Hydrolysis of lactose in skim milk by immobilized beta-galactosidase in a spiral flow reactor

beta-galactosidase from Aspergillus Oryzae immobilized in a spiral flow reactor was used to effect the hydrolysis of the lactose component of skim milk. Residence time distribution measurements were used to assess the amount of longitudinal dispersion occurring as a consequence of the spiral flow pattern and the semiporous nature of the polymeric material used to construct the spiral. It was possible to model the flow conditions as tubular flow with a Peclet number that was a linear function of the reactor space time. Nonlinear regression methods were used to determine the kinetic parameters of three proposed enzymatic rate expressions. The best fit of the data was obtained using a rate expression containing separate terms for competitive inhibition of the reaction by both the a and beta anomers of galactose. This kinetic model also incorporates the kinetics of the mutarotation between these forms. At 30 degrees C and a space time of 7 minutes, 80% of the lactose present in skim milk can be converted to glucose and galactose.     

Immobilized preparation of cold-adapted and halotolerant Antarctic beta-galactosidase as a highly stable catalyst in lactose hydrolysis

A cold-active beta-galactosidase of Antarctic marine bacterium Pseudoalteromonas sp. 22b was synthesized by an Escherichia coli transformant harboring its gene and immobilized on glutaraldehyde-treated chitosan beads. Unlike the soluble enzyme the immobilized preparation was not inhibited by glucose, its apparent optimum temperature for activity was 10 degrees C higher (50 vs. 40 degrees C, respectively), optimum pH range was wider (pH 6-9 and 6-8, respectively) and stability at 50 degrees C was increased whilst its pH-stability remained unchanged. Soluble and immobilized preparations of Antarctic beta-galactosidase were active and stable in a broad range of NaCl concentrations (up to 3 M) and affected neither by calcium ions nor by galactose. The activity of immobilized beta-galactosidase was maintained for at least 40 days of continuous lactose hydrolysis at 15 degrees C and its shelf life at 4 degrees C exceeded 12 months. Lactose content in milk was reduced by more than 90% over a temperature range of 4-30 degrees C in continuous and batch systems employing the immobilized enzyme.     

Molecular cloning of lactose genes in dairy lactic streptococci: the phospho-beta-galactosidase and beta-galactosidase genes and their expression products

The mesophilic (S. lactis and S. cremoris) and thermophilic (S. thermophilus) dairy lactic streptococci, which are used in industrial dairy fermentations, contain two different lactose hydrolysing enzymes, a phospho-beta-galactosidase and a beta-galactosidase. The central role of these enzymes in the pathways used for lactose transport and degradation is discussed along with their properties and distributions in lactic streptococci. In addition, recent results on the cloning, expression and sequence organization of the genes for the mesophilic phospho-beta-galactosidase and thermophilic beta-galactosidase are reviewed. Original data are presented concerning heterologous gene expression in the study of lactose hydrolysis in lactic streptococci. These include 1) the purification of the S. lactis phospho-beta-galactosidase from an overproducing Escherichia coli, and 2) the expression of the E. coli beta-galactosidase (lacZ) gene in S. lactis employing a lactic streptococcal expression vector.     

Pore diffusion model for a two-substrate enzymatic reaction: Application to galactose oxidase immobilized on porous glass particles

An analysis of the pore diffusion model involving a two-substrate enzymatic reaction is presented. The resulting equations have been applied to the case of galactose oxidase catalyzed oxidation of galactose when the enzyme is immobilized on porous glass particles. The physical constants of the system were obtained by theoretical predictions and the enzyme concentration in the porous medium was derived from the experimental results. The calculations were performed with the assumption that the kinetic parameters of the enzyme remain unchanged upon immobilization. The theoretically calculated effectiveness factors were compared with the experimental effectiveness factors determined from the batch kinetic experiments and were found to be in agreement. The results are presented as effectiveness factor plots graphed as functions of bulk galactose and oxygen concentrations. The model was extended in order to study the effect of external mass transfer coefficients and pore enzyme concentrations on the effectiveness factors.     

