Immobilization of β-galactosidases from Thermus aquaticus YT-1 for oligosaccharides synthesis

Summary -galactosidases of Thermus aquaticus YT-1, exhibiting a galactosyl transferase activity, were immobilized using different techniques. Entrapment in agarose or gellan gum beads was unsuitable for enzyme immobilization due to enzyme leakage. A technique that efficiently immobilized the enzymes was developed using glutaraldehyde co-crosslinking of -galactosidases with bovine serum albumin, followed by entrapment in agarose beads.     

Sustainable synthesis of uridine-5′-monophosphate analogues by immobilized uracil phosphoribosyltransferase from Thermus thermophilus

Nowadays enzymatic synthesis of nucleic acid derivatives is gaining momentum over traditional chemical synthetic processes. Biotransformations catalyzed by whole cells or enzymes offer an ecofriendly and efficient alternative to the traditional multistep chemical methods, avoiding the use of chemical reagents and organic solvents that are expensive and environmentally harmful. Herein we report for the first time the covalent immobilization a uracil phosphoribosyltransferase (UPRT). In this sense, UPRT from Thermus thermophilus HB8 was immobilized onto glutaraldehyde-activated MagReSyn®Amine magnetic iron oxide porous microparticles (MTtUPRT). According to the catalyst load experiments, MTtUPRT3 was selected as optimal biocatalyst for further studies. MTtUPRT3 was active and stable in a broad range of temperature (70–100 °C) and in the pH interval 6–8, displaying maximum activity at 100 °C and pH 7 (activity 968 IU/g_support, retained activity 100%). In addition, MTtUPRT3 could be reused up to 8 times in the synthesis of uridine-5′-monophosphate (UMP). Finally, MTtUPRT3 was successfully applied in the sustainable synthesis of different 5-modified uridine-5′-monophosphates at short times. Taking into account these results, MTtUPRT3 would emerge as a valuable biocatalyst for the synthesis of nucleoside monophosphates through an efficient and environmentally friendly methodology.     

Synthesis of propyl-β-d-galactoside with free and immobilized β-galactosidase from Aspergillus oryzae

Synthesis of propyl-β-galactoside catalyzed by Aspergillus oryzae β-galactosidase in soluble form was optimized using response surface methodology (RSM). Temperature and 1-propanol concentration were selected as explanatory variables; yield and productivity were chosen as response variables. Optimal reaction conditions were determined by weighing the responses through a desirability function. Then, synthesis of propyl-β-galactoside was evaluated at the optimal condition previously determined, with immobilized β-galactosidase in glyoxyl-agarose and amino-glyoxyl-agarose, and with cross-linked aggregates (CLAGs). Yields of propyl-β-galactoside obtained with CLAGs, amino-glyoxyl-agarose and glyoxyl-agarose enzyme derivatives were 0.75, 0.81 and 0.87 mol/mol and volumetric productivities were 5.2, 5.6 and 5.9 mM/h, respectively, being significantly higher than the corresponding values obtained with the soluble enzyme: 0.47 mol/mol and 4.4 mM/h. As reaction yield was increased twofold with the glyoxyl-agarose derivative, this catalyst was chosen for evaluating the synthesis of propyl-β-galactoside in repeated batch operations. Then, after ten sequential batches, the efficiency of catalyst use was 115% higher than obtained with the free enzyme. Enzyme immobilization also favored product recovery, allowing catalyst reuse, and avoiding browning reactions. Propyl-β-galactoside was recovery by extraction in 90%v/v acetone with a purity higher than 99% and its synthesis was confirmed by mass spectrometry.     

