Design of β‐galactosidase/silica biocatalysts: Impact of the enzyme properties and immobilization pathways on their catalytic performance

The use of heterogeneous biocatalysis in industrial applications is advantageous and the enzyme stability improvement is a continuous challenge. Therefore, we designed β‐galactosidase heterogeneous biocatalysts by immobilization, involving the support synthesis and enzyme selection (from Bacillus circulans, Kluyveromyces lactis, and Aspergillus oryzae). The underivatized, tailored, macro‐mesoporous silica exhibited high surface area, offered high enzyme immobilization yields and activity. Its chemical activation with glyoxyl groups bound the enzyme covalently, which suppressed lixiviation and conferred higher pH and thermal stability (120‐fold than for the soluble enzyme), without observable reduction of activity/stability due to the presence of silica. The best balance between the immobilization yield (68%), activity (48%), and stability was achieved for Bacillus circulans β‐galactosidase immobilized on glyoxyl‐activated silica, without using stabilizing agents or modifying the enzyme. The enzyme stabilization after immobilization in glyoxyl‐activated silica was similar to that observed in macroporous agarose‐glyoxyl support, with the reported microbiological and mechanical advantages of inorganic supports. The whey lactolysis at pH 6.0 and 25°C by using this catalyst (1 mg ml^?1, 290 UI g^?1) was still 90%, even after 10 cycles of 10 min, in batch process but it could be also implemented on continuous processes at industrial level with similar results.     

Immobilization of a thermostable α‐amylase by covalent binding to an alginate matrix increases high temperature usability

Thermostable α‐amylase was covalently bound to calcium alginate matrix to be used for starch hydrolysis at liquefaction temperature of 95°C. 1‐ethyl‐3‐(3‐dimethylamino‐propyl) carbodiimide hydrochloride (EDAC) was used as crosslinker. EDAC reacts with the carboxylate groups on the calcium alginate matrix and the amine groups of the enzyme. Ethylenediamine tetraacetic acid (EDTA) treatment was applied to increase the number of available carboxylate groups on the calcium alginate matrix for EDAC binding. After the immobilization was completed, the beads were treated with 0.1 M calcium chloride solution to reinstate the bead mechanical strength. Enzyme loading efficiency, activity, and reusability of the immobilized α‐amylase were investigated. Covalently bound thermostable α‐amylase to calcium alginate produced a total of 53 g of starch degradation/mg of bound protein after seven consecutive starch hydrolysis cycles of 10 min each at 95°C in a stirred batch reactor. The free and covalently bound α‐amylase had maximum activity at pH 5.5 and 6.0, respectively. The Michaelis‐Menten constant (K_m) of the immobilized enzyme (0.98 mg/mL) was 2.5 times greater than that of the free enzyme (0.40 mg/mL). The maximum reaction rate (V_max) of immobilized and free enzyme were determined to be 10.4‐mg starch degraded/mL min mg bound protein and 25.7‐mg starch degraded/mL min mg protein, respectively. The high cumulative activity and seven successive reuses obtained at liquefaction temperature make the covalently bound thermostable α‐amylase to calcium alginate matrix, a promising candidate for use in industrial starch hydrolysis process. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Characterization of microbial endo-β-glucanase immobilized in alginate beads

Endo-β-glucanase (endo-β-1,4-glucano-glucanase EC 3.2.1.4), isolated from Trichoderma reesei, was immobilized in calcium alginate beads, retaining 75% of its original activity. The polyanionic moiety surrounding the immobilized enzyme displaced the pH-activity profile to alkaline regions with respect to that of the free enzyme. The enzyme was inhibited by carboxymethylcellulose, but this inhibition appeared to be decreased by immobilizatíon. The enzyme immobilized in alginate beads showed a K_m value (1.02% w/v) lower than that of the enzyme (1.31%). The apparent V_max of immobilized cellulase preparations (238.3 μmol glucose/ml × h) decreased by a factor of 0.59 with respect to that of the soluble enzyme. The optimum temperature (60°C) of the free and entrapped enzymes remained unaltered. In contrast, the half-life of the endoglucanase immobilized in calciumalginate beads was 4.6 h at 55°C and 5.4 h at 60°C, while that of the free enzyme was 3.0 h at 55°C and 1.2 h at 60°C. A technological application of the immobilized enzymes was tested using wheat straw as a source of fermentable sugars. The hydrolytic degradation of straw, by means of a crude extract of free and immobilized cellulases and β-glucosidase, released a large amount of reducing sugars from wheat straw after 48 h (between 250–720 mg glucose/g straw), carrying out more than a 90% saccharification. A mixture of immobilized β-glucosidase and free cellulases maintained 80% of the activity of the soluble counterparts, and the co-immobilization of both types of enzymes reduced by hydrolytic efficiency to half.     