Effects of temperature on the hydrolysis of lactose by immobilized beta-galactosidase in a capillary bed reactor

The effects of temperature on the hydrolysis of lactose by immobilized beta-galactosidase were studied in a continuous flow capillary bed reactor. Temperature affects the rates of enzymatic reactions in two ways. Higher temperatures increase the rate of the hydrolysis reaction, but also increase the rate of thermal deactivation of the enzyme. The effect of temperature on the kinetic parameters was studied by performing lactose hydrolysis experiments at 15, 20, 25, 30, and 40 degrees C. The kinetic parameters were observed to follow an Arrhenius-type temperature dependence. Galactose mutarotation has a significant impact on the overall rate of lactose hydrolysis. The temperature dependence of the mutarotation of galactose was effectively modelled by first-order reversible kinetics. The thermal deactivation characteristics of the immobilized enzyme reactor were investigated by performing lactose hydrolysis experiments at 52, 56, 60, and 64 degrees C. The thermal deactivation was modelled effectively as a first order decay process. Based on the estimated thermal deactivation rate constants, at an operating temperature of 40 degrees C, 10% of the enzyme activity would be lost in one year.     

Process development for the production of prebiotic galacto-oligosaccharides from lactose using beta-galactosidase from Lactobacillus sp

Galacto-oligosaccharides (GOS) are formed from lactose in discontinuous mode of conversion using beta-galactosidase from Lactobacillus sp. (beta-gal). The discontinuous process was optimized for technical application with regard to GOS yield, enzyme preparation, reaction temperature and substrate source. It proved to be advantageous to directly apply the crude cell-free enzyme extract for the conversion, since similar GOS yields and composition were obtained as when using the pure enzyme preparation, but expensive purification could be avoided. Reaction temperature was lowered to 17 degrees C to limit microbial contamination when using technical substrates. Thereby GOS yield decreased from 30% to 28% of total sugars and enzyme demand increased 2.7-fold. Whey permeate was compared to buffered lactose solution as a substrate source. The initial reaction rate was found to be 1.8 times higher for the whey permeate substrate; however, GOS yield was slightly lower (approximately 25% of total sugar at 17 degrees C) mainly due to smaller amounts of allolactose[beta-D-Galp-(1-->6)-D-Glc] and the trisaccharide beta-D-Galp-(1-->6)-D-Lac formed.     

The preparation of immobilized lactase and its use in the enzymatic hydrolysis of acid whey

The enzyme β-galactosidase (lactose) obtained from several microbial sources was immobilized on zirconia-coated porous glass particles. The immobilized enzymes were characterized by determining pH profiles, kinetic constants, thermal profiles, and operationalhalf-lives in lactose and whey ultrafiltrate solutions. Studies were carried out on continuous reactor performance, and enzyme requirements for scale-up were estimated. Lactose or whey hydrolyzed by this technique could find use commercially as a sweetener in a number of dairy products.     

Influence of lactose hydrolysis and solids concentration on alcohol production by yeast in acid whey ultrafiltrate

Alcohol yields of 6.5% were obtained with Saccharomyces cerevisiae in lactasehydrolyzed acid whey permeate containing 30–35% total solids. Maximum alcohol yields obtained with Kluyveromyces fragilis were 4.5% in lactase-hydrolyzed acid whey permeate at a solids concentration of 20% and 3.7% in normal permeate at a solids concentration of 10%. Saccharomyces cerevisiae efficiently converted the glucose present in lactase-hydrolyzed whey permeates containing 5–30% total solids (2–13% glucose) to alcohol. However, the galactose, which comprised about half the available carbohydrate in lactase-hydrolyzed whey, was not utilized by S. cerevisiae, so that even though alcohol yields were higher when this organism was used, the process was wasteful in that a substantial proportion of the substrate was not fermented. For the process to become commercially feasible, an efficient means of rapidly converting both the galactose and glucose to alcohol must be found.     

Use of whey lactose from dairy industry for economical kefiran production by Lactobacillus kefiranofaciens in mixed cultures with yeasts

