Hydrolysis of β-galactosides using polymer-entrapped lactase. A study towards producing lactose-free milk

A yeast lactase, Maxilact, was immobilized in crosslinked polyacrylamide using a bead-polymerization technique. The polymer beads obtained, containing the entrapped enzyme, were used for the preparation of lactose-free milk. The binding yield of the enzyme and residual enzymic activity in the “enzyme beads” were studied as a function of the amounts of monomeric acrylamide and cysteine and bovine serum albumin present as protecting agents in the monomer-enzyme solution prior to polymerization. A maximum of about 75% of the enzyme could be immobilized using a 20% (w/v) solution of acrylamide plus N, N′-methylenebis-acrylamide, whereas the highest activity quotient (bound to free) of about 60% was observed on using a 25% solution. The presence of cysteine increased the activity by up to 30% and that of serum albumin up to about 15%.     

Magnetic chitosan beads for covalent immobilization of nucleoside 2′-deoxyribosyltransferase: application in nucleoside analogues synthesis

Cross-linked magnetic chitosan beads were prepared in presence of epichlorohydrin under alkaline conditions, and subsequently incubated with glutaraldehyde in order to obtain an activated support for covalent attachment of nucleoside 2′-deoxyribosyltransferase from Lactobacillus reuteri (LrNDT). Changing the amount of magnetite (Fe₃O₄) and epichlorohydrin (EPI) led to different macroscopic beads to be used as supports for enzyme immobilization, whose morphology and properties were characterized by scanning electron microscopy, spin electron resonance (ESR), and vibrating sample magnetometry (VSM). Once activated with glutaraldehyde, the best support was chosen after evaluation of immobilization yield and product yield in the synthesis of thymidine from 2′-deoxyuridine and thymine. In addition, optimal conditions for highest activity of immobilized LrNDT on magnetic chitosan were determined by response surface methodology (RSM). Immobilized biocatalyst retained 50 % of its maximal activity after 56.3 h at 60 °C, whereas 100 % activity was observed after storage at 40 °C for 144 h. This novel immobilized biocatalyst has been successfully employed in the enzymatic synthesis of 2′-deoxyribonucleoside analogues as well as arabinosyl-nucleosides such as vidarabine (ara-A) and cytarabine (ara-C). Furthermore, this is the first report which describes the enzymatic synthesis of these arabinosyl-nucleosides catalyzed by an immobilized nucleoside 2′-deoxyribosyltransferase. Finally, the attached enzyme to magnetic chitosan beads could be easily recovered and recycled for 30 consecutive batch reactions with negligible loss of catalytic activity in the synthesis of 2,6-diaminopurine-2′-deoxyriboside and 5-trifluorothymidine.     

Hydrolysis of lactose in whey permeate by immobilized β-galactosidase from Penicillium notatum

A new low-cost β-galactosidase (lactase) preparation for whey permeate saccharification was developed and characterized. A biocatalyst with a lactase activity of 10 U/mg, a low transgalactosylase activity and a protein content of 0.22 mg protein/mg was obtained from a fermenter culture of the fungus Penicillium notatum. Factors influencing the enzymatic hydrolysis of lactose, such as reaction time, pH, temperature and enzyme and substrate concentration were standardized to maximize sugar yield from whey permeate. Thus, a 98.1% conversion of 5% lactose in whey permeate to sweet (glucose-galactose) syrup was reached in 48 h using 650 β-galactosidase units/g hydrolyzed substrate. After the immobilization of the acid β-galactosidase from Penicillium notatum on silanized porous glass modified by glutaraldehyde binding, more than 90% of the activity was retained. The marked shifts in the pH value (from 4.0 to 5.0) and optimum temperatures (from 50°C to 60°C) of the solid-phase enzyme were observed and discussed. The immobilized preparation showed high catalytic activity and stability at wider pH and temperature ranges than those of the free enzyme, and under the best operating conditions (lactose, 5%; β-galactosidase, 610–650 U/g lactose; pH 5.0; temperature 55°C), a high efficiency of lactose saccharification (84–88%) in whey permeate was achieved when lactolysis was performed both in a batch process and in a recycling packed-bed bioreactor. It seems that the promising results obtained during the assays performed on a laboratory scale make this immobilizate a new and very viable preparation of β-galactosidase for application in the processing of whey and whey permeates.     

