Glyoxyl-agarose Supports Research Articles

Acid Phosphatase Immobilization and Production Study by Trichoderma spp. in Soybean Molasses

AbstractAcid phosphatase production was carried out by Trichoderma spp. using treated soybean molasses at different concentrations. Results showed higher acid phosphatase activity when the soybean molasses concentration increased for the microorganisms Trichoderma harzianum and Trichoderma reesei. The acid phosphatase activity performance of Trichoderma asperellum was also evaluated. In general, the polyethyleneimine glyoxyl agarose support showed much better performance and final recovery of activity than DEAE Toyopearl 650s.

Effect of Acyl Acceptor on Biodiesel Production from Soybean Cooking Oil Waste Using Immobilized Aspergillus Oryzae Lipase on Glyoxyl-Agarose Support and Characterization of the Produced Biodiesel

Effect of Acyl Acceptor on Biodiesel Production from Soybean Cooking Oil Waste Using Immobilized Aspergillus Oryzae Lipase on Glyoxyl-Agarose Support and Characterization of the Produced Biodiesel

Immobilization of the Peroxygenase from Agrocybe aegerita. The Effect of the Immobilization pH on the Features of an Ionically Exchanged Dimeric Peroxygenase

This paper outlines the immobilization of the recombinant dimeric unspecific peroxygenase from Agrocybe aegerita (rAaeUPO). The enzyme was quite stable (remaining unaltered its activity after 35 h at 47 °C and pH 7.0). Phosphate destabilized the enzyme, while glycerol stabilized it. The enzyme was not immobilized on glyoxyl-agarose supports, while it was immobilized albeit in inactive form on vinyl-sulfone-activated supports. rAaeUPO immobilization on glutaraldehyde pre-activated supports gave almost quantitative immobilization yield and retained some activity, but the biocatalyst was very unstable. Its immobilization via anion exchange on PEI supports also produced good immobilization yields, but the rAaeUPO stability dropped. However, using aminated agarose, the enzyme retained stability and activity. The stability of the immobilized enzyme strongly depended on the immobilization pH, being much less stable when rAaeUPO was adsorbed at pH 9.0 than when it was immobilized at pH 7.0 or pH 5.0 (residual activity was almost 0 for the former and 80% for the other preparations), presenting stability very similar to that of the free enzyme. This is a very clear example of how the immobilization pH greatly affects the final biocatalyst performance.

One-step purification and immobilization of recombinant proteins using SpyTag/SpyCatcher chemistry.

Based on the specific and spontaneous formation of isopeptide bonds by SpyCatcher/SpyTag, we have developed a one-step method for purification and immobilization of recombinant proteins. The procedure is to immobilize SpyCatcher on glyoxyl agarose gels, and then the SpyCatcher immobilisate can be used to immobilize the SpyTag-fused protein in the crude extract selectively. A mutant of SpyCatcher (mSC), in which a peptide (LysGlyLysGlyLysGly) was added to the C-terminus of SpyCatcher and three lysine residues around the SpyTag/SpyCatcher binding domain were replaced with arginine, was designed to improve the attachment of SpyCatcher to the support. Compared with wild-type SpyCatcher, mSC can be immobilized on the glyoxyl-agarose support more efficiently, which enables the obtained mSC derivative a high binding capacity of the SpyTag-fused protein. The results showed that the target proteins in the crude enzyme extract were purified and immobilized in one step, and the thermal stability of the immobilized target proteins was also remarkably improved.

Synthesis of Lactulose in Continuous Stirred Tank Reactor With β-Galactosidase of Apergillus oryzae Immobilized in Monofunctional Glyoxyl Agarose Support.

