Adsorption features of reduced aminated supports modified with glutaraldehyde: Understanding the heterofunctional features of these supports.

Immobilization of enzymes on aminated supports using the glutaraldehyde chemistry may involve three different interactions, cationic, hydrophobic, and covalent interactions. To try to understand the impact this heterofunctionality, we study the physical adsorption of the beta-galactosidase from Aspergillus niger, on aminated supports (MANAE) and aminated supports with one (MANAE-GLU) or two molecules of glutaraldehyde (MANAE-GLU-GLU). To eliminate the chemical reactivity of the glutaraldehyde, the supports were reduced using sodium borohydride. After enzyme adsorption, the release of the enzyme from the supports using different NaCl concentrations, Triton X100, ionic detergents (SDS and CTAB), or different temperatures (4 °C to 55 °C) was studied. Using MANAE support, at 0.3 M NaCl almost all the immobilized enzyme was released. Using MANAE-GLU, 0.3 M, and 0.6 M NaCl similar results were obtained. However, incubation at 1 M or 2 M NaCl, many enzyme molecules were not released from the support. For the MANAE-GLU-GLU support, none of the tested concentrations of NaCl was sufficient to release all enzyme bound to the support. Only using high temperatures, 0.6 M NaCl, and 1 % CTAB or SDS, could the totality of the proteins be released from the support. The results shown in this paper confirm the heterofunctional character of aminated supports modified with glutaraldehyde.

Immobilization and Application of the Recombinant Xylanase GH10 of Malbranchea pulchella in the Production of Xylooligosaccharides from Hydrothermal Liquor of the Eucalyptus (Eucalyptus grandis) Wood Chips.

Xylooligosaccharides (XOS) are widely used in the food industry as prebiotic components. XOS with high purity are required for practical prebiotic function and other biological benefits, such as antioxidant and inflammatory properties. In this work, we immobilized the recombinant endo-1,4-ß-xylanase of Malbranchea pulchella (MpXyn10) in various chemical supports and evaluated its potential to produce xylooligosaccharides (XOS) from hydrothermal liquor of eucalyptus wood chips. Values >90% of immobilization yields were achieved from amino-activated supports for 120 min. The highest recovery values were found on Purolite (142%) and MANAE-MpXyn10 (137%) derivatives, which maintained more than 90% residual activity for 24 h at 70 °C, while the free-MpXyn10 maintained only 11%. In addition, active MpXyn10 derivatives were stable in the range of pH 4.0−6.0 and the presence of the furfural and HMF compounds. MpXyn10 derivatives were tested to produce XOS from xylan of various sources. Maximum values were observed for birchwood xylan at 8.6 mg mL−1 and wheat arabinoxylan at 8.9 mg mL−1, using Purolite-MpXyn10. Its derivative was also successfully applied in the hydrolysis of soluble xylan present in hydrothermal liquor, with 0.9 mg mL−1 of XOS after 3 h at 50 °C. This derivative maintained more than 80% XOS yield after six cycles of the assay. The results obtained provide a basis for the application of immobilized MpXyn10 to produce XOS with high purity and other high-value-added products in the lignocellulosic biorefinery field.

Prebiotic effect of sorghum biomass xylooligosaccharides employing immobilized endoxylanase from Thermomyces lanuginosus PC7S1T.

Purified endoxylanase from Thermomyces lanuginosus PC7S1T was immobilized in calcium alginate, resulting in a yield of 78.5% and a reusability for 11 cycles. The stability of the immobilized enzyme was given for a pH range of 4 to 9 for 96 h. Endoxylanase immobilized in calcium alginate at 65 °C exhibited thermal stability equal to the soluble enzyme for 5 h, and at high temperatures of 75 °C and 85 °C showed half-lives of 4 and 3 h, respectively. Both soluble endoxylanase and immobilized forms were able to hydrolyze hemicellulose, obtained from low-lignin sorghum biomass pretreated with 5% H2O2 and 2% NaOH, after 1 h of incubation at 65 °C, releasing a mixture of short-chain xylooligosaccharides (X2-X6). The highest amounts of XOS generated were those for X5 (24 to 40%), X4 (33 to 39%), and X3 (11 to 22%). These XOS acted as prebiotics, promoting the growth of the probiotic L. acidophilus, similar to glucose in the MRS broth. These results show the potential of low-lignin sorghum to generate XOS with prebiotic activity, suggesting the application of these compounds in the food industry.

Effect of Tris Buffer in the Intensity of the Multipoint Covalent Immobilization of Enzymes in Glyoxyl-Agarose Beads.