Urease Immobilization on Arylamine Glass Beads and its Characterization

Jack bean urease has been immobilized on arylamine glass beads (200–400 mesh size, 75–100 Å pore size) and its properties compared with soluble enzyme. The binding of urease was 13.71 mg per gram beads. The K_m for soluble and immobilized urease for urea was 4.20 mM and 8.81 mM, respectively. V_max values of urease decreased from 200 to 43.48 μmol of ammonia formed per min per mg protein at 37°C on immobilization. Both pH and buffer ions influenced the activities of soluble as well as immobilized urease. Soluble urease exhibited pH optima at 5.5 and 8.0. However, immobilized urease showed one additional pH optimum at 6.5. In comparison to phosphate buffer, citrate buffer was inhibitory to urease activity. Immobilization of urease on arylamine glass beads resulted in improved thermal, storage and operational stability. Because of inertness of support and stability of immobilized urease, the preparation can find applications in ‘artificial kidney’ and urea estimation in biological fluids viz., blood, milk etc.     

Biosynthesis of sucrose-6-acetate catalyzed by surfactant-coated Candida rugosa lipase immobilized on sol–gel supports

Sucrose-6-acetate is an important intermediate in the preparation of sucralose (a finest sweetener). In our study, Candida rugosa lipase coated with surfactant was firstly immobilized on sol–gel supports. Then, the immobilized enzyme was used in the regioselective synthesis of sucrose-6-acetate by transesterification of sucrose and vinyl acetate. The screening results revealed that Tween 80 was an ideal surfactant to coat lipase immobilized in sol–gel and exhibited the highest yield of sucrose-6-acetate. Other factors that influenced the yield during the preparation process were also studied. Under optimal conditions, the yield of sucrose-6-acetate could reach up to 78.68 %, while free lipase was easily inactivated in polar solvent. Thermal and operational stabilities were also improved significantly. Surfactant-coated lipase immobilized in sol–gel remained stable when the temperature was higher than 60 °C. Moreover, they could maintain high catalytic activity after six recycles. This strategy is economical, convenient and promising for the food industry.     

Fibrous polymer-grafted chitosan/clay composite beads as a carrier for immobilization of papain and its usability for mercury elimination

Papain, which is an industrially important enzyme, has been immobilized on fibrous polymer-modified composite beads, namely poly(methacrylic acid)-grafted chitosan/clay. Characterization studies have been done using FTIR and SEM analysis. Operating parameters such as pH and initial concentration of papain have been varied to obtain the finest papain immobilized polymer-modified composite beads. The immobilization capacity of composite beads has been determined as 34.47 ± 1.18 (n = 3) mg/g. The proteolytic activity of immobilized papain was operated using bovine serum albumin (BSA) and maximum velocity (V _max) and Michaelis–Menten constant (K_m) values of the free and immobilized enzymes were determined using Lineweaver–Burk and Eadie–Hofstee equations. Usability of papain immobilized polymer-modified composite beads as adsorbents for the elimination of mercury was investigated. The maximum removal capacity of PIPMC beads has been found to be 4.88 ± 0.21 mg Hg/g when the initial metal concentration and weight of polymer-modified composite beads were 50 mg/L and 0.04 g at pH 7, respectively. Mercury removal performance of the papain immobilized polymer-modified composite beads was investigated in conjunction with Cu (II), Zn (II) and Cd (II) ions. The mercury adsorption capacity of papain immobilized polymer-modified composite beads was a slight reduction from 1.15 to 0.89 mg/g in presence of multiple metal salts.     