Lactulose synthesis from fructose and lactose in continuous stirred tank (CSTR) reactor operation with glyoxyl-agarose immobilized Aspergillus oryzae β-galactosidase is reported for the first time. The effect of operational variables: inlet concentrations of sugar substrates, temperature, feed substrate molar ratio, enzyme loading and feed flow rate was studied on reactor performance. Even though the variation of each one affected to a certain extent lactulose yield (YLactulose), specific productivity (πLactulose) and selectivity of the reaction (lactulose/transgalactosylated oligosaccharides molar ratio) (SLu/TOS), the most significant effects were obtained by varying the inlet concentrations of sugar substrates and the feed substrate molar ratio. Maximum YLactulose of 0.54 g⋅g–1 was obtained at 50°C, pH 4.5, 50% w/w inlet concentrations of sugar substrates, feed flowrate of 12 mL⋅min–1, fructose/lactose molar ratio of 8 and reactor enzyme load of 29.06 IUH⋅mL–1. At such conditions SLu/TOS was 3.7, lactose conversion (XLactose) was 0.39 and total transgalactosylation yield was 0.762 g⋅g–1, meaning that 76% of the reacted lactose corresponded to transgalactosylation and 24% to hydrolysis, which is a definite advantage of this mode of operation. Even though XLactose in CSTR was lower than in other reported modes of operation for lactulose synthesis, transgalactosylation was more favored over hydrolysis which reduced the inhibitory effect of galactose on β-galactosidase.

New Strategy for the Immobilization of Lipases on Glyoxyl-Agarose Supports: Production of Robust Biocatalysts for Natural Oil Transformation.

Immobilization on Glyoxyl–agarose support (Gx) is one of the best strategies to stabilize enzymes. However, the strategy is difficult to apply at neutral pH when most enzymes are stable and, even when possible, produces labile derivatives. This work contributes to overcoming this hurdle through a strategy that combines solid-phase amination, presence of key additives, and derivative basification. To this end, aminated industrial lipases from Candida artarctica (CAL), Thermomyces lunuginosus (TLL), and the recombinant Geobacillus thermocatenulatus (BTL2) were immobilized on Gx for the first time at neutral pH using anthranilic acid (AA) or DTT as additives (immobilization yields >70%; recovered activities 37.5–76.7%). The spectroscopic evidence suggests nucleophilic catalysis and/or adsorption as the initial lipase immobilization events. Subsequent basification drastically increases the stability of BTL2–glyoxyl derivatives under harsh conditions (t1/2, from 2.1–54.5 h at 70 °C; from 10.2 h–140 h in 80% dioxane). The novel BTL2-derivatives were active and selective in fish oil hydrolysis (1.0–1.8 μmol of polyunsaturated fatty acids (PUFAs) min−1·g−1) whereas the selected TLL-derivative was as active and stable in biodiesel production (fatty ethyl esters, EE) as the commercial Novozyme®-435 after ten reaction cycles (~70% EE). Therefore, the potential of the proposed strategy in producing suitable biocatalysts for industrial processes was demonstrated.

Effects of Triton X-100 and PEG on the Catalytic Properties and Thermal Stability of Lipase from Candida Rugosa Free and Immobilized on Glyoxyl-Agarose.