Stabilization of Aspergillus parasiticus cytosine deaminase by immobilization on calcium alginate beads improved enzyme operational stability

Cytosine deaminase (CD) from Aspergillus parasiticus, which has half-life of 1.10?h at 37°C, was stabilized by immobilization on calcium alginate beads. The immobilized CD had pH and temperature optimum of 5 and 50°C respectively. The immobilized enzyme also stoichiometrically deaminated Cytosine and 5-fluorocytosine (5-FC) with the apparent K_M values of 0.60?mM and 0.65?mM respectively, displaying activation energy of 10.72 KJ/mol. The immobilization of native CD on calcium alginate beads gave the highest yield of apparent enzymatic activity of 51.60% of the original activity and the enzymatic activity was lost exponentially at 37°C over 12?h with a half-life of 5.80?h. Hence, the operational stability of native CD can be improved by immobilization on calcium alginate beads.     

Immobilization of goat brain aminopeptidase B in calcium alginate beads

Aminopeptidase B, an arginyl aminopeptidase, was purified from goat brain with a purification factor of ～280 and a yield of 2.7%. It was entrapped in calcium alginate together with bovine serum albumin. The optimal conditions for immobilization for maximum activity yield were 1% CaCl₂ and 2.5% alginate. The immobilized enzyme retained ～62% of its initial activity and could be used for five successive batch reactions with retention of 30% of the initial activity. The pH and temperature optima of the free and immobilized enzyme were pH 7.4, 45°C and pH 7.8, 50°C respectively, while the pH and thermal stability as well as the stability of the enzyme in organic solvents were improved significantly after entrapment. The K_m value for the immobilized enzyme was about twofold higher than that of the soluble enzyme. Because of this increased stability, the immobilized enzyme may be useful in the meat processing industry.     

Novel grafted agar disks for the covalent immobilization of β‐D‐galactosidase

Novel grafted agar disks were prepared for the covalent immobilization of β‐D‐galactosidase (β‐gal). The agar disks were activated through reacting with ethylenediamine or different molecular weights of Polyethyleneimine (PEI), followed by glutaraldehyde (GA). The modification of the agar gel and the binding of the enzyme were verified by Fourier Transform Infrared (FTIR) and elemental analysis. Moreover, the agar's activation process was optimized, and the amount of immobilized enzyme increased 3.44 folds, from 38.1 to 131.2 U/g gel, during the course of the optimization process. The immobilization of β‐gal onto the activated agar disks caused its optimum temperature to increase from 45°C to 45–55°C. The optimum pH of the enzyme was also shifted towards the acidic side (3.6–4.6) after its immobilization. Additionally, the Michaelis‐Menten constant (K_m) increased for the immobilized β‐gal as compared to its free counterpart whereas the maximum reaction rate (V_max) decreased. The immobilized enzyme was also shown to retain 92.99% of its initial activity after being used for 15 consecutive times. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 675–684, 2015.     