Purification and properties of neutral β-galactosidases from human liver

Two neutral β-galactosidase isozymes were purified from human liver. The initial step of purification was removal of the acidic β-galactosidases by adsorption on concanavalin A-Sepharose 4B conjugate. Subsequent purification steps included ammonium sulfate precipitation, diethylaminoethyl cellulose column chromatography, Sephadex G-100 gel filtration, and preparative polyacrylamide-gel isoelectric focusing. The final step of purification was affinity chromatography of the separated isoelectric forms on ?-aminocaproyl-β-d-galactosylamine-Sepharose 4B conjugate. The purified β-galactosidase isozymes had activity toward both β-d-galactoside and β-d-glucoside derivatives of 4-methylumbelliferone and p-nitrophenol with a pH optimum around 6.2. These enzyme forms were also found to possess lactosylceramidase II activity with a pH optimum in the range of 5.4 to 5.6, but not lactosylceramidase I activity and no activity toward galactosylceramide or GM₁-ganglioside. The molecular weight was found to be in the range of 37,500–39,500 for the two neutral isozymes and they had similar K_m and V values; the more acidic form (designated β-galactosidase N₁) was more heat stable than the other form (designated β-galactosidase N₂). Antibodies evoked against the N₁ and N₂ β-galactosidases gave identical precipitin lines retaining enzymatic activity. No cross-reactivity was observed between the neutral and the acidic isozymes when examined with the respective antisera.     

New Sulfated Oligosaccharides Produced by Pseudomonas β-Agarase from Gracilaria verrucosa Polysaccharide

Polysaccharide (partially sulfated agarose) with macrophage-stimulation activity, derived from Gracilaria verrucosa, was decomposed by two types of β-agarase (agarases II and IV) from Pseudomonas sp. O-148. The hydrolysates were fractionated with ethanol precipitation and anion-exchange chromatography. The resulting anionic oligosaccharides with sulfate groups were investigated by ¹³C-NMR spectroscopy. While the spectra of oligosaccharides produced by agarase IV showed identical patterns with those by β-agarase I from Pseudomonas atlantica and indicated the location of a sulfated saccharide unit on the non-reducing end, another new type of saccharide was found in the products by agarase II. The novel oligosaccharides by agarase II had a neoagarobiose unit on their non-reducing end and had sulfated units internally. This indicated the novelty of agarase II in cleavage fashion.     

Enzymatic synthesis of γ-glutamylmethylamide from L-glutamylhydrazine and methylamine catalysed by immobilized recombinant γ-glutamyltranspeptidase

In many organisms, γ-glutamylmethylamide is a significant amino acid constituent. In this research, a novel method of γ-glutamylmethylamide synthesis is presented. The synthesis of γ-glutamylmethylamide was catalysed by immobilized recombinant γ-glutamyltranspeptidase and used L-glutamylhydrazine as an economical substrate. The optimal enzymes and γ-glutamyltranspeptidase reaction conditions for the production of γ-glutamylmethylamide were 200?mM L-glutamylhydrazine, 1?M methylamine, and 0.1?g/ml immobilized γ-glutamyltranspeptidase cells at pH 10 and 37?°C for 10?h. The immobilized γ-glutamyltranspeptidase cells were used for 10 reactions, and the average conversion ratio from L-glutamylhydrazine to γ-glutamylmethylamide reached 93.2%. The activity of immobilized recombinant γ-glutamyltransferase was not inhibited by 200?mM L-glutamylhydrazine. The immobilized γ-glutamyltranspeptidase cells exhibited favourable operational stability.     

Picrocrocin hydrolysis by immobilized β-glucosidase

Summary -Glucosidase from sweet almond was immobilized onto a nylon support and used to hydrolyze picrocrocin, the glycoside precursor of the saffron essential volatile oil, safranal. The nylon support was derivatized as hydrazide and the enzyme attached through Schiff base to bonds. The coupling efficiency was 46.8%, the immobilization yield 29.5%, and the derivative showed 24.2 and 4.0 U/mg activity for p-nitrophenylglucoside and picrocrocin, respectively, as substrates. Kinetic parameters of the immobilized derivative were determined, with picrocrocin as substrate, showing K_M=7.2 mM and V_max=4,0 U/mg. Glucose behaved as a competitive inhibitor (K_i=95.0 mM). The immobilized derivative was thermally stable up to 45°C; from that temperature onwards thermoinactivation occured. The operational deactivation showed a biphasic pattern, t_1/2 being 4.2 days for the first four days of continuous operation, and 20.1 days from that point on. The immobilized enzyme lost a 50% of its initial activity after 30.7 days of storage at 4°C.     