To evaluate the feasibility of producing kefiran industrially, whey lactose, a by-product from dairy industry, was used as a low cost carbon source. Because the accumulation of lactic acid as a by-product of Lactobacillus kefiranofaciens inhibited cell growth and kefiran production, the kefir grain derived and non-derived yeasts were screened for their abilities to reduce lactic acid and promote kefiran production in a mixed culture. Six species of yeasts were examined: Torulaspora delbrueckii IFO 1626; Saccharomyces cerevisiae IFO 0216; Debaryomyces hansenii TISTR 5155; Saccharomyces exiguus TISTR 5081; Zygosaccharomyces rouxii TISTR 5044; and Saccharomyces carlsbergensis TISTR 5018. The mixed culture of L. kefiranofaciens with S. cerevisiae IFO 0216 enhanced the kefiran production best from 568 mg/L in the pure culture up to 807 and 938 mg/L in the mixed cultures under anaerobic and microaerobic conditions, respectively. The optimal conditions for kefiran production by the mixed culture were: whey lactose 4%; yeast extract 4%; initial pH of 5.5; and initial amounts of L. kefiranofaciens and S. cerevisiae IFO 0216 of 2.1×10(7) and 4.0×10(6)CFU/mL, respectively. Scaling up the mixed culture in a 2L bioreactor with dissolved oxygen control at 5% and pH control at 5.5 gave the maximum kefiran production of 2,580 mg/L in batch culture and 3,250 mg/L in fed-batch culture.     

Acetate production from whey lactose using co-immobilized cells of homolactic and homoacetic bacteria in a fibrous-bed bioreactor

Acetate was produced from whey lactose in batch and fed-batch fermentations using co-immobilized cells of Clostridium formicoaceticum and Lactococcus lactis. The cells were immobilized in a spirally wound fibrous sheet packed in a 0.45-L column reactor, with liquid circulated through a 5-L stirred-tank fermentor. Industrial-grade nitrogen sources, including corn steep liquor, casein hydrolysate, and yeast hydrolysate, were studied as inexpensive nutrient supplements to whey permeate and acid whey. Supplementation with either 2.5% (v/v) corn steep liquor or 1.5 g/L casein hydrolysate was adequate for the cocultured fermentation. The overall acetic acid yield from lactose was 0.9 g/g, and the productivity was 0.25 g/(L h). Both lactate and acetate at high concentrations inhibited the homoacetic fermentation. To overcome these inhibitions, fed-batch fermentations were used to keep lactate concentration low and to adapt cells to high-concentration acetate. The final acetate concentration obtained in the fed-batch fermentation was 75 g/L, which was the highest acetate concentration ever produced by C. formicoaceticum. Even at this high acetate concentration, the overall productivity was 0.18 g/(L h) based on the total medium volume and 1.23 g/(L h) based on the fibrous-bed reactor volume. The cells isolated from the fibrous-bed bioreactor at the end of this study were more tolerant to acetic acid than the original culture used to seed the bioreactor, indicating that adaptation and natural selection of acetate-tolerant strains occurred. This cocultured fermentation process could be used to produce a low-cost acetate deicer from whey permeate and acid whey.     

Continuous production of itaconic acid by Aspergillus terreus immobilized in a porous disk bioreactor

Summary Aspergillus terreus NRRL 1960 was grown on porous disks rotating intermittently in and out of the liquid phase. This immobilized fungal cell bioreactor was used to produce itaconic acid from glucose in a continuous operation. The effect of temperature, pH, disk rotation speed, and feed rate on the itaconic acid concentration and volumetric productivity were studied. The highest itaconic acid concentration and volumetric productivity obtained were 18.2 g/l and 0.73 g/l·h, respectively, under the following conditions: temperature at 36°C, pH 3.0, disk rotation speed at 8 rpm, and feed rate at 60 ml/h. These results are better than those by conventional fermentation or by other immobilized method.Nomenclature F feed rate (l/h) - K _1s saturation constant for immobilized cells (g/l) - K _2s saturation constant for suspended cells (g/l) - M ₁ increased mass of immobilized cells (g) - M ₂ total mass of immobilized cells (g) - P concentration of itaconic acid (g/l) - S substrate concentration in and out of the reactor (g/l) - S ₀ substrate concentration in the feed (g/l) - V liquid volume of the reactor (1) - X concentration of the suspended cells (g/l) - Y ₁ apparent yield of the immobilized cells (g cells/g substrate) - Y ₂ apparent yield of the suspended cells (g cell/g substrate) - Y ₃ apparent yield of itaconic acid (g itaconic acid/g substrate) - m ₁ maintenance and by-products coefficient of the immobilized cells (g substrate/g cell·h) - m ₂ maintenance and by-products coefficient of the suspended cells (g substrate/g cell·h) - µ_1max maximum specific growth rate of the immobilized cells (h^-1) - µ_2max maximum specific growth rate of the suspended cells (h^-1)     

The production of cellulase in a spouted bed fermentor using cells immobilized in biomass support particles