Modification of Immobead 150 support for protein immobilization: Effects on the properties of immobilized Aspergillus oryzae β‐galactosidase

We studied the modification of Immobead 150 support by either introducing aldehyde groups using glutaraldehyde (Immobead‐Glu) or carboxyl groups through acid solution (Immobead‐Ac) for enzyme immobilization by covalent attachment or ion exchange, respectively. These two types of immobilization were compared with the use of epoxy groups that are now provided on a commercial support. We used Aspergillus oryzae β‐galactosidase (Gal) as a model protein, immobilizing it on unmodified (epoxy groups, Immobead‐Epx) and modified supports. Immobilization yield and efficiency were tested as a function of protein loading (10–500 mg g^?1 support). Gal was efficiently immobilized on the Immobeads with an immobilization efficiency higher than 75% for almost all supports and protein loads. Immobilization yields significantly decreased when protein loadings were higher than 100 mg g^?1 support. Gal immobilized on Immobead‐Glu and Immobead‐Ac retained approximately 60% of its initial activity after 90 days of storage at 4°C. The three immobilized Gal derivatives presented higher half‐lifes than the soluble enzyme, where the half‐lifes were twice higher than the free Gal at 73°C. All the preparations were moderately operationally stable when tested in lactose solution, whey permeate, cheese whey, and skim milk, and retained approximately 50% of their initial activity after 20 cycles of hydrolyzing lactose solution. The modification of the support with glutaraldehyde provided the most stable derivative during cycling in cheese whey hydrolysis. Our results suggest that the Immobead 150 is a promising support for Gal immobilization. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:934–943, 2018     

Immobilization and characterization of lipase from an indigenous Bacillus aryabhattai SE3-PB isolated from lipid-rich wastewater

Abstract

Extracellular lipase from an indigenous Bacillus aryabhattai SE3-PB was immobilized in alginate beads by entrapment method. After optimization of immobilization conditions, maximum immobilization efficiencies of 77%?±?1.53% and 75.99%?±?3.49% were recorded at optimum concentrations of 2% (w/v) sodium alginate and 0.2?M calcium chloride, respectively, for the entrapped enzyme. Biochemical properties of both free and immobilized lipase revealed no change in the optimum temperature and pH of both enzyme preparations, with maximum activity attained at 60?°C and 9.5, respectively. In comparison to free lipase, the immobilized enzyme exhibited improved stability over the studied pH range (8.5–9.5) and temperature (55–65?°C) when incubated for 3?h. Furthermore, the immobilized lipase showed enhanced enzyme-substrate affinity and higher catalytic efficiency when compared to soluble enzyme. The entrapped enzyme was also found to be more stable, retaining 61.51% and 49.44% of its original activity after being stored for 30 days at 4?°C and 25?°C, respectively. In addition, the insolubilized enzyme exhibited good reusability with 18.46% relative activity after being repeatedly used for six times. These findings suggest the efficient and sustainable use of the developed immobilized lipase for various biotechnological applications.     

Characterization of an ethanol‐tolerant 1,4‐β‐xylosidase produced by Pichia membranifaciens

Aims: The purification and biochemical properties of the 1,4‐β‐xylosidase of an oenological yeast were investigated. Methods and Results: An ethanol‐tolerant 1,4‐β‐xylosidase was purified from cultures of a strain of Pichia membranifaciens grown on xylan at 28°C. The enzyme was purified by sequential chromatography on DEAE cellulose and Sephadex G‐100. The relative molecular mass of the enzyme was determined to be 50 kDa by SDS‐PAGE. The activity of 1,4‐β‐xylosidase was optimum at pH 6·0 and at 35°C. The activity had a K_m of 0·48 ± 0·06 mmol l^?1 and a V_max of 7·4 ± 0·1 μmol min^?1 mg^?1 protein for p‐nitrophenyl‐β‐d ‐xylopyranoside. Conclusions: The enzyme characteristics (pH and thermal stability, low inhibition rate by glucose and ethanol tolerance) make this enzyme a good candidate to be used in enzymatic production of xylose and improvement of hemicellulose saccharification for production of bioethanol. Significance and Impact of the Study: This study may be useful for assessing the ability of the 1,4‐β‐xylosidase from P. membranifaciens to be used in the bioethanol production process.     

