Continuous Hydrolysis Of Lactose Research Articles

Immobilization of Aspergillus oryzae β galactosidase on concanavalin A-layered calcium alginate-cellulose beads and its application in lactose hydrolysis in continuous spiral bed reactors

Immobilization of Aspergillus oryzae β galactosidase on concanavalin A-layered calcium alginate-cellulose beads and its application in lactose hydrolysis in continuous spiral bed reactors In this study, Aspergillus oryzae β galactosidase was immobilized on concanavalin A layered calcium alginate-cellulose beads as a bioaffinity support. Immobilized enzyme showed a remarkable broadening in temperature-activity profiles as compared to the native enzyme and exhibited 65% activity in the presence of 5% galactose. Michaelis constant (Km) was 2.57 mM and 5.38 mM for the free and the immobilized β galactosidase, respectively. Crosslinked β galactosidase showed greater catalytic activity in the presence of Mg2+ and was more stable during storage at 4°C for 6 weeks. Immobilized enzyme hydrolyzed 67% lactose in milk in 8 h and 85% lactose in whey in 9 h in the stirred batch process at 50°C. The continuous hydrolysis of lactose by crosslinked β galactosidase in spiral bed reactor exhibited 93% and 88% hydrolysis of lactose at flow rate of 20 ml/h and 30 ml/h, after 1 month operation, respectively.

Calcium alginate entrapped preparations of Aspergillus oryzae β galactosidase: Its stability and applications in the hydrolysis of lactose

Insoluble concanavalin A–β galactosidase complex was obtained by using jack bean extract and this complex was crosslinked with glutaraldehyde, in order to maintain the integrity of complex in the presence of its substrate or products. Concanavalin A–β galactosidase complex retained 92% of the initial enzyme activity whereas crosslinked complex showed 88% activity. Entrapment of concanavalin A–β galactosidase complex into calcium alginate beads provided suitability to use this preparation in reactors. Temperature- and pH-optima of the various immobilized β galactosidase preparations were the same as its soluble counterpart. Entrapped crosslinked concanavalin A–β galactosidase complex retained more than 50% activity after 1 h exposure with 4.0 M urea at room temperature. Moreover, entrapped crosslinked concanavalin A–β galactosidase complex retained 81 and 62% of the original enzymatic activity in the presence of 5% calcium chloride and 5% galactose, respectively. Entrapped crosslinked concanavalin A–β galactosidase complex preparation was more superior in the continuous hydrolysis of lactose in a batch process as compared to the other entrapped preparations. This entrapped crosslinked concanavalin A–β galactosidase complex retained 95% activity after seventh repeated use and 93% of its original activity even after 2 months storage at 4 °C.

Development of an ultrahigh‐temperature process for the enzymatic hydrolysis of lactose. III. Utilization of two thermostable β‐glycosidases in a continuous ultrafiltration membrane reactor and galacto‐oligosaccharide formation under steady‐state conditions

Hydrolysis of lactose by hyperthermophilic beta-glycosidases from the archaea Sulfolobus solfataricus (SsbetaGly) and Pyrococcus furiosus (CelB) was carried out at 70 degrees C in a continuous stirred-tank reactor (CSTR) coupled to a 10-kDa cross-flow ultrafiltration module to recycle the enzyme. Recirculation rates of > or =1 min(-1), reaction of proteins with reducing sugars, and enzyme adsorption onto the membrane are major "operational" factors of enzyme inactivation in the CSTR. They cause the half-life times of SsbetaGly and CelB to be reduced two- and eight-fold, respectively, the average value for both enzymes now being approximately 5 to 7 days. Using lactose at initial concentrations of 45 and 170 g/L, the CSTR was operated at a constant conversion level of approximately 80% for more than 2 weeks without the occurrence of microbial contamination. The productivities for the SsbetaGly-catalyzed conversion of lactose were determined at different dilution rates and initial substrate concentrations, and exceed by a factor of < or =2 those observed with CelB under otherwise identical conditions. This difference reflects the approximately eight-fold stronger product inhibition of CelB by D-glucose. While the maximum total galacto-oligosaccharide production (90-100 mM) at 170 g/L lactose in the CSTR was not different from that in the batch reactor (CelB) or was greater by approximately 25% (SsbetaGly), continuous and batchwise reactions with both enzymes differed markedly with regard to relative proportions of the individual saccharide components present at 80% substrate conversion. The CSTR yielded an up to four-fold greater ratio of disaccharides to trisaccharides concomitant with a 5- to 30-fold larger relative proportion of beta-D-Galp-(1-->3)-D-Glc in the product mixture. The results show that apart from continuous hydrolysis of lactose at 70 degrees C, a CSTR charged with SsbetaGly or CelB and operated at steady-state conditions could be a useful reaction system for the production of galacto-oligosaccharides in which composition is narrower and more easily programmable, in terms of the individual components contained, as compared to the batchwise reaction.