Bacterial cellulose-chitosan composite hydrogel beads for enzyme immobilization

In this work, we report the preparation of bacterial cellulose (BC)-chitosan composite hydrogel beads by co-dissolution of BC and chitosan in 1-ethyl-3-methylimidazolium acetate and subsequent reconstitution with distilled water. The BC-chitosan hydrogel beads were used as enzyme supports for immobilizing Candida rugosa lipase by physical adsorption and covalent cross-linking. BC-chitosan hydrogel beads immobilized lipase more efficiently than microcrystalline cellulose (MCC)-chitosan hydrogel beads. The amount of protein adsorbed onto BCchitosan beads was 3.9 times higher than that adsorbed onto MCC-chitosan beads, and the catalytic activity of lipase was 1.9 times higher on the BC-chitosan beads. The lipase showed the highest thermal and operational stability when covalently cross-linked on BC-chitosan hydrogel beads. The half-life time of the lipase cross-linked on BC-chitosan bead at 60°C was 22.7 times higher than that of free lipase. Owing to their inherent biocompatibility and biodegradability, the BC-chitosan composite hydrogel beads described here could be used to immobilize proteins for various biomedical, environmental, and biocatalytic applications.     

Use of a glass bead-containing liquid medium for efficient production of a soil-free culture with polychlorinated biphenyl-dechlorination activity

We established a soil-free culture capable of dechlorinating polychlorinated biphenyls (PCBs) in Kanechlor-300 and Kanechlor-400 by establishing a PCB-dechlorinating soil culture in liquid medium containing 0.5 mm glass beads. PCB-dechlorination activity in liquid cultures with glass beads appeared to depend on the size of the glass beads, and soil-free cultures with 0.05-, 1.0- or 2.0 mm glass beads did not dechlorinate PCBs. Soil-free culture without glass beads also failed to dechlorinate PCBs. The soil-free culture containing 0.5 mm glass beads dechlorinated 42.6 ± 12.0 mol% in total PCBs. This soil-free culture was more effective than soil culture for dechlorinating PCBs ranging from dichlorinated PCBs to tetrachlorinated PCBs. Clone analysis of the 16S rRNA gene sequences showed that one of the predominant groups of microorganisms in the soil-free culture comprised heat-tolerant and spore-forming bacteria from the phylum Firmicutes. Heat treatment (100 °C, 10 min) did not destroy the PCB-dechlorination activity of the soil-free culture with glass beads. These results suggest that unknown species of the phylum Firmicutes were involved in PCB dechlorination in the soil-free culture. In this study, we succeeded in using a liquid medium containing glass beads as an inorganic soil substitute and showed that such a medium enhances PCB-dechlorination activity. Our study provides valuable information for developing PCB-bioremediation techniques using dechlorinating bacteria in anoxic contaminated soils and sediments.     

Correlation of G/A -22018 single-nucleotide polymorphism with lactase activity and its usefulness in improving the diagnosis of adult-type hypolactasia among North Indian children

Adult-type hypolactasia (AtH or lactase non-persistence) is the physiological decline in lactase activity that manifests in majority of the world’s population after weaning. Recently, various single-nucleotide polymorphisms (SNPs) upstream of lactase gene (LCT) have been suggested to be associated with AtH or the lactase persistent trait in different human populations. C/T -13910 SNP was found be completely associated with AtH in Finnish population, and G/A -22018 SNP was found to be strongly, but not completely, associated with AtH. The aim of this study was to correlate G/A -22018 SNP with intestinal lactase activity in North Indian children. These children were also genotyped for C/T -13910 SNP. We also examined the differences in milk consumption and milk-related clinical symptoms in children with different genotypes of G/A -22018 and C/T -13910 SNPs. Intestinal biopsies were obtained from 231 children aged 2–16 years undergoing routine endoscopy for various abdominal complaints. The biopsies were assayed for lactase, sucrase, and maltase activities and genotyped for G/A -22018 and C/T -13910 SNPs using restriction fragment length polymorphism and DNA sequencing analysis. There was a significant correlation between lactase activity and different genotypes of G/A -22018 SNP. Children with G/G -22018 genotype had low lactase activity. With a reference value of <10 U/g protein (lactase activity) to be indicative of AtH, the sensitivity and specificity of genetic test based on G/A -22018 SNP was 94.4 and 94.1 %, respectively. Furthermore, the consumption of milk was lower in children with G/G -22018 genotype. Flatulence was the only symptom significantly more frequent among the children with G/G -22018 genotype compared to those with G/A and A/A -22018 genotypes. However, most of the children with G/G -22018 genotype seem to tolerate small amounts of milk without any significant difference in gastrointestinal symptoms from those with G/A and A/A -22018 genotypes.     