Purification and characterization of an endoxylanase from the culture broth of <Emphasis Type="Italic">Bacillus cereus</Emphasis> BSA1

An extracellular xylanase from the fermented broth of Bacillus cereus BSA1 was purified and characterized. The enzyme was purified to 3.43 fold through ammonium sulphate precipitation, DEAE cellulose chromatography and followed by gel filtration through Sephadex-G-100 column. The molecular mass of the purified xylanse was about 33 kDa. The enzyme was an endoxylanase as it initially degraded xylan to xylooligomers. The purified enzyme showed optimum activity at 55°C and at pH 7.0 and remained reasonably stable in a wide range of pH (5.0–8.0) and temperature (40–65°C). The K _m and V _max values were found to be 8.2 mg/ml and 181.8 μmol/(min mg), respectively. The enzyme had no apparent requirement of cofactors, and its activity was strongly inhibited by Cu²⁺, Hg²⁺. It was also a salt tolerant enzyme and stable upto 2.5 M of NaCl and retained its 85% activity at 3.0 M. For stability and substrate binding, the enzyme needed hydrophobic interaction that revealed when most surfactants inhibited xylanase activity. Since the enzyme was active over wide range of pH, temperature and remained active in higher salt concentration, it could find potential uses in biobleaching process in paper industries.     

In situ immobilization of β‐galactosidase from Bacillus circulans in silica by sol‐gel process: Application in prebiotic synthesis

The enzyme encapsulation is a very well‐known stabilization pathway. However, there are some challenges in order to avoid the enzyme denaturation under encapsulation conditions. The β‐galactosidase from Bacillus circulans was immobilized through sol‐gel encapsulation route assisted by Triton X‐100 surfactant and sugars. The effects of sugar presence in the immobilization process and the gelation time on the biocatalyst activity/stability were explained taking into account the characteristics of the formed silica matrix and the changes of the enzyme environment. The enzyme was effectively immobilized by this strategy, with high immobilization yield in terms of activity (29%) and expressed activity (47 IU/g). The immobilization through silica sol‐gel in the presence of 1×10^?3 M Triton X‐100 and fructose conferred 28.4‐fold higher stability to the enzyme compared with the soluble form. This is an advantage for its use in the synthesis of the galacto‐oligosaccharides at 50ºC. The total lactose conversion to galacto‐oligosaccharides was 26%wt, which is comparable with that reported in the literature. The obtained biocatalyst is useful for the synthesis of galacto‐oligosaccharides and its catalytic behavior is rationalized in this work.     

Purification and characterization of intracellular and extracellular α‐glucosidases from Geobacillus toebii strain E134

Two different α‐glucosidase‐producing thermophilic E134 strains were isolated from a hot spring in Kozakli, Turkey. Based on the phenotypic, phylogenetic and chemotaxonomic evidence, the strain was proposed to be a species of G. toebii. Its thermostable exo‐α‐1,4‐glucosidases also were characterized and compared, which were purified from the intracellular and extracellular fractions with estimated molecular weights of 65 and 45 kDa. The intracellular and extracellular α‐glucosidases showed optimal activity at 65 °C, pH 7·0, and at 70 °C, pH 6·8, with 3·65 and 0·83 K_m values for the pNPG substrate, respectively. Both enzymes remained active over temperature and pH ranges of 35–70 °C and 4·5–11·0. They retained 82 and 84% of their activities when incubated at 60 °C for 5 h. Their relative activities were 45–75% and 45–60% at pH 4·5 and 11·0 values for 15 h at 35 °C. They could hydrolyse the α‐1,3 and α‐1,4 bonds on substrates in addition to a high transglycosylation activity, although the intracellular enzyme had more affinity to the substrates both in hydrolysis and transglycosylation reactions. Furthermore, although sodium dodecyl sulfate behaved as an activator for both of them at 60 °C, urea and ethanol only increased the activity of the extracellular α‐glucosidase. By this study, G. toebii E134 strain was introduced, which might have a potential in biotechnological processes when the conformational stability of its enzymes to heat, pH and denaturants were considered. Copyright © 2011 John Wiley & Sons, Ltd.     

