 6 times (from 3.54 to 23.79), beta-CD yield decreased 6 times for the first, and second reaction media with 3% (v/v) ethanol, and only approx 3 times for SFC (from 11.5 to 3.73 mM), showing that this process is less sensitive to variations in the DE.

Production and purification of CGTase of alkalophylic Bacillus isolated from Brazilian soil.

Alkalophylic bacilli that produce cyclodextringlycosyltransferase (CGTase) were isolated from Brazilian soil, with a scheme of two plating steps. In the first step, the bacterial isolate forms a halo in the cultivation medium that contains gamma-cyclodextrin (CD) complexing dyes. The CGTase of an isolate was purified 157-fold by biospecific affinity chromatography, with beta-CD showing a mol wt of 77,580 Daltons. It produces a gamma- to beta-CD ratio of 0.156 and a small amount of alpha-CD, using maltodextrin 10% as substrate, at 50 degrees C, pH 8.0 and 22 h reaction time, reaching 21.4% conversion of the substrate to cyclodextrins. In the second screening step, the isolates chosen give larger halos with beta-CD complexing dyes, and smaller halos with beta-CD complexing dyes, leading to a 30% improvement in gamma-CD selectivity, although at lower total yield for cyclodextrins (11.5%).

Modeling fixed and fluidized reactors for cassava starch saccharification with immobilized enzyme.

Cassava starch saccharification in fixed-and fluidized-bed reactors using immobilized enzyme was modeled in a previous paper using a simple model in which all dextrins were grouped in a single substrate. In that case, although good fit of the model to experimental data was obtained, physical inconsistency appeared as negative kinetic constants. In this work, a multisubstrate model, developed earlier for saccharification with free enzyme, is adapted for immobilized enzyme. This latter model takes into account the formation of intermediate substrates, which are dextrins competing for the catalytic site of the enzyme, reversibility of some reactions, inhibition by substrate and product, and the formation of isomaltose. Kinetic parameters to be used with this model were obtained from initial velocity saccharification tests using the immobilized enzyme and different liquefied starch concentrations. The new model was found to be valid for modeling both fixed- and fluidized-bed reactors. It did not present inconsistencies as the earlier one had and has shown that apparent glucose inhibition is about seven times higher in the fixed-bed than in fluidized-bed reactor.