Immobilization and characterization of cellulase on concanavalin A (Con A)-layered calcium alginate beads

Cellulase has been immobilized on hybrid concanavalin A (Con A)-layered calcium alginate–starch beads. Immobilized cellulase retained about 82% of its activity. Con A was extracted from jack bean and the obtained crude protein was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The immobilized beads showed high mechanical and storage stability; immobilized cellulase retained 100% and 85% activity at 4°C and 30°C, respectively, over one month. The immobilized cellulase retained about 70% of its activity after five cycles of use. The immobilized cellulase retained 70% activity after 120-min exposure to 60°C, whereas the soluble form only retained about 20%, showing that immobilization improved thermal stability. Surface morphology and elemental analysis of immobilized cellulase were examined using scanning electron microscope equipped with energy-dispersive X-ray. Based on the enzyme stability and reuse, this method of immobilization is both convenient and cheap.     

Immobilization of nitrilase on bioinspired silica for efficient synthesis of 2-hydroxy-4-(methylthio) butanoic acid from 2-hydroxy-4-(methylthio) butanenitrile

In this paper, a simple and effective method using sodium metasilicate as precursor and amine as additive was first reported to immobilize recombinant nitrilase, for efficient production of 2-hydroxy-4-(methylthio) butanoic acid from 2-hydroxy-4-(methylthio) butanenitrile. High immobilization recovery of enzyme activity (above 90 %) was achieved. The immobilized enzyme displayed better thermal stability, pH stability and shelf life compared to free nitrilase. Moreover, it showed excellent reusability and could be recycled up to 16 batches without significant loss in activity. 200 mM 2-hydroxy-4-(methylthio) butanenitrile was completely converted by the immobilized enzyme within 30 min, and the accumulation amount of 2-hydroxy-4-(methylthio) butanoic acid reached 130 mmol/g of immobilized beads after 16 batches. These encouraging results demonstrated the efficiency of the new technology for nitrilase immobilization, which has great potential in preparation of 2-hydroxy-4-(methylthio) butanoic acid.     

Bioconversion of phosphatidylcholine to phosphatidylserine using immobilized enzyme mini-columns

Phospholipase-d, immobilized on controlled porosity glass beads, was used for the preparation of phosphatidylserine. The synthesis of phosphatidylserine was monitored using immobilized mini-reactors of choline oxidase and serine dehydratase isolated from rat liver. The immobilized enzymes showed good stability, and no deterioration in enzyme activity was recorded after use for 4 months.     

Stabilization of kappa-carrageenan gel with polymeric amines: Use of immobilized cells as biocatalysts at elevated temperatures

Spherical beads of kappa-carrageenan containing entrapped cells were prepared in a two-step process. First, the beads were formed by dispersing a warm carrageenan cell suspension into stirring oil. After cooling (gelation) the beads were cured by treatment with amines. Ten amines of various sizes and structures were tested. We evaluated the mechanical strength and the applicability of aminetreated gels as immobilization matrices. The results of critical compression tests indicate that linear and branched polyethylenimines (PEI) are both good curing agents. PEI treated carrageenan beads also exhibited superior resistance to heat and abrasion. Furthermore, PEI polymers were demonstrated to be effective in stabilizing the lactase activity of the free and immobilized Bacillus stearothermophilus cells. The immobilized cell preparations of Saccharomyces cerevisiae, B. stearothermophilus, and Flavobacterium sp. were treated with branched PEI and evaluated for the activity of invertase (EC 3.2.1.26), lactase (EC 3.2.1.23), and glucose isomerase (EC 5.3.1.18), respectively, in a packed bed reactor at 60 degrees C. The apparent half-lives were 108, 39, and 64 days, respectively.     