Downstream processing,characterization, and structure–function relationship of solvent‐, detergent‐, psychro‐, thermo‐, alkalistable metalloprotease from metal‐, solvent‐tolerant psychrotrophic Pseudomonas putida SKG‐1 isolate

The purification and characterization of psychro‐thermoalkalistable protease from psychrotrophic Pseudomonas putida isolate is being reported for the first time. A ～53 kDa protease was purified 21.4‐folds with 57.2% recovery by ultrafiltration and hydrophobic interaction chromatography. Kinetic analyses revealed the K_m and V_max to be 1.169 mg mL^?1 and 0.833 mg mL^?1 min^?1, respectively. The k_cat value of 3.05 × 10² s^?1 indicated high affinity and catalytic efficiency toward casein. The protease was most active at pH 9.5 and 40°C, with 100% stability in pH and temperature range of 6.0–11.0 and 10–40°C, respectively. Presence of Zn²⁺ increased the thermostability of protease (at 70°C) by 433%. Ethylene diamine tetra acetic acid (EDTA) and 1,10‐phenanthroline were inhibitory, whereas phenyl methyl sulfonyl fluoride (PMSF), p‐chloro mercuric benzoate (PCMB), and β‐mercaptoethanol were ineffective, revealing the enzyme to be a metalloprotease. Zinc, calcium, iron, nickel, and copper at 1 mM increased the enzyme activity (102–134%). Complete reversion of enzyme inhibition (caused by Ethylene diamine tetra acetic acid [EDTA]) by Zn²⁺ affirmed this enzyme as zinc‐dependent metalloprotease. At 0.1% concentration, Triton X‐100 and Tween 80 slightly increased, while SDS and H₂O₂ reduced the protease activity. In the presence of 0.1% commercial detergents, the enzyme was fairly stable (54–81%). In the presence of organic solvent, the protease was remarkably stable exhibiting 72–191% activities. In contrast, savinase exhibited good stability in the presence of hydrophilic solvents, while chymotrypsin showed elevated activities with benzene, toluene, and xylene only. Circular dichroism analysis revealed the protease as a β‐rich protein, having large fraction (～40%) of β‐sheets. Presence of different environmental conditions altered the β‐content, which accordingly affected the protease activity. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Purification, immobilization and characterization of linoleic acid isomerase on modified palygorskite

Linoleic acid isomerase from Lactobacillus delbrueckii subsp. bulgaricus 1.1480 was purified by DEAE ion-exchange chromatography and gel filtration chromatography. An overall 5.1% yield and purification of 93-fold were obtained. The molecular weight of the purified protein was ~41 kDa which was analyzed by SDS-PAGE. The purified enzyme was immobilized on palygorskite modified with 3-aminopropyltriethoxysilane. The immobilized enzyme showed an activity of 82 U/g. The optimal temperature and pH for the activity of the free enzyme were 30 °C and pH 6.5, respectively; whereas those for the immobilized enzyme were 35 °C and pH 7.0, respectively. The immobilized enzyme was more stable than the free enzyme at 30–60 °C, and the operational stability result showed that more than 85% of its initial activity was retained after incubation for 3 h. The K _m and V _max values of the immobilized enzyme were found to be 0.0619 mmol l⁻¹ and 0.147 mmol h⁻¹ mg⁻¹, respectively. The immobilized enzyme had high operational stability and retained high enzymatic activity after seven cycles of reuse at 37 °C.     

Effect of calcium on immobilization of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) peroxidase for bioassays in sodium alginate and Agarose gel