Tris is an extensively used buffer that presents a primary amine group on its structure. In the present work trypsin, chymotrypsin and penicillin G acylase (PGA) were immobilized/stabilized on glyoxyl agarose in presence of different concentrations of Tris (from 0 to 20 mM). The effects of the presence of Tris during immobilization were studied analyzing the thermal stability of the obtained immobilized biocatalysts. The results indicate a reduction of the enzyme stability when immobilized in the presence of Tris. This effect can be observed in inactivations carried out at pH 5, 7, and 9 with all the enzymes assayed. The reduction of enzyme stability increased with the Tris concentration. Another interesting result is that the stability reduction was more noticeable for immobilized PGA than in the other immobilized enzymes, the biocatalysts prepared in presence of 20 mM Tris lost totally the activity at pH 7 just after 1 h of inactivation, while the reference at this time still kept around 61 % of the residual activity. These differences are most likely due to the homogeneous distribution of the Lys groups in PGA compared to trypsin and chymotrypsin (where almost 50% of Lys group are in a small percentage of the protein surface). The results suggest that Tris could be affecting the multipoint covalent immobilization in two different ways, on one hand, reducing the number of available glyoxyl groups of the support during immobilization, and on the other hand, generating some steric hindrances that difficult the formation of covalent bonds.

Effect of Concentrated Salts Solutions on the Stability of Immobilized Enzymes: Influence of Inactivation Conditions and Immobilization Protocol.

This paper aims to investigate the effects of some salts (NaCl, (NH4)2SO4 and Na2SO4) at pH 5.0, 7.0 and 9.0 on the stability of 13 different immobilized enzymes: five lipases, three proteases, two glycosidases, and one laccase, penicillin G acylase and catalase. The enzymes were immobilized to prevent their aggregation. Lipases were immobilized via interfacial activation on octyl agarose or on glutaraldehyde-amino agarose beads, proteases on glyoxyl agarose or glutaraldehyde-amino agarose beads. The use of high concentrations of salts usually has some effects on enzyme stability, but the intensity and nature of these effects depends on the inactivation pH, nature and concentration of the salt, enzyme and immobilization protocol. The same salt can be a stabilizing or a destabilizing agent for a specific enzyme depending on its concentration, inactivation pH and immobilization protocol. Using lipases, (NH4)2SO4 generally permits the highest stabilities (although this is not a universal rule), but using the other enzymes this salt is in many instances a destabilizing agent. At pH 9.0, it is more likely to find a salt destabilizing effect than at pH 7.0. Results confirm the difficulty of foreseeing the effect of high concentrations of salts in a specific immobilized enzyme.

Effect of amine length in the interference of the multipoint covalent immobilization of enzymes on glyoxyl agarose beads.

Trypsin, chymotrypsin, penicillin G acylase and ficin extract have been stabilized by immobilization on glyoxyl agarose, adding different aliphatic compounds bearing a primary amine group during the immobilization: ethyl amine, butyl amine, hexyl amine (at concentrations ranging from 0 to 20 mM) and octyl amine (from 0 to 10 mM) to analyze their effects on the immobilized enzyme stability. As expected, the presence of amines reduced the intensity of the enzyme-support multipoint covalent attachment, and therefore the enzyme stability. However, it is clear that this effect is higher using octyl amine for all enzymes (in some cases the enzyme immobilized in the presence of 10 mM octyl amine was almost inactivated while the reference kept over 50 % of the initial activity). This way, it seems that the most important effect of the presence of aminated compounds came from the generation of steric hindrances to the enzyme/support multi-reaction promoted by the ammines that are interacting with the aldehyde groups. In some instances, just 1 mM of aminated compounds is enough to greatly decrease enzyme stability. The results suggested that, if the composition of the enzyme extract is unknown, to eliminate small aminated compounds may be necessary to maximize the enzyme-support reaction.

Preparation of immobilized/stabilized biocatalysts of ß-glucosidases from different sources: Importance of the support active groups and the immobilization protocol.

ß-Glucosidases from two different commercial preparations, Pectinex Ultra SP-L and Celluclast® 1.5L, were immobilized on divinylsulfone (DVS) supports at pH 5.0, 7.0, 9.0, and 10. In addition, the biocatalysts were also immobilized in agarose beads activated by glyoxyl, and epoxide as reagent groups. The best immobilization results were observed using higher pH values on DVS-agarose, and for Celluclast® 1.5L, good results were also obtained using the glyoxil-agarose immobilization. The biocatalyst obtained using Pectinex Ultra SP-L showed the highest thermal stability, at 65°C, and an operational stability of 67% of activity after 10 reuses cycles when immobilized on DVS-agarose immobilized at pH 10 and blocked with ethylenediamine. The