Background: Candida rugosa Lipase (CRL) shows a very low alkaline stability that comprises its immobilization on glyoxyl-agarose, which requires pH above 10. In this way, an adaptation from the original method was used; an enzyme solution at pH 7 was slowly added at a suspension of glyoxyl-agarose prepared in bicarbonate buffer, pH 10. This change of protocol was enough for allowing the preparation of derivatives actives of CRL on glyoxyl-agarose and verifying the effect of this modified procedure on the properties of the immobilized enzyme. The effect of the additives Triton-X-100 and polyethylene glycol (PEG) on the enzymatic activity recovery and immobilized enzyme stability was evaluated.Methods:The glyoxyl-agarose support was prepared by etherification of 6% agarose beads with glycidol and further oxidation with sodium periodate. CRL was immobilized covalently on glyoxyl-agarose support in the absence and presence of 1% (w/v) Triton-X-100 or 5 g L-1 polyethylene glycol (PEG). The lipolysis activity of the free and immobilized enzyme was determined at 37ºC and pH 7.0, using p-nitrophenyl palmitate (p-NPP) as substrate. Profiles of temperature-activity (37-65ºC, pH 7.0) and pH-activity (6.0-9.5, 37ºC) were evaluated as well as thermal (45ºC and pH 8.0) and operational (15 min batches of p-NPP hydrolysis at 50ºC and pH 8.0) stabilities of free and immobilized CRL.Results:Using a single modification of the original protocol, the CRL poorly stable under alkaline conditions could be immobilized on glyoxyl-agarose in its active conformation (recovered activity varying from 10.3 to 30.4%). Besides, the presence of a detergent (Triton-X-100) and an enzyme stabilizer (PEG) contributed to the preparation of more active and more stable biocatalysts, respectively. CRL immobilized on glyoxyl-agarose in the presence of PEG was around 5 times more stable than the free CRL and around 3 times more stable than the CRL immobilized on glyoxyl-agarose in absence of PEG. The higher stability of the CRL-glyoxyl derivative prepared in the presence of PEG allowed its reuse in four successive 15 min-batches of p-nitrophenyl palmitate hydrolysis at 50ºC and pH 8.0.Conclusion:The technique of immobilizing enzymes covalently on glyoxyl-agarose showed promising results for Candida rugosa lipase (CRL). The derivatives prepared in the presence of the additives retained two to three times more activity than those prepared in the absence of additives. The enzyme immobilized in presence of PEG was about three times more stable than the enzyme immobilized in absence of this additive. Maximum catalytic activity of the immobilized CRL (in absence of additives) was observed in a temperature 10ºC above that for the free enzyme and the pH of the maximum activity was maintained in the range 6.5-7.5 for free and immobilized CRL.

Immobilization-stabilization of a complex multimeric sucrose synthase from Nitrosomonas europaea. Synthesis of UDP-glucose.

Sucrose synthases (SuSys) can be used to synthesize cost-effective uridine 5'-diphosphate glucose (UDP-glc) or can be coupled to glycosyltransferases (GTs) for the continuous recycling of UDP-glc. In this study, we present the first report of the immobilization-stabilization of a SuSy by multipoint covalent attachment. This stabilization strategy is very complex for multimeric enzymes because a very intense multipoint attachment can promote a dramatic loss of activity and/or stability. The homotetrameric SuSy from Nitrosomonas europaea (SuSyNe) was immobilized on a glyoxyl agarose support through two different orientations. The first occurred at pH 8.5 through the surface area containing the greatest number of amino termini from several enzyme subunits. The second orientation occurred at pH 10 through the region of the whole enzyme containing the highest number of Lys residues. The multipoint covalent immobilization of SuSy on glyoxyl agarose at pH 10 provided a very significant stabilization factor under reaction conditions (almost 1000-fold more stable than soluble enzyme). Unfortunately, this important enzyme rigidification led to a dramatic loss of catalytic activity. A less stabilized conjugate, which was 65-fold more stable than the soluble form, preserved 64% of its initial catalytic activity. This derivative could be used for 3 reaction cycles and yielded approximately 210mM of UDP-glc per cycle. This optimal biocatalyst was modified with a polycationic polymer, polyethyleneimine (PEI), increasing its stability in the presence of the organic co-solvents necessary to glycosylate apolar antioxidants by GTs coupled to SuSy.

Oriented immobilization and characterization of a poly-lysine-tagged cephalosporin C acylase on glyoxyl agarose support.

Cephalosporin C acylase (CCA), an important industrial enzyme for the production of 7-aminocephalosporanic acid, has very limited and scattered surface lysine residues. A mutant of cephalosporin C acylase (mCCA) has been designed to fuse a poly-lysine tag to the C-terminal of the β-subunit, which is far away from the active site. The free mCCA showed a near equal specific activity with the wild-type CCA, while a much higher activity recovery was obtained for the mCCA than its wild-type counterpart after immobilization on glyoxyl agarose support (73.3 versus 53.3 %). The mCCA's oriented immobilization enables it to obtain a higher substrate affinity and even a higher thermal stability than the wild-type enzyme. The improvement of stability might be attributed to the multipoint covalent attachment by the oriented enzyme immobilization via the adhered poly-lysine tag, which prevents the dissociation of the β-subunit of CCA from the support.