The direct immobilization of soluble peroxidase isolated and partially purified from shoots of rice seedlings in calcium alginate beads and in calcium agarose gel was carried out. Peroxidase was assayed for guaiacol oxidation products in presence of hydrogen peroxide. The maximum specific activity and immobilization yield of the calcium agarose immobilized peroxidase reached 2,200 U mg⁻¹ protein (540 mU cm⁻³ gel) and 82%, respectively. In calcium alginate the maximum activity of peroxidase upon immobilization was 210 mU g⁻¹ bead with 46% yield. The optimal pH for agarose immobilized peroxidase was 7.0 which differed from the pH 6.0 for soluble peroxidase. The optimum temperature for the agarose immobilized peroxidase however was 30°C, which was similar to that of soluble peroxidase. The thermal stability of calcium agarose immobilized peroxidase significantly enhanced over a temperature range of 30∼60°C upon immobilization. The operational stability of peroxidase was examined with repeated hydrogen peroxide oxidation at varying time intervals. Based on 50% conversion of hydrogen peroxide and four times reuse of immobilized gel, the specific degradation of guaiacol for the agarose immobilized peroxidase increased three folds compared to that of soluble peroxidase. Nearly 165% increase in the enzyme protein binding to agarose in presence of calcium was noted. The results suggest that the presence of calcium, ions help in the immobilization process of peroxidase from rice shoots and mediates the direct binding of the enzyme to the agarose gel and that agarose seems to be a better immobilization matrix for peroxidase compared to sodium alginate.     

Stabilization of immobilized cathepsin L in non-aqueous medium

A lysosomal cysteine protease cathepsin L (3.4.22.15) purified from goat brain has been immobilized in calcium alginate beads in the presence of BSA through entrapment. Most favorable conditions for the entrapment were standardized as 3.0%(w/v) alginate and 1.5%(w/v) calcium chloride. Comparing the properties of free and immobilized enzyme using Z-Phe-Arg-4mβNA as chromogenic substrate, it was found that the immobilized enzyme could retain～70% of the original activity after five successive batch reactions. Vis-à-vis the free enzyme, immobilization conferred high stability to the enzyme both in the acidic and alkaline range, the enzyme lost no activity up to 60°C (Temperature stability for free enzyme is only up to 50°C). The pH optima for the enzyme shifted from 6.2 to 6.6 on entrapment. The increase in activity and stability of the enzyme in immobilized form even in the presence of high concentration of DMSO and ethanol is surprising and may make it useful for catalyzing organic reactions like trans-esterification and trans-amidation.     

Immobilization of the α-amylase of Bacillus amyloliquifaciens TSWK1-1 for the improved biocatalytic properties and solvent tolerance

The α-amylase of Bacillus amyloliquifaciens TSWK1-1 (GenBank Number, GQ121033) was immobilized by various methods, including ionic binding with DEAE cellulose, covalent coupling with gelatin and entrapment in polyacrylamide and agar. The immobilization of the purified enzyme was most effective with the DEAE cellulose followed by gelatin, agar and polyacrylamide. The K _m increased, while V _max decreased upon immobilization on various supports. The temperature and pH profiles broadened, while thermostability and pH stability enhanced after immobilization. The immobilized enzyme exhibited greater activity in various non-ionic surfactants, such as Tween-20, Tween-80 and Triton X-100 and ionic surfactant, SDS. Similarly, the enhanced stability of the immobilized α-amylase in various organic solvents was among the attractive features of the study. The reusability of the immobilized enzyme in terms of operational stability was assessed. The DEAE cellulose immobilized α-amylase retained its initial activity even after 20 consequent cycles. The DEAE cellulose immobilized enzyme hydrolyzed starch with 27 % of efficiency. In summary, the immobilization of B. amyloliquifaciens TSWK1-1 α-amylase with DEAE cellulose appeared most suitable for the improved biocatalytic properties and stability.     

A novel enzymatic microreactor with Aspergillus oryzae β‐galactosidase immobilized on silicon dioxide nanosprings

The use of silicon dioxide (SiO₂) nanosprings as supports for immobilized enzymes in a continuous microreactor is described. A nanospring mat (2.2 cm² × 60 μm thick) was functionalized with γ‐aminopropyltriethoxysilane, then treated with N‐succinimidyl‐3‐(2‐pyridyldithio)‐propionate (SPDP) and dithiothreitol (DTT) to produce surface thiol (? SH) groups. SPDP‐modified β‐galactosidase from Aspergillus oryzae was immobilized on the thiolated nanosprings by reversible disulfide linkages. The enzyme‐coated nanospring mat was placed into a 175‐μm high microchannel, with the mat partially occluding the channel. The kinetics and steady‐state conversion of hydrolysis of o‐nitrophenyl β‐D ‐galactosylpyranoside at various substrate flow rates and concentrations were measured. Substantial flow was observed through the nanosprings, for which the Darcy permeability κ ≈ 3 × 10^?6 cm². A simple, one‐parameter numerical model coupling Navier‐Stokes and Darcy flow with a pseudo‐first‐order reaction was used to fit the experimental data. Simulated reactor performance was sensitive to changes in κ and the height of the nanospring mat. Permeabilities lower than 10^?8 cm² practically eliminated convective flow through the nanosprings, and substantially decreased conversion. Increasing the height of the mat increased conversion in simulations, but requires more enzymes and could cause sealing issues if grown above channel walls. Preliminary results indicate that in situ regeneration by reduction with DTT and incubation with SPDP‐modified β‐galactosidase is possible. Nanosprings provide high solvent‐accessible surface area with good permeability and mechanical stability, can be patterned into existing microdevices, and are amenable to immobilization of biomolecules. Nanosprings offer a novel and useful support for enzymatic microreactors, biosensors, and lab‐on‐chip devices. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Improvement of covalent immobilization procedure of β‐galactosidase from Kluyveromyces lactis for galactooligosaccharides production: Modeling and kinetic study

Galactooligosaccharides (GOS) are prebiotics produced from lactose through an enzymatic reaction. Employing an immobilized enzyme may result in cost reductions; however, the changes in its kinetics due to immobilization has not been studied. This study experimentally determined the optimal reaction conditions for the production of GOS from lactose by β‐galactosidase (EC 3.2.1.23) from Kluyveromyces lactis covalently immobilized to a polysiloxane‐polyvinyl alcohol (POS‐PVA) polymer activated with glutaraldehyde (GA), and to study the transgalactosylation kinetics. Yield immobilization was 99 ± 1.1% with 78.5 ± 2.4% enzyme activity recovery. An experimental design 24 with 1 center point and 2 replicates was used. Factors were lactose [L], enzyme concentration [E], pH and temperature (T). Response variables were glucose and galactose as monosaccharides [G1], residual lactose [Lac]r and GOS as disaccharides [G2] and trisaccharides [G3]. Best conditions were pH 7.1, 40 °C, 270 gL^?1 initial lactose concentration and 6 U mL^?1 enzyme concentration, obtaining 25.46 ± 0.01 gL^?1 yield of trisaccharides. Although below the HPLC‐IR detection limit, tetrasaccharides were also identified after 115 min of reaction. The immobilization protocol was then optimized by diminishing total reactant volumes : support ratio, resulting in improved enzyme activity synthesizing 43.53 ± 0.02 gL^?1 of trisaccharides and 13.79 ± 0.21 gL^?1 of tetrasaccharides, and after four cycles remaining relative activity was 94%. A reaction mechanism was proposed through which a mathematical model was developed and rate constants were estimated, considering a pseudo steady‐state hypothesis for two concomitant reactions, and from this simplified analysis, the reaction yield could eventually be improved. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1568–1578, 2017     

Purification and enzymatic properties of α-galactosidase from Penicillium janthinellum

α-Galactosidase (E.C.3.2.1.22) from Penicillium janthinellum was purified by precipitation and fractionation with ammonium sulphate, cold acetone or ethanol, calcium phosphate gel, and column chromatographies on Sephadex G-100 and G-200. The enzyme was purified about 110.39-fold when Sephadex G-100 was used. α-Galactosidase exhibited the optimum pH and temperature at 4.5 and 60°C, respectively. The optimum enzyme stability was obtained at pH 3.5 for 24 h (at room temperature). The enzyme was found to be thermostable below 65°C up to 40 minutes and was gradually inactivated by increasing the temperature above this degree. The MICHAELIS constant was 0.55 mM for p-nitrophenyl-α-D-galactoside. The α-galactosidase activity was strongly inhibited by Hg⁺⁺ and slightly activated by Mn⁺⁺. The results show the possibility of producing a thermostable enzyme from a low-priced agricultural product, for instance, lupine.