Conventional and non-conventional applications of β-galactosidases

β-Galactosidase is one of the most important industrial enzymes, that has been used for many decades in the dairy industry. The main application of β-galactosidase is related to the production of low-lactose and lactose-free milk and dairy products, which are now common consumer goods in supermarket shelves. This is a well-established market that is expected to keep on growing as these products become more accessible to mid-income people worldwide. However, a fresh air has come into the β-galactosidase business as non-conventional applications arose in recent decades based on its transgalactosylation activity. This capacity is certainly a major asset for a commodity enzyme that can be used now as a catalyst for the upgrading of readily available and cheap lactose into high added-value glycosides in processes of organic synthesis in tune with green chemistry principles within the framework of sustainability. This is a reflection of a paradigm shift, where enzymes are now being considered as apt catalysts for the synthesis of valuable organic compounds. This article reviews the main applications of β-galactosidase, going from its conventional use related to its hydrolytic activity to the ongoing non-conventional applications in the synthesis of high added-value oligosaccharides based on its transgalactosylation activity.     

A comparison of the kinetic properties of free and immobilized Aspergillus oryzae β-galactosidase

The objective of this work was to compare the properties of free and immobilized β-galactosidase (Aspergillus oryzae), entrapped in alginate–gelatin beads and cross-linked with glutaraldehyde. The free and immobilized forms of the enzyme showed no decrease in enzyme activity when incubated in buffer solutions in pH ranges of 4.5–7.0. The kinetics of lactose hydrolysis by the free and immobilized enzymes were studied at maximum substrate concentrations of 90 g/L and 140 g/L, respectively, a temperature of 35 °C and a pH of 4.5. The Michaelis–Menten model with competitive inhibition by galactose fit the experimental results for both forms. The K_m and V_m values of the free enzyme were 52.13 ± 2.8 mM and 2.56 ± 0.3 g_glucose/L min mg_enzyme, respectively, and were 60.30 ± 3.3 mM and 1032.07 ± 51.6 g_lactose/min m³_catalyst, respectively, for the immobilized form. The maximum enzymatic activity of the soluble form of β-galactosidase was obtained at pH 4.5 and 55 °C. Alternatively, the immobilized form was most active at pH 5.0 at 60 °C. The free and immobilized enzymes presented activation energies of 6.90 ± 0.5 kcal/mol and 7.7 ± 0.7 kcal/mol, respectively, which suggested that the immobilized enzyme possessed a lower resistance to substrate transfer.     

Amino acid ester synthesis by immobilized α-chymotrypsin

Summary A screening of immobilized -chymotrypsin preparations suitable for the synthesis of N-acetyl-L-tyrosine ethyl ester from N-acetyl-L-tyrosine and up to 99% ethanol was carried out. -Chymotrypsin adsorbed to Sepharose LH-20 or covalently bound to Sepharose 4B (tresyl chloride activation) was found to be an efficient catalyst. A column packed with immobilized enzyme retained 60% of its initial activity after 6 days of operation in a cyclohexane-ethanol medium.     

Detailed kinetic model describing new oligosaccharides synthesis using different β-galactosidases

The production of prebiotic galactooligosaccharides (GOS) from lactose has been widely studied whereas the synthesis of new prebiotic oligosaccharides with improved properties as those derived from lactulose is receiving an increasing interest. Understanding the mechanism of enzymatic oligosaccharides synthesis from lactulose would help to improve the quality of the products in a rational way as well as to increase the production efficiency by optimally selecting the operating conditions. A detailed kinetic model describing the enzymatic transgalactosylation reaction during lactulose hydrolysis is presented here for the first time. The model was calibrated with the experimental data obtained in batch assays with two different β-galactosidases at various temperatures and concentrations of substrate. A complete system identification loop, including model selection, robust estimation of the parameters by means of a global optimization method and computation of confidence intervals was performed. The kinetic model showed a good agreement between experimental data and predictions for lactulose conversion and provided important insights into the mechanism of formation of new oligosaccharides with potential prebiotic properties.     