Purification and improvement of the functional properties of Rhizopus oryzae lipase using immobilization techniques

The adsorption/immobilization of Rhizopus oryzae lipase (ROL) has permitted the development of several strategies to improve the properties of this industrial enzyme. The enzyme can be purified, immobilized, hyperactivated and stabilized, though its enantioselectivity could still be improved. A moderately hydrophobic support (e.g., phenyl-Toyopearl) allows the almost-quantitative immobilization of the enzyme via selective hydrophobic adsorption, while any contaminant proteins are not adsorbed. A further desorption of the adsorbed enzyme with surfactants (e.g., 0.5% sucrose laurate) allows the complete purification of the enzyme in only one step, with a 90% purification yield and an 8-fold purification factor. By using a more hydrophobic support, the ROL can be immobilized and hyperactivated. The enzyme is completely adsorbed on octyl-Sepharose and C18-Sepabeads. The pure enzyme (when adsorbed on these supports) becomes hyperactivated to approximately 250% of the activity of the soluble enzyme. In this study, two protocols for the covalent immobilization of ROL were developed: a one-point immobilization on CNBr-activated Sepharose and a multipoint covalent immobilization on highly activated glyoxyl-agarose supports. The multipoint attached enzyme decreased in activity to 50% of the activity of the soluble enzyme, but the derivatives were 300-fold more stable than the soluble enzyme. The hydrolysis of racemic 2-O-butyryl-2-phenylacetic acid was modulated by the different immobilized derivatives. In fact, the most active and selective preparation was demonstrated to be the C18-SB-ROL derivative (0.05UI/mg and E=22) when compared with the CNBr-ROL enzyme preparation (0.0073UI/mg and E=3.5).

Continuous production of xylooligosaccharides in a packed bed reactor with immobilized–stabilized biocatalysts of xylanase from Aspergillus versicolor

The production of xylooligosaccharides (XOS) using a packed-bed enzymatic reactor was studied at lab-scale. For this, a xylanase from Aspergillus versicolor was immobilized on different supports. The optimal derivative was xylanase immobilized on glyoxyl-agarose supports. This derivative preserved 85% of its catalytic activity; it was around 700-fold more stable than the soluble enzyme after incubation at 60 °C and was able to be reused for at least 10 1 h-cycles retaining full catalytic activity. About 18% of oligosaccharides with prebiotic interest (X2–X6) were produced by the glyoxyl derivative in batch hydrolysis. The production of xylobiose was 2.5-fold higher using the immobilized preparation than with soluble enzyme and small concentrations of xylose (<0.1%) were observed only at the end of the reaction. The derivative was employed on a packed bed reactor, and the continuous operation with no recirculation reached 56% and 70% of the end of reaction with flow rates of 60 mL/h and 12 mL/h, respectively. In continuous operation with recirculation at a flow rate of 60 mL/h, the reaction was completed after four hours.

Production of xylo-oligosaccharides by immobilized-stabilized derivatives of endo-xylanase from Streptomyces halstedii

An endoxylanase from Streptomyces halstedii was stabilized by multipoint covalent immobilization on glyoxyl-agarose supports. The immobilized enzyme derivatives preserved 65% of the catalytic activity corresponding to the one of soluble enzyme that had been immobilized. These immobilized derivatives were 200 times more stable 200 times more stable than the one-point covalently immobilized derivative in experiments involving thermal inactivation at 60°C. The activity and stability of the immobilized enzyme was higher at pH 5.0 than at pH 7.0. The optimal temperature for xylan hydrolysis was 10°C higher for the stabilized derivative than for the non-stabilized derivative. On the other hand, the highest loading capacity of activated 10% agarose gels was 75mg of enzyme per mL of support. To prevent diffusional limitations, low loaded derivatives (containing 0.2mg of enzyme per mL of support) were used to study the hydrolysis of xylan at high concentration (close to 1% (w/v)). 80% of the reducing sugars were released after 3h at 55°C. After 80% of enzymatic hydrolysis, a mixture of small xylo-oligosaccharides was obtained (from xylobiose to xylohexose) with a high percentage of xylobiose and minimal amounts of xylose. The immobilized-stabilized derivatives were used for 10 reaction cycles with no loss of catalytic activity.