Evaluation of the catalytic specificity,biochemical properties,and milk clotting abilities of an aspartic peptidase from <Emphasis Type="Italic">Rhizomucor miehei</Emphasis>

In this study, we detail the specificity of an aspartic peptidase from Rhizomucor miehei and evaluate the effects of this peptidase on clotting milk using the peptide sequence of k-casein (Abz-LSFMAIQ-EDDnp) and milk powder. Molecular mass of the peptidase was estimated at 37 kDa, and optimum activity was achieved at pH 5.5 and 55 °C. The peptidase was stable at pH values ranging from 3 to 5 and temperatures of up 45 °C for 60 min. Dramatic reductions in proteolytic activity were observed with exposure to sodium dodecyl sulfate, and aluminum and copper (II) chloride. Peptidase was inhibited by pepstatin A, and mass spectrometry analysis identified four peptide fragments (TWSISYGDGSSASGILAK, ASNGGGGEYIFGGYDSTK, GSLTTVPIDNSR, and GWWGITVDRA), similar to rhizopuspepsin. The analysis of catalytic specificity showed that the coagulant activity of the peptidase was higher than the proteolytic activity and that there was a preference for aromatic, basic, and nonpolar amino acids, particularly methionine, with specific cleavage of the peptide bond between phenylalanine and methionine. Thus, this peptidase may function as an important alternative enzyme in milk clotting during the preparation of cheese.     

Improving of Catalase Stability Properties by Encapsulation in Alginate/Fe3O4 Magnetic Composite Beads for Enzymatic Removal of H2O2

The aim of this study was enhancing of stability properties of catalase enzyme by encapsulation in alginate/nanomagnetic beads. Amounts of carrier (10–100 mg) and enzyme concentrations (0.25–1.5 mg/mL) were analyzed to optimize immobilization conditions. Also, the optimum temperature (25–50°C), optimum pH (3.0–8.0), kinetic parameters, thermal stability (20–70°C), pH stability (4.0–9.0) operational stability (0–390 min), and reusability were investigated for characterization of the immobilized catalase system. The optimum pH levels of both free and immobilized catalase were 7.0. At the thermal stability studies, the magnetic catalase beads protected 90% activity, while free catalase maintained only 10% activity at 70°C. The thermal profile of magnetic catalase beads was spread over a large area. Similarly, this system indicated the improving of the pH stability. The reusability, which is especially important for industrial applications, was also determined. Thus, the activity analysis was done 50 times in succession. Catalase encapsulated magnetic alginate beads protected 83% activity after 50 cycles.     

Evaluation of immobilized lipases on poly-hydroxybutyrate beads to catalyze biodiesel synthesis

Five microbial lipase preparations from several sources were immobilized by hydrophobic adsorption on small or large poly-hydroxybutyrate (PHB) beads and the effect of the support particle size on the biocatalyst activity was assessed in the hydrolysis of olive oil, esterification of butyric acid with butanol and transesterification of babassu oil (Orbignya sp.) with ethanol. The catalytic activity of the immobilized lipases in both olive oil hydrolysis and biodiesel synthesis was influenced by the particle size of PHB and lipase source. In the esterification reaction such influence was not observed. Geobacillus thermocatenulatus lipase (BTL2) was considered to be inadequate to catalyze biodiesel synthesis, but displayed high esterification activity. Butyl butyrate synthesis catalyzed by BTL2 immobilized on small PHB beads gave the highest yield (≈90 mmol L(-1)). In biodiesel synthesis, the catalytic activity of the immobilized lipases was significantly increased in comparison to the free lipases. Full conversion of babassu oil into ethyl esters was achieved at 72 h in the presence of Pseudozyma antarctica type B (CALB), Thermomyces lanuginosus lipase (Lipex(?) 100 L) immobilized on either small or large PHB beads and Pseudomonas fluorescens (PFL) immobilized on large PHB beads. The latter preparation presented the highest productivity (40.9 mg of ethyl esters mg(-1) immobilized protein h(-1)).     

Immobilization of tomato (Lycopersicon esculentum) pectinmethylesterase in calcium alginate beads and its application in fruit juice clarification

Clarity of fruit juices is desirable to maintain an aesthetically pleasing quality and international standards. The most commonly used enzymes in juice industries are pectinases. A partially-purified pectinmethylesterase from tomato was entrapped in calcium alginate beads and used for juice clarification. The activity yield was maximum at 1 % (w/v) CaCl₂ and 2.5 % (w/v) alginate. The immobilized enzyme retained ~55 % of its initial activity (5.7 × 10^?2 units) after more than ten successive batch reactions. The K_m, pH and temperature optima were increased after immobilization. The most effective clarification of fruit juice (%T₆₂₀ ~60 %) by the immobilized enzyme was at 4 °C with a holding time of 20 min. The viscosity dropped by 56 % and the filterability increased by 260 %. The juice remains clear after 2 months of storage at 4 °C.     