Distinct functions of regions 1.1 and 1.2 of RNA polymerase σ subunits from Escherichia coli and Thermus aquaticus in transcription initiation

RNA polymerase (RNAP) from thermophilic Thermus aquaticus is characterized by higher temperature of promoter opening, lower promoter complex stability, and higher promoter escape efficiency than RNAP from mesophilic Escherichia coli. We demonstrate that these differences are in part explained by differences in the structures of the N-terminal regions 1.1 and 1.2 of the E. coli σ(70) and T. aquaticus σ(A) subunits. In particular, region 1.1 and, to a lesser extent, region 1.2 of the E. coli σ(70) subunit determine higher promoter complex stability of E. coli RNAP. On the other hand, nonconserved amino acid substitutions in region 1.2, but not region 1.1, contribute to the differences in promoter opening between E. coli and T. aquaticus RNAPs, likely through affecting the σ subunit contacts with DNA nucleotides downstream of the -10 element. At the same time, substitutions in σ regions 1.1 and 1.2 do not affect promoter escape by E. coli and T. aquaticus RNAPs. Thus, evolutionary substitutions in various regions of the σ subunit modulate different steps of the open promoter complex formation pathway, with regions 1.1 and 1.2 affecting promoter complex stability and region 1.2 involved in DNA melting during initiation.     

Enzymatic epoxidation of β-caryophyllene using free or immobilized lipases or mycelia from the Amazon region

The chemo-enzymatic epoxidation of the terpene β-caryophyllene is reported herein. This compound can form two products, the mono-epoxide 2 and the di-epoxide 3. Different experimental conditions, varying the source of the lipases (including mycelia from the Amazon region), the oxidizing agents (H₂O₂ aq. (AHP) or urea-hydrogen peroxide (UHP)) and the substituted acyl donors on the alkyl chain (bromide and alkyl), along with the influence of organic medium, were evaluated. Depending on the experimental conditions the formation of a single product could be obtained. CAL-B was the most efficient catalyst (conv. >99%). When using the commercial lipases product 2 was obtained in conversions of 16–27%, and using the native lipases 2 was obtained in conversions of 20–23%. With the use of mycelia UEA_06 and UEA_53 the conversions were 16 and 21%, respectively. When the 2-bromo alkylated and 2-ethylhexanoic acids were used as acyl donors only the mono-epoxide 2 was obtained in conversions of 14–54% (24 h). AHP was found to be a better oxidizing agent than UHP, a shorter time and lower amount being required to obtain 2 or 3 as the sole product in good conversions (60 up to >99%). The organic solvents were also selective. When using n-hexane the preferred formation of 2 was observed with >99% conversion, and when ethyl acetate or toluene were used the conversion to 3 was also >99% (in 8 and 24 h, respectively).     

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%.     

Preparation of salidroside with n-butyl β-D-glucoside as the glycone donor via a two-step enzymatic synthesis catalyzed by immobilized β-glucosidase from bitter almonds

β-Glucosidase from bitter almonds was immobilized on epoxy group-functionalized beads for catalyzing salidroside synthesis in a two-step process with n-butyl-β-D-glucoside (BG) as the glucosyl donor. The formation of salidroside ((0.59?±?0.02) M) at a yield of 39.04%±1.25% was accomplished in 8?h by the transglucosylation of immobilized β-glucosidase at pH 8.0 and 50?°C when the ratio of BG to tyrosol was 1:2 (mol/mol). A study on the influence of different glycosyl acceptors demonstrated that the yield of the glucosylation reaction of phenylmethanol and cyclohexanol was higher than that of either phenol or cyclohexanol. This may account for the selectivity of the immobilized enzyme towards the alcoholic hydroxyl group of tyrosol in the salidroside synthesis reaction. A study on the synthesis of BG via the reverse hydrolysis of immobilized β-glucosidase showed that a yield of 78.04%±2.2% BG can be obtained with a product concentration of (0.23?±?0.015) M.     

Regioselective synthesis of neo-erlose by the β-fructofuranosidase from Xanthophyllomyces dendrorhous

The β-fructofuranosidase from the yeast Xanthophyllomyces dendrorhous (Xd-INV) catalyzes the synthesis of neo-fructooligosaccharides (neo-FOS of the ⁶G-series), which contain a β(2 → 6) linkage between a fructose and the glucosyl moiety of sucrose. In this work we demonstrate that the enzyme is also able to fructosylate other carbohydrates that contain glucose, in particular disaccharides (maltose, isomaltulose, isomaltose, trehalose) and higher oligosaccharides (maltotriose, raffinose, maltotetraose), but not monosaccharides (glucose, fructose, galactose). With maltose as acceptor, the reaction in the presence of Xd-INV proceeded with high regioselectivity; the product was purified and chemically characterized, and turned out to be 6′-O-β-fructosylmaltose (neo-erlose). Using 100 g/L sucrose as fructosyl donor and 300 g/L maltose as acceptor, the maximum concentration of neo-erlose was 38.3 g/L. Thus, novel hetero-fructooligosaccharides with potential applications in the functional food and pharmaceutical industries can be obtained with Xd-INV.     