Reactivation of a thermostable lipase by solid phase unfolding/refolding: Effect of cysteine residues on refolding efficiency

Lipase from Geobacillus thermocatenulatus (BTL2) was immobilized in two different matrixes. In one derivative, the enzyme was immobilized on agarose activated with cyanogen bromide (CNBr-BTL2) via its most reactive superficial amino group, whereas the other derivative was covalently immobilized on glyoxyl agarose supports (Gx-BTL2). The latter immobilization protocol leads to intense multipoint covalent attachment between the lysine richest region of enzyme and the glyoxyl groups on the support surface. The resulted solid derivatives were unfolded by incubation under high concentrations of guanidine and then resuspended in aqueous media under different experimental conditions. In both CNBr-BTL2 and Gx-BTL2 derivatives, the oxidation of Cys residues during the unfolding/refolding processes led to inefficient folding for the enzyme because only 25–30% of its initial activity was recovered after 3 h in refolding conditions. Dithiothreitol (DTT), a very mild reducing agent, prevented Cys oxidation during the unfolding/refolding process, greatly improving activity recovery in the refolded forms. In parallel, other variables such as pH, buffer composition and the presence of polymers and other additives, had different effects on refolding efficiencies and refolding rates for both derivatives. In the case of solid derivatives of BTL2 immobilized on CNBr-agarose, the surface's chemistry was crucial to guarantee an optimal protein refolding. In this way, uncharged protein vicinities resulted in better refolding efficiencies than those charged ones.

Improvement of Enzyme Properties with a Two-Step Immobilizaton Process on Novel Heterofunctional Supports

Novel heterofunctional glyoxyl-agarose supports were prepared. These supports contain a high concentration of groups (such as quaternary ammonium groups, carboxyl groups, and metal chelates) that are capable of adsorbing proteins, physically or chemically, at neutral pH as well as a high concentration of glyoxyl groups that are unable to immobilize covalently proteins at neutral pH. By using these supports, a two-step immobilization protocol was developed. In the first step, enzymes were adsorbed at pH 7.0 through adsorption of surface regions, which are complementary to the adsorbing groups on the support, and in the second step, the immobilized derivatives were incubated under alkaline conditions to promote an intramolecular multipoint covalent attachment between the glyoxyl groups on the support and the amino groups on the enzyme surface. These new derivatives were compared with those obtained on a monofunctional glyoxyl support at pH 10, in which the region with the greatest number of lysine residues participates in the first immobilization step. In some cases, multipoint immobilization on heterofunctional supports was much more efficient than what was achieved on the monofunctional support. For example, derivatives of tannase from Lactobacillus plantarum on an amino-glyoxyl heterofunctional support were 20-fold more stable than the best derivative on a monofunctional glyoxyl support. Derivatives of lipase from Geobacillus thermocatenulatus (BTL2) on the amino-glyoxyl supports were two times more active and four times more enantioselective than the corresponding monofunctional glyoxyl support derivative.

Immobilization–stabilization of glucoamylase: Chemical modification of the enzyme surface followed by covalent attachment on highly activated glyoxyl-agarose supports

Commercial glucoamylase immobilizes very slowly on highly activated glyoxyl-agarose supports. The resulting derivatives were only 6-fold more stable than soluble enzyme. The unmodified glucoamylase, highly glycosylated, seems to have a low number of Lys able to react with glyoxyl groups on the support. Thus, the enzyme surface was highly enriched in amino groups by chemical modification of carboxyl groups (activated with carbodiimide) with ethylenediamine. The aminated enzyme preserves a good percentage of activity (80%) and it exhibits the same stability than unmodified enzyme. The aminated enzyme was immobilized very rapidly on highly activated glyoxyl-agarose support. The new resulting derivatives preserved 50% of activity and were more than 500-fold more stable than soluble enzyme in experiments of thermal inactivation.