Continuous cellulase production by Trichoderma viride QM 9123, immobilized in 6 mm diameter, spherical, stainless steel biomass support particles, has been achieved using a medium containing glucose as the main carbon source. Experiments were carried out in a 10-L spouted bed fermentor. In this type of reactor-recycled broth is used to create a jet at the base of a bed of particles, causing the particles to spout and circulate. During the circulation, particles pass through a region of high shear near the jet inlet. This effectively prevents a buildup of excess biomass and thus enables steady-state conditions to be achieved during continuous operation. Continuous production of cellulase was achieved at significantly higher yield and productivity than in conventional systems. At a dilution rate of 0.15 h(-1) (nominal washout rate for freely suspended cells is 0.012 h(-1)), the yield of cellulase on glucose was 31% higher than that measured during batch operation, while the volumetric productivity (31.5 FPA U/L. h) was 53% greater than in the batch system. The specific cellulase productivity of the immobilized cells was more than 3 times that of freely suspended cells, showing that diffusional limitations can be beneficial. This offers significant opportunity for the further development of biomass support particles and associated bioreactors.     

Preparation of immobilized β-galactosidase by poly(vinylpyrrolidone) and the continuous hydrolysis of lactose in acid whey

Immobilized β-galactosidase gel was prepared using poly(vinylpyrrolidone) (PVP) under β-ray irradiation. In contrast to the gelation of N-vinylpyrrolidone monomer–enzyme solution, the gelation of PVP-β-galactosidase solution (PVP content: 10%) was almost completely uneffected by the dose rate and amount of phosphate present. PVP-enzyme solution was gelled by irradiation with 3.0 Mrad. The expressed activity of the PVP-enzyme gel was about 30% of the initial activity and added activity was almost totally entrapped. No leakage of enzyme from these gels could be detected. Leakage was, however, detected in the case of the gelation of PVP-enzyme solution containing more than 1% of enzyme protein. When the general properties of the gel were compared with those of the native enzyme, the gel proved to be slightly inferior to the native enzyme with respect to optimum temperature, heat stability, pH activity, and pH stability. Continuous hydrolysis of lactose in acid whey could be carried out at 50°C using a column packed with the gel and sawdust and the degree of hydrolysis was found to be almost, constant for 12 days. The merits of using PVP in the immobilization of enzymes include the simplicity of the procedure and the fact that the PVP-enzyme gel can be used in the food industry without anxiety because of its high degree of compatibility with living organisms.     

Isolation of a Xanthomonas campestris strain with elevated beta-galactosidase activity for direct use of lactose in xanthan gum production

AIMS: To isolate a Xanthomonas campestris strain that can use lactose directly for xanthan gum production. METHODS AND RESULTS: The presence of indigenous beta-galactosidase gene in the wild-type Xc17 was detected by PCR and Southern hybridization. Treatment of Xc17 with nitrous acid resulted in the isolation of Xc17L with a 3.5-fold elevation of beta-galactosidase activity capable of growing in lactose-based medium. Xc17L is stable for at least 100 generations in terms of beta-galactosidase expression. The amounts of xanthan produced by Xc17L in lactose-based medium are comparable to those in glucose-based medium. CONCLUSIONS: Xc17L is potentially useful for xanthan production from whey, a waste containing lactose. SIGNIFICANCE AND IMPACT OF THE STUDY: A lactose-utilizing strain of X. campestris strain can be constructed without incorporation of any exotic DNA or antibiotic resistance gene and therefore concern of a gene-modified organism and fear of a spread of an antibiotic-resistant gene are avoided.     

A novel procedure for the preparation and characterization of catalytically active fatty acid synthetase immobilized on sepharose beads

A novel procedure for immobilization of enzymatically active fatty acid synthetase is presented. The enzyme is coupled to a Sepharose 4B matrix containing covalently attached antibodies which recognize, and bind specifically to, the thioesterase domain of this polyfunctional enzyme. A continuous flow system is described for assay of the immobilized enzyme. Fatty acid synthetase activity apparently is not limited by movement of substrates through the Nernst diffusion layer surrounding the matrix particles, since normal Michaelis-Menten kinetics are observed and reaction rates are independent of flow rate. The Km values for acetyl-CoA and malonyl-CoA, the pH/activity profile, and the reaction products are essentially the same as for the freely soluble enzyme, although the specific activity is lower by about 55%. The preparation and characterization of immobilized subunits of the enzyme could provide a valuable approach for studying the role of structural and functional subunit interactions in the enzyme. In addition, the immobilized enzyme offers a model for studying the properties of this enzyme in a highly structured environment such as might exist in vivo, permitting study of both physical and functional interactions of fatty acid synthetase with other lipogenic enzymes.     

