Microbial production,immobilization and applications of β‐D‐galactosidase

β‐D ‐Galactosidase (β‐D ‐galactoside galactohydrolase, E.C. 3.2.1.23), most commonly known as lactase, is one of the most important enzymes used in food processing, which catalyses the hydrolysis of lactose to its constituent monosaccharides, glucose and galactose. The enzyme has been isolated and purified from a wide range of microorganisms but most commonly used β‐D ‐galactosidases are derived from yeasts and fungal sources. The major difference between yeast and fungal enzyme is the optimum pH for lactose hydrolysis. The application of β‐D ‐galactosidase for lactose hydrolysis in milk and whey offers nutritional, technological and environmental applications to human life. In this review, the main emphasis has been given to elaborate the various techniques used in recent times for the production, purification, immobilization and applications of β‐D ‐galactosidase. Copyright © 2006 Society of Chemical Industry     

β‐Galactosidase immobilization into poly(hydroxyethyl methacrylate) membrane and performance in a continuous system

The activity of β‐galactosidase immobilized into a poly(2‐hydroxyethyl methacrylate) (pHEMA) membrane increased from 1.5 to 10.8 U/g pHEMA upon increase in enzyme loading. The K_m values for the free and the entrapped enzyme were found to be 0.26 and 0.81 mM, respectively. The optimum reaction temperatures for the free and the entrapped β‐galactosidase were both found to be 50°C. Similarly, the optimum reaction pH was 7.5 for both the free and the entrapped enzyme. The immobilized β‐galactosidase was characterized in a continuous system during lactose hydrolysis and the operational inactivation rate constant (k_iop) of the entrapped enzyme was found to be 3.1 × 10⁻⁵ min⁻¹. © 1999 John Wiley & Sons, Inc. J Appl Polym Sci 72: 1367–1373, 1999     

Production of β‐galactosidase from recombinant Saccharomyces cerevisiae grown on lactose

Improved productivity and costs reduction in fermentation processes may be attained by using flocculating cell cultures. The production of extracellular heterologous β‐galactosidase by recombinant flocculating Saccharomyces cerevisiae cells, expressing the lacA gene (coding for β‐galactosidase) of Aspergillus niger under the ADHI promotor and terminator in a bioreactor was studied. The effects of lactose concentration and yeast extract concentration on β‐galactosidase production in a semi‐synthetic medium were analysed. The extracellular β‐galactosidase activity increased linearly with increasing initial lactose concentrations (5–150 g dm^?3). β‐Galactosidase production also increased with increased yeast extract concentration. During the entire fermentation, no accumulation of the hydrolysed sugars, glucose and galactose, was observed. The catabolic repression of the recombinant strain when cultured in a medium containing equal amounts of glucose and galactose was confirmed. In complete anaerobiosis, the fermentation of lactose resulted in a very slow fermentation pattern with lower levels of β‐galactosidase activity. The bioreactor operation together with optimisation of culture conditions (lactose and yeast extract concentration) led to a 21‐fold increase in the extracellular β‐galactosidase activity produced when compared with preliminary Erlenmeyer fermentations. Copyright © 2004 Society of Chemical Industry     

Application of chitosan‐entrapped β‐galactosidase in a packed‐bed reactor system

The model enzyme β‐galactosidase was entrapped in chitosan gel beads and tested for hydrolytic activity and its potential for application in a packed‐bed reactor. The chitosan beads had an enzyme entrapment efficiency of 59% and retained 56% of the enzyme activity of the free enzyme. The Michaelis constant (K_m) was 0.0086 and 0.011 μmol/mL for the free and immobilized enzymes, respectively. The maximum velocity of the reaction (V_max) was 285.7 and 55.25 μmol mL^?1 min^?1 for the free and immobilized enzymes, respectively. In pH stability tests, the immobilized enzyme exhibited a greater range of pH stability and shifted to include a more acidic pH optimum, compared to that of the free enzyme. A 2.54 × 16.51‐cm tubular reactor was constructed to hold 300 mL of chitosan‐immobilized enzyme. A full‐factorial test design was implemented to test the effect of substrate flow (20 and 100 mL/min), concentration (0.0015 and 0.003M), and repeated use of the test bed on efficiency of the system. Parameters were analyzed using repeated‐measures analysis of variance. Flow (p < 0.05) and concentration (p < 0.05) significantly affected substrate conversion, as did the interaction progressing from Run 1 to Run 2 on a bed (p < 0.05). Reactor stability tests indicated that the packed‐bed reactor continued to convert substrate for more than 12 h with a minimal reduction in conversion efficiency. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 91: 1294–1299, 2004     

Effect of hydrolysis products and Mg2+, Mn2+ and Ca2+ ions on whey lactose hydrolysis and β‐galactosidase stability

BACKGROUND: Enzyme inhibition is one of the constraints of reactions catalysed by enzymes, and information is required on the inhibition mechanisms that affect the process yield. Therefore the aim of the present study was to investigate the effect of hydrolysis products and ions on the hydrolysis of lactose recovered from whey and enzyme inactivation during the reaction. The experiments were carried out in 250 mL of 25 mmol L^?1 phosphate buffer solution using β‐galactosidase from Kluyveromyces marxianus lactis in a batch reactor system. RESULTS : The degree of lactose hydrolysis (%) and the residual enzyme activity (%) in the presence and absence of lactose over time were investigated versus hydrolysate amount, glucose and galactose concentrations and Mg²⁺, Mn²⁺ and Ca²⁺ ion concentrations. The hydrolysis degree decreased with the addition of all hydrolysis products, as enzyme inhibition occurred. The residual enzyme activity increased with the addition of hydrolysate and glucose but decreased with the addition of galactose. It was observed that Mn²⁺ and Mg²⁺ ions activated the enzyme. It was also found that the hydrolysis degree was not affected by the addition of Mn²⁺ ions. On the other hand, the hydrolysis degree decreased with the addition of Ca²⁺ ions, as the enzyme was inactivated. CONCLUSION: Evaluation of the experimental data showed that both β‐galactosidase activity and lactose hydrolysis were affected by the addition of hydrolysis products and ions. Moreover, mathematical models proposed to predict the residual lactose concentration and residual enzyme activity were confirmed by the experimental results. Copyright © 2008 Society of Chemical Industry     

Purification of β‐Galactosidase by Ion Exchange Chromatography: Elution Optimization Using an Experimental Design

β‐Galactosidase is an enzyme industrially used to hydrolyze milk lactose, generating dairy products destined for people intolerant to this sugar. Its importance is due to its galactosiltransferase activity. The effects of elution pH and salt gradient volume were evaluated for purification of β‐galactosidase by ion exchange chromatography using an experimental design and response surface techniques. The best conditions for purification of β‐galactosidase were pH 5.5 with an elution volume of 62.8 mL, obtaining a yield of 85.5 % and a 12‐fold increase in the purification factor in a one‐step chromatography process. Purification of β‐galactosidase by application of a single stage of ion exchange and evaluation of the important process parameters using an experimental design provided good results in the recovery and purification factor that could subsequently be scaled up.     

Application of tris(hydroxymethyl)phosphine as a coupling agent for β‐galactosidase immobilized on chitosan to produce galactooligosaccharides

β‐Galactosidase was immobilized on chitosan using tris(hydroxymethyl)phosphine (THP) as a coupling agent to produce galactooligosaccharides (GOS) from lactose. Both the THP‐immobilized and the free enzymes were maximally achieved at pH 5.0 and the optimal temperature was 55 °C. The residual activities for the THP‐immobilized enzyme and the free enzyme were 75 and 25%, respectively, after being incubated in 0.1 mol dm^?3 sodium acetate buffer (pH 5.0) at 55 °C for 13 days. The formation of GOS was catalyzed by free and THP‐immobilized β‐galactosidase from lactose. The yield of GOS produced by the free enzyme from the lactose solution (36%, w/v) at 55 °C was 43% on a dry weight basis, which was similar to the 41% GOS yield produced by the THP‐immobilized enzyme system. Copyright © 2005 Society of Chemical Industry     

Mathematical modeling and simulation of steady state plug flow for lactose-lactase hydrolysis in fixed bed

A detailed mathematical model for evaluating lactose hydrolysis with immobilized enzyme in a packed bed tubular reactor is presented. The model accounts for axial and radial dispersion effects, chemical reaction and external mass transfer resistances but is void of significant internal diffusion resistances of the particles. The comprehensive model was then simplified to a plug flow model for lactose-lactose hydrolysis in fixed bed. The resulting plug flow model was solved by using Runge-Kutta-Gill method via employing different kinetics for lactose hydrolysis. The reliability of model simulations was tested using experimental data from a laboratory packed bed column, where the β-galactosidase of Kluyveromyces fragilis was immobilized on spherical chitosan beads. Comparison of the simulated results with experimental exit conversion show that the plug flow model incorporating Michaelis-Menten kinetics with competitive product (galactose) inhibition are appropriate to interpret the experimental results and simulate the process of lactose hydrolysis in a fixed bed when the mass transfer resistance was reduced by a factor of 34.5.     

Hydrolysis of Penicillin G by Penicillin G Acylase Immobilized on Chitosan Microbeads in Different Reactor Systems

The kinetic parameters for penicillin G hydrolysis in systems with penicillin G acylase from Escherichia coli (free and immobilized on activated chitosan microbeads produced by electrostatic extrusion) were determined. The obtained kinetic results indicated that both systems (free and immobilized) are inhibited by high concentrations of the substrate (penicillin G) as well as by products of the reaction (6‐aminopenicillanic acid and phenylacetic acid). The microbeads appeared convenient for penicillin G acylase immobilization reducing negative inhibitory effects. The hydrolysis was also investigated in a packed bed reactor. The derived kinetic model predicted good hydrolysis rates in the reactor while the system with recirculation of the reaction mixture proved to be a potentially favorable solution providing operation at low shear stresses and possibly higher hydrolysis rates than in the packed bed reactor alone.     

Galacto-oligosaccharide production with immobilized β-galactosidase in a packed-bed reactor vs. free β-galactosidase in a batch reactor

We report here that the usage of immobilized enzyme in a continuous packed bed reactor (PBR) can be a good alternative for GOS production instead of the traditional use of free enzyme in a batch reactor. The carbohydrate composition of the product of the PBR with immobilized enzyme was comparable to that of the batch reactor with free enzyme. The stability of the immobilized enzyme at a lactose concentration of 38% (w/v) and at 50 °C was very high: the half-life time of the immobilized enzyme was approximately 90 days. The enzymatic productivity of GOS production using immobilized enzyme in a PBR can be more than six times higher than that of GOS production with free enzyme in a batch reactor. Besides, when aiming for an equal volumetric productivity to the batch process in designing a PBR, the volume of the PBR can be much smaller than that of the batch reactor, depending on the enzyme dosage and the run time of a single batch.     

Development of a new two‐enzyme biosensor based on poly(pyrrole‐co‐3,4‐ethylenedioxythiophene) for lactose determination in milk

A new lactose biosensor was developed by preparing a suitable copolymer of polypyrrole and poly(3,4‐ethylenedioxythiophene) synthesized using the electropolymerization method. Pyrrole and 3,4‐ethylenedioxythiophene monomers were deposited in the presence of sodium dodecylbenzene sulphonic acid on a platinum disc electrode, which was used as the working electrode. The sensor is based on the serial reactions of β‐galactosidase and galactose oxidase immobilized on a copolymer‐modified platinum disc electrode. Successful synthesis of the enzyme‐immobilized copolymer was confirmed by FT‐IR spectrometry, SEM, and electrochemical analysis. The response of the enzyme electrode to lactose was determined by cyclic voltammetry at + 0.40 V. The response time of the biosensor was found to be from 8 to 10 s, and the upper limit of the linear working portion was found to be at a lactose concentration of 2.30 mM with a detection limit of 1.4 × 10^?5 M. The apparent Michaelis–Menten constant was found to be 0.65 mM of lactose. The effects of interferents were also investigated. Lactose concentrations determined by the biosensor were in good agreement with those measured by the reference methods. Our results show that the developed biosensor has a significant potential to the determination of lactose concentration in milk. POLYM. ENG. SCI., 58:839–848, 2018. © 2017 Society of Plastics Engineers     

Stability of β‐galactosidase immobilized on composite microspheres of artemisia seed gum and chitosan

In this work, composite microspheres were prepared by using artemisia seed gum and chitosan as a source. The composite microspheres have activated aldehyde groups by using glutaraldehyde. β‐Galactosidase was covalently bound on these activated microspheres. The properties of the immobilized enzyme were investigated and compared with those of the free enzyme, for which o‐nitrophenol β‐D ‐galactopyranoside (ONPG) was chosen as a substrate. The results showed that the pH and thermal stability of the immobilized β‐galactosidase were higher than those of the soluble one. Apart from these, the Michaelis constant K_m was evaluated for the immobilized β‐galactosidase and the soluble enzyme. The immobilized β‐galactosidase exhibited better environmental adaptability and reusability than the soluble one. POLYM. COMPOS., 29:9–14, 2008. © 2007 Society of Plastics Engineers     

Covalent immobilization of β‐galactosidase onto electrospun nanofibers of poly (AN‐co‐MMA) copolymer

Immobilization of β‐galactosidase in poly (acrylonitrile‐co‐methyl methacrylate) poly (AN‐co‐MMA) Nanofibers was studied by electrospinning, and a spacer‐arm i.e., (Polyethyleneimine (PEI)) was covalently attached by the reaction of carbonyl groups of Poly (AN‐co‐MMA) nanofibers. β‐galactosidase was then covalently immobilized through the spacer‐arm of the Poly (AN‐co‐MMA) nanofibers by using glutaraldehyde (GA) as a coupling agent. Nanofibers mode of interaction was proven by FTIR and thermal gravimetric analysis and supported by morphological changes recognized through SEM examination. Factors affecting the modification process such as PEI concentration, reaction time, and reaction temperature have been studied. Its influence on the amount of coupled PEI was consequently correlated to the changes of the catalytic activity and the retained activity of immobilized enzyme, the main parameters judging the success of the immobilization process. Evidences of Poly (AN‐co‐MMA) nanofibers modification were extracted from morphological changes recognized through SEM examination. The maximum activity (V_max) and michaelis constant (K_m) of immobilized enzyme were found to be 8.8 μmole/min mg protein and 236.7 mM, respectively. Stabilities of the immobilized β‐galactosidase were obviously improved. The optimum temperature for β‐galactosidase immobilized on the spacer‐arm attached nanofiber was 5°C higher than that of the free enzyme and was also significantly broader. The immobilized β‐galactosidase had better resistance to temperature inactivation than did the free form. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013     

Preparation,properties and uses of polymer-enzyme conjugates

Enzymes can be immobilized by gel entrapment, by microencapsulation, by physical or ionic adsorption, by covalent binding to inorganic or organic carriers, or by whole cell immobilization. Of particular interest is the large number of chemical reactions developed for the covalent binding of enzymes via their nonessential functional groups to inorganic carriers such as glass, ceramics and iron, to natural polymers such as cellulose and Sepharose, and to synthetic polymers such as nylon, polyacrylamide, and other vinyl polymers and copolymers possessing reactive chemical groups. The stability of certain enzymes is markedly increased on their immobilization. It was thus possible to transform the biologically active polymer derivatives into active enzyme beads, enzyme capsules, enzyme columns and enzyme membranes and these enabled the construction of enzyme reactors such as the batch-stirred tank reactors, the continuous packed bed reactors, and fluidized bed reactors. So far mainly immobilized hydralases and isomerases are being used in industry on a large scale. It seems likely, however, that once adequate techniques become available for cofactor recycling, the use of immobilized enzymes will be extended to other organic reactions, particularly those involving stereospecific synthesis of simple or complex organic molecules. Among the industrial processes in which immobilized enzymes are being used, it is worth mentioning the industrial-scale continuous production of fructose enriched syrup from glucose by immobilized glucose-isomerase, the batch process for the production of 6-aminopenicillanic acid (6-APA) from penicillin G with the aid of immobilized penicillin amidase; the production of aspartame from aspartic acid and phenylalanine by immobilized thermoase; the large scale production of optically active amino acids with immobilized amino acid acylase; and the large scale production and application of immobilized lactase for the hydrolysis of lactose. The recently developed process for acrylamide production using immobilized nitrilase containing microbial cells should also be referred to. The successful use of an NAD-polyethylene glycol conjugate (NAD-PEG) as a nondialyzable water-soluble coenzyme derivative in the enzymic synthesis of leucine from α-ketoisocaproic acid and ammonia, in a membrane-enclosed reactor containing L-leucine dehydrogenase, NAD-PEG, formate and formate dehydrogenase, illustrates the new possibilities opened up by making use of cofactor-polymer conjugates. The use of enzyme-polymer conjugates in analytical and clinical is also illustrated.     

Production of optically pure L‐valine in fluidized and packed bed reactors with immobilized L‐aminoacylase

A process to obtain L ‐valine has been developed using fluidized and packed bed reactors with L ‐aminoacylase (from hog kidney) immobilized by covalent binding. L ‐Valine production using the immobilized derivative of L ‐aminoacylase in fluidized and packed bed reactors was studied at three different substrate concentrations and two different flow rates. Higher productions were obtained in the packed bed reactor in all cases. The different solubilities of L ‐valine and acetyl‐D ‐valine in ethanol were used to purify L ‐amino acid from the reactor effluents. The amount of added ethanol did not influence the separation yields, although the purity of L ‐valine was strongly affected by this parameter. The last step involved was racemization of the unhydrolyzed acetyl‐D ‐valine, which was then used as substrate in a new reaction cycle. © 1999 Society of Chemical Industry     

Influence of TiO2‐Layer Thickness of Spray‐Coated Glass Beads on Their Photocatalytic Performance

TiO₂ is a suitable catalyst for potential photocatalytic processes, e.g., in wastewater treatment. For a technical realization of such processes, the application of immobilized TiO₂ in a continuous process would be desirable. However, since UV radiation has a limited penetration depth into a packed bed of pure TiO₂, supporting it on UV‐transparent glass beads offers the possibility to implement continuous photocatalytic processes in a fixed‐bed reactor. Considering this fact, glass beads were coated with TiO₂ powder in a fluidized‐bed reactor. The coated glass beads with varying TiO₂ layer thickness were tested in the photocatalytic degradation of methylene blue, and the influence of an addition of methyl cellulose during the coating process on the photocatalytic performance was investigated.     

Aspergillus oryzae Lipase-Catalyzed Synthesis of Dioleoyl; Palmitoyl-Rich Triacylglycerols in Two Reactors

Dioleoyl; palmitoyl‐rich triacylglycerols (OPO‐rich TAG) were synthesized through Aspergillus oryzae lipase (AOL)‐catalyzed acidolysis of palm stearin with commercial oleic acid by a one‐step process in a stirred tank reactor and continuous packed bed reactor to evaluate the feasibility of using immobilized AOL. AOL was found to be valuable for the synthesis of OPO‐rich TAG when compared with commercial lipase from Thermomyces lanuginose (Lipozyme^® TL IM; Novozymes A/S, Bagsvaerd, Denmark). The C52 (triglycerides with a carbon number of 52, stands for OPO, OPL, LPL and their isomers) content of AOL was higher (45.65 %), and the intensity of treatment (IOT: lipase amount × reaction time/TAG amount) of AOL was just 6.25 % of that of Lipozyme^® TL IM under similar reaction conditions in the stirred tank reactor. Response surface methodology were used to optimize the reaction conditions of the AOL‐catalyzed acidolysis is reaction in the packed bed reactor. The optimal point for the set of experimental conditions generated were as follows: residence time 3.0 h; temperature 62.09 °C; substrate molar ratio 7.13 mol/mol. The highest C52 content obtained was 48.60 ± 2.36 %, with 57.46 ± 1.72 % total palmitic acid at the sn‐2 position and 74.21 ± 2.45 % oleic acid at the sn‐1,3 positions. The half‐life of AOL was 24 h in the stirred tank reactor and 140 h in the packed bed reactor. The immobilized AOL achieved similar conversion and selectivity to commercial lipases for the catalyzed synthesis of OPO‐rich TAG and may offer a cheaper alternative.     

Production of L‐methionine by immobilized pellets of Aspergillus oryzae in a packed bed reactor

Production of L ‐methionine by immobilized pellets of Aspergillus oryzae in a packed bed reactor was investigated. Based on the determination of relative enzymatic activity in the immobilized pellets, the optimum pH and temperature for the resolution reaction were 8.0 and 60 °C, respectively. The effects of substrate concentration on the resolution reaction were also investigated and the kinetic constants (K_m and V_m) of immobilized pellets were found to be 7.99 mmol dm^?3 and 1.38 mmol dm^?3 h^?1, respectively. The maximum substrate concentration for the resolution reaction without inhibition was 0.2 mol dm^?3. The L ‐methionine conversion rate reached 94% and 78% when substrate concentrations were 0.2 and 0.4 mol dm^?3, respectively, at a flow rate of 7.5 cm³ h^?1 using the small‐scale packed bed reactor developed. The half‐life of the L ‐aminoacylase in immobilized pellets was 70 days in continuous operation. All the results obtained in this paper exhibit a practical potential of using immobilized pellets of Aspergillus oryzae in the production of L ‐methionine. © 2002 Society of Chemical Industry     

Purification of Glucose Oxidase and β‐Galactosidase by Partitioning in a PEG‐Salt Aqueous Two‐Phase System in the Presence of PEG‐Derivatives

Abstract

Purification of glucose oxidase from Aspergillus niger and that of β‐galactosidase from Kluyveromyces lactis have been attempted using poly(ethylene glycol) (PEG)‐sodium sulfate aqueous two phase system (ATPS) in the presence of PEG‐derivatives, i.e. PEG‐Coomassie brilliant blue G‐250 and PEG‐benzoate, PEG‐palmitate and PEG‐TMA, respectively. The enzymes showed poor partitioning towards the PEG phase in comparison with other proteins in ATPS containing no ligands. Selective partitioning of other proteins was observed towards the PEG phase in the presence of PEG‐benzoate and PEG‐palmitate enriching β‐galactosidase in the salt phase whereas in the case of glucose oxidase, PEG‐Coomassie brilliant blue G‐250 derivative worked as a better affinity ligand for other proteins. A 19‐fold purification was obtained with the PEG dye derivative after 5 stage cross extractions with 80% recovery of glucose oxidase and an enrichment factor upto ～7 for β‐galactosidase with the PEG‐TMA derivative. The interaction of PEG‐benzoate and PEG‐TMA ligands with the active site of β‐galactosidase has been evaluated by molecular modeling. The effect of the molecular weight of glucose oxidase on its partitioning was confirmed as the molecular simulation shows strong affinity interaction of PEG‐glucoside with the enzyme.