Production of Brassica carinata Protein Hydrolyzates with a High Fischer's Ratio Using Immobilized Proteases

Brassica carinata protein isolates were hydrolyzed using the digestive enzymes trypsin, chymotrypsin, and carboxypeptidase A in order to obtain hydrolyzates with a high Fischer's ratio. The proteases were immobilized using two glyoxyl-agarose supports of different porosity, 4 and 10% agarose gels, in order to evaluate the effect of substrate diffusion into the support containing the enzyme on the hydrolytic process. Reaction time, substrate concentration, and the enzyme to substrate ratio were optimized in an attempt to increase the Fischer's ratio in the resulting hydrolyzates. Gel filtration chromatography of a hydrolyzate with a degree of hydrolysis of 36% yielded a fraction that represented 31% of the total hydrolyzed proteins and had a Fischer's ratio of 28.3 with a phenylalanine + tyrosine content below 1.5%. This material could be used for preparing special diets when there is a need to increase the supply of branched amino acids and/or reduce the intake of aromatic amino acids.

Evaluation of different immobilization strategies to prepare an industrial biocatalyst of formate dehydrogenase from Candida boidinii

Different immobilization strategies have been issued to prepare a stable and active biocatalyst of the formate dehydrogenase from Candida boidinii: immobilization on glutaraldehyde, epoxy, amino-epoxy, glyoxyl or treatment of enzymes adsorbed on aminated supports with glutaraldehyde. The best results in terms of stability were achieved using amino-epoxy supports (by a 12-fold factor compared to soluble and BrCN immobilized enzyme) and glyoxyl agarose supports (by a 150-fold factor), although in both cases activity recovery was just over 15%. The use of milder conditions permitted to improve the recovered activity, but decreased in a parallel way the enzyme stability, until being almost identical to the soluble enzyme. Alternatively, ionic adsorption of the enzyme was assayed. Supports coated with PEI permitted to keep full activity of the enzyme, permitted a very strong adsorption and the reutilization of the support after enzyme inactivation. Moreover, all the immobilization techniques prevent enzyme inactivation under strong stirring, necessary to keep the pH value and eliminate the CO 2 formed during the reaction. In this way, a battery of solutions to immobilize this enzyme has been proposed, fulfilling the different requirements that the use of this NADH recycling enzyme may have, depending on the particular reaction.

Some special features of glyoxyl supports to immobilize proteins

This paper described that glyoxyl activated supports are only able to quantitatively immobilize proteins at alkaline pH values and using highly activated supports (otherwise, immobilization is negligible). Furthermore, the immobilization rate depends on the surface density of glyoxyl groups and not on the total concentration of groups in the suspension. Moreover, the temperature presented a dramatic effect on enzyme immobilization rate and different enzymes become immobilized at very different rates. Finally, it was not possible to detect immobilization of mono aminated compounds at neither alkaline nor neutral pH values. In fact, these supports may be used to purify enzymes due to these great differences in immobilization rate (e.g., glutamate racemase). These special features are not shared with other supports usually utilized for covalent immobilization of enzymes (e.g., glutaraldehyde, cyanogen bromide supports) that are able to immobilize proteins by just one very stable bond and suggesting an unique mechanism for glyoxyl agarose supports. In fact, all these results suggested that each individual amino-glyoxyl bond is very weak. Therefore, the first immobilization of proteins on supports activated with glyoxyl groups, in absence of reducing reagents, needs the simultaneous establishment of, at least, two attachments between the protein and the support. This mechanism promotes that proteins become immobilized by the area/s where the highest lysine residues density is located and explain the high enzyme stabilization usually achieved by using this immobilization technique.