Hydrolysis of lactose by beta-glycosidase CelB from hyperthermophilic archaeon Pyrococcus furiosus: comparison of hollow-fiber membrane and packed-bed immobilized enzyme reactors for continuous processing of ultrahigh temperature-treated skim milk.

Recombinant beta-glycosidase CelB from the hyperthermophilic archaeon Pyrococcusfuriosus was produced through expression of the plasmid-encoded gene in Escherichia coli. Bioreactor cultivations of E. coli in the presence of the inductor isopropyl-1-thio-beta-D-galactoside (0.1 mM) gave approx 100,000 U of enzyme activity/L of culture medium after 8 h of growth. A technical-grade enzyme for the hydrolysis of lactose was prepared by precipitating the mesophilic protein at 80 degrees C. A hollow-fiber membrane reactor was developed, and its performance during continuous processing of ultrahigh temperature-treated (UHT) skim milk at 70 degrees C was analyzed regarding long-term stability, productivity, and diffusional limitation thereof. CelB was covalently attached onto Eupergit C in yields of 80%, and a packed-bed immobilized enzyme reactor was used for the continuous hydrolysis of lactose in UHT skim milk at 70 degrees C. The packed-bed reactor was approximately 10-fold more stable and gave about the same productivity at 80% substrate conversion as the hollow-fiber reactor at 60% substrate conversion. The marked difference in the stability of free and immobilized CelB seems to reflect mainly binding of the soluble enzyme to the membrane surface of the hollow-fiber module. Under these bound conditions, CelB is essentially inactive. CelB is essentially inactive. Microbial contamination of the reactors did not occur during reaction times of up to 39 d, given that UHT skim milk and not pasteurized skim milk was used as the substrate.

Batch and Continuous Hydrolysis of Lactose Using P-Galactosidase Immobilized on Gelatin-Cmc

β-Galactosidase enzyme (E.C. 3.2.1.23) was immobilized onto a gelatin-carboxymethylcellulose carrier system by using cross-linker chromium acetate. A relative activity increase of 33% was achieved by adding carboxymethylcellulose to gelatin. The effect of operation temperature and pH on relative activity and reusability of the immobilized enzyme was investigated. A fluidized-bed reactor with a down flow mechanism was used for continuous process hydrolysis, and a constant glucose production rate was obtained.

Lactose hydrolysis in aqueous two-phase system by whole-cellβ-galactosidase of Kluyveromyces marxianus

Semicontinuous and continuous hydrolysis of lactose in aqueous two-phase systems (polyethylene glycol 20000/dextran 40) with whole-cellβ-galactosidase ofK. marxianus were studied. Both phase polymers had no effect onβ-galactosidase activity confined in cells. Good operational stability of the biocatalyst during 55 cycles of semicontinuous process was observed without appreciable decrease in product concentration. Continuous hydrolysis of lactose was performed in the stirred bioreactor, connected with the phase separator. The satisfactory degree of hydrolysis (between 82–88%) and volumetric productivity (21.6 g/l/h) were reached during 72 hours of continuous hydrolysis of 5% (w/w) lactose.

Lactose hydrolysis by β-galactosidase in aqueous two-phase systems

Lactose hydrolysis was studied in aqueous two-phase systems with β-galactosidases from bacteria ( Escherichia coli), yeast ( Saccharomyces fragilis), and fungi ( Aspergillus oryzae). Salts such as potasium phosphate, ammonium sulfate, and sodium citrate used at concentrations forming phase systems could severely inhibit β-galactosidase activity from yeast and inhibit E. coli enzyme activity to a lesser extent, but had no effect on fungal β-galactosidase activity. Polymers such as polyethylene glycol (PEG) and a modified starch (Reppal PES) could increase the enzyme activity toward hydrolyzing o- nitrophenyl-β- d-galactopyranoside . E. coli β-galactosidase showed a partition coefficient of 127 in a PEG/Reppal PES system to provide an efficient system for carrying out extractive hydrolysis of lactose. A. oryzae β-galactosidase showed a partition coefficient of 0.0042 in a PEG/sodium citrate system. Continuous hydrolysis of lactose in this system with a mixer-settler enzyme reactor could be operated for a period of 84 h with only 10% loss of enzyme activity. Slower reaction rates in two-phase systems than in buffer systems may arise from preferential distributions of both enzyme and inhibitory products into the same phase.

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%.

Yeast neutral lactase and fungal acid lactase immobilized to weakly basic anion exchange resins chemically modified by 2-carboxymethylamino-4,6-dichloro-s-triazine.

Both neutral lactase partially purified from yeast and acid lactase prepared from fungi were immobilized to weakly basic anion exchange resin (Duolite A 7) chemically modified by a bifunctional reagent, 2-carboxymethylamino-4, 6-dichloro-s-triazine. These immobilized enzymes could carry out a continuous hydrolysis of lactose in milk and whey, respectively, at appreciably high flow rates. The immobilized enzymes obtained were stable over broad ranges of pH and temperature and were protected from the attack by protease and bacteria.

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

AbstractImmobilized 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.