Continous production of 1-kestose by β-fructofuranosidase immobilized onshirasu porous glass

Summary 1-Kestose was produced continously and selectively from 40% (w/v) sucrose solution at fast flow rate by a column packed with an immobilized -fructofuranosidase onshirasu porous glass.     

β-1-3- and β-1-4-glucan synthesis by membrane fractions from the fungus Saprolegnia

The -glucan synthetase activity of the fungus Saprolegnia monoica was assayed by supplying UDP-glucose to membrane fractions of mycelial homogenate. The analysis of glucan products by hydrolysis with various -glucanases and by chromatography show that both -1-3- and -1-4-linkages are formed at high substrate concentrations. In the absence of MgCl₂, -1-3-linked glucans are mainly produced. By increasing MgCl₂ concentrations the total synthesis activity and -1-3-linkages production are reduced. At low substrate concentrations in the presence of MgCl₂, -1-4-linked glucans are the only polysaccharide synthesized. Electron microscopy of radioactive products, synthesized by original membrane fractions or by membrane fractions isolated from continuous sucrose density gradients, shows microfibrils when the assays are conducted at high substrate concentrations in the absence of MgCl₂.Abbreviations G.S. I glucan synthetase I - G.S. II glucan synthetase II - Dol. P dolichol phosphate     

Biodegradation of cellulose by β-glucosidase and cellulase immobilized on a pH-responsive copolymer

Biodegradation of cellulose involves synergistic action of the endoglucanases, exoglucanases and β-glucosidases in cellulase. However, the yield of glucose is limited by the lack of β-glucosidase to hydrolyze cellobiose into glucose. In this study, β-glucosidase as a supplemental enzyme along with cellulase are co-immobilized on a pHresponsive copolymer, poly (MAA-co-DMAEMA-co-BMA) (abbreviated P_MDB, where MAA is α-methacrylic acid, DMAEMA is 2-dimethylaminoethyl methacrylate and BMA is butyl methacrylate). The thermal and storage stabilities of PMDB with immobilized enzymes are improved greatly, compared with those of free cellulase. Biodegradation of cellulose is carried out in a pH-responsive recyclable aqueous two-phase system composed of poly (AA-co- DMAEMA-co-BMA) (abbreviated P_{ADB 3.8}, where AA is acrylic acid) and P_MDB. Insoluble substrate and P_MDB with immobilized cellulase and β-glucosidase (Celluclast 1.5L FG and Novozyme 188, respectively) were biased to the bottom phase, while the product was partitioned to the top phase in the presence of 40 mM (NH₄)₂SO₄. When the degradation reaction of cellulose is carried out with PMDB containing immobilized cellulase and β-glucosidase, the concentration of glucose reaches 4.331 mg/mL after 108 h. The yield of glucose is 50.25% after P_MDB containing the immobilized enzymes is recycled five times.     

Hydrolysis of lactose by β-galactosidase immobilized in polyvinylalcohol

Summary Fungal -galactosidase was immobilized in polyvinylalcohol gel formed in pores of contton material. Temperature and pH effects on the activity of free and immobilized enzymes were studied. The optimum temperatures of free and immobilized enzymes were 60° C and 55° C respectively. The pH optimum ranged from 4.5 to 5.0 for both enzymes. The thermal stability of the immobilized -galactosidase was slightly higher. The K_m values for soluble and immobilized enzymes were respectively 1.9 mM and 2.5 mM. The optimization of conditions for a highly effective hydrolysis of 4% lactose solution and reusability of the immobilized enzyme resulted in 75% hydrolysis after 5–6 h. The degree of conversion decreased to 50% after 30 repeated runs. The capacity of the immobilized enzyme to hydrolyze lactose in whey was also studied.