Oriented and selective enzyme immobilization on functionalized silica carrier using the cationic binding module Z basic2: design of a heterogeneous D-amino acid oxidase catalyst on porous glass

D-amino acid oxidase from Trigonopsis variabilis (TvDAO) is applied in industry for the synthesis of pharmaceutical intermediates. Because free TvDAO is extremely sensitive to exposure to gas-liquid interfaces, biocatalytic processing is usually performed with enzyme immobilizates that offer enhanced stability under bubble aeration. We herein present an "Immobilization by Design" approach that exploits engineered charge complementarity between enzyme and carrier to optimize key features of the immobilization of TvDAO. A fusion protein between TvDAO and the positively charged module Z(basic2) was generated, and a corresponding oppositely charged carrier was obtained by derivatization of mesoporous glass with 3-(trihydroxysilyl)-1-propane-sulfonic acid. Using 250 mM NaCl for charge screening at pH 7.0, the Z(basic2) fusion of TvDAO was immobilized directly from E. coli cell extract with almost absolute selectivity and full retention of catalytic effectiveness of the isolated enzyme in solution. Attachment of the homodimeric enzyme to the carrier was quasi-permanent in low-salt buffer but fully reversible upon elution with 5 M NaCl. Immobilized TvDAO was not sensitive to bubble aeration and received substantial (≥ tenfold) stabilization of the activity at 45°C as compared to free enzyme, suggesting immobilization via multisubunit oriented interaction of enzyme with the insoluble carrier. The Z(basic2) enzyme immobilizate was demonstrated to serve as re-usable heterogeneous catalyst for D-amino acid oxidation. Z(basic2) -mediated binding on a sulfonic acid group-containing glass carrier constitutes a generally useful strategy of enzyme immobilization that supports transition from case-specific empirical development to rational design.     

Flow injection analysis of lactose using covalently immobilized beta-galactosidase, mutarotase, and glucose oxidase/peroxidase on a 2-fluoro-1-methylpyridinium salt-activated Fractogel support

Milk samples were analyzed for their lactose content using flow injection analysis and incorporating immobilized beta-galactosidase or beta-galactosidase/mutarotase and glucose oxidase/peroxidase bioreactors. These enzymes were immobilized, under mild conditions, on to a 2-fluoro-1-methylpyridinium salt-activated Fractogel support. The use of a phosphate buffer (0.15 M) was found to facilitate the rapid mutarotation of alpha-D-glucose and hence could obviate the need for the more expensive mutarotase. The chromogenic agents of choice for monitoring the reaction were 3-methyl-2-benzothiazolinone hydrazone and 3-dimethylaminobenzoic acid. Linearity was observed over the concentration range 16-160 micrograms/ml using lactose standards (r = 0.996). Between 30 and 40 milk samples/h can be analyzed. Comparisons are made with existing HPLC and alkaline methylamine methods for a range of milk matrices. The FIA method consistently gives the lowest standard deviations and coefficient of variation for the various milk matrices analyzed.     

Menstrum for culture preservation and medium for seed preparation in a tetanus toxin production process containing no animal or dairy products

AIMS: To completely eliminate animal and dairy products from the lyophilization menstrum and the seed medium used to produce tetanus toxin with Clostridium tetani. METHODS AND RESULTS: Tetanus toxin production in a recently developed fermentation medium lacking animal and dairy products was studied with different seed media. It was found that soy peptone could completely replace the beef heart infusion plus animal peptone previously used as seed medium. In addition, we found that cells lyophilized in soy milk could replace the usual type of cells lyophilized in cow's milk. CONCLUSIONS: We have now developed a complete tetanus toxin production process containing no animal and dairy products. SIGNIFICANCE AND IMPACT OF THE STUDY: Toxoid preparations made from toxin produced with animal and dairy products can contain undesirable contaminants such as the prion causing bovine spongiform encephalopathy (Mad Cow's Disease) or antigenic peptides that stimulate anaphylactic reactions and other undesirable immune reactions in immunized hosts. The new vegetable-based process described here avoids such unfortunate possibilities.