Preparation of immobilized lactase. Continued studies on the enzymatic hydrolysis of lactose

A soluble fungal lactase (lactase-W) of greater activity that the previously available fungal lactase (lactase-M) has been covalently coupled to ZrO₂-coated porous glass particles and 1 mm diameter porous TiO₂ particles. The immobilized lactase-W appears to give results similar to the lactase-M except for the operational half-life. At 30°C the half-life of the lactase-M appears to exceed that of the lactase-W by approximately 100 days under operational conditions.     

Enzymatic synthesis of <Emphasis Type="Italic">S</Emphasis>-adenosylhomocysteine: immobilization of recombinant <Emphasis Type="Italic">S</Emphasis>-adenosylhomocysteine hydrolase from <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> (ATCC 13032)

Recombinant S-adenosylhomocysteine hydrolase from Corynebacterium glutamicum (CgSAHase) was covalently bound to Eupergit® C. The maximum yield of bound protein was 91% and the catalytic efficiency was 96.9%. When the kinetic results for the immobilized enzyme were compared with those for the soluble enzyme, no decrease in the catalytic efficiency of the former was detected. Both soluble and immobilized enzymes showed similar optimum pH and temperature ranges. The reuse of immobilized CgSAHase caused a loss of synthetic activity due to NAD⁺ release, although the binding to the support was sufficiently strong for up to 5 cycles with 95% conversion efficiency. The immobilized enzyme was incubated every 3 cycles with 100 μM NAD⁺ to recover the loss of activity after 5 cycles. This maintained the activity for another 50 cycles. The purification of S-adenosylhomocysteine (SAH) provided an overall yield of 76% and 98% purity as determined by HPLC and NMR analyses. The results indicate the suitability of immobilized CgSAHase for synthesizing SAH and other important S-nucleosidylhomocysteine.     

Cobra venom phospholipase A2 immobilized to porocus glass beads.

Phospholipase A₂ (EC 3.1.1.4) from cobra venom (Naja naja naja) has been covalently immobilized to aryl amine porous glass beads by diazo coupling. The attachment of the enzyme to the glass beads is apparently through tyrosine. The activity of the immobilized enzyme toward phospholipid substrate has been monitored using the Triton X-100/phospholipid mixed micelle assay system. The activity of the immobilized phospholipase A₂ toward phosphatidylcholine is about 160 μmol min^?1 ml^?1 of glass beads, and the specific activity is about 13 μmol min^?1 mg^?1 of protein in this assay system. The pH rate profile and apparent pK_a in 10 mm Ca²⁺ of the immobilized enzyme parallels that of the soluble enzyme. The substrate specificity of the immobilized enzyme toward individual phospholipid species in mixed micelles is phosphatidylcholine ? phosphatidylethanolamine. In binary lipid mixtures in mixed micelles containing phosphatidylcholine and phosphatidylethanolamine together, a reversal in specificity is observed, and phosphatidylethanolamine is the preferred substrate. This unusual specificity reversal in binary mixtures is also observed for the soluble enzyme. The activity of the immobilized enzyme toward phospholipid inserted in mixed micelles is the same as toward a synthetic phospholipid which forms monomers, while a 20-fold decrease in activity toward monomeric substrate is observed for the soluble enzyme. The immobilized enzyme is stable at temperatures of 90 °C as is the soluble enzyme. However, p-bromphenacyl bromide, a reagent which inactivates the soluble enzyme, does not inactivate the immobilized enzyme. The immobilized enzyme can be stored frozen for several months and is reusable. The mechanism of action of immobilized phospholipase A₂ from cobra venom and the potential usefullness of the bound enzyme as a probe for phospholipids in surfaces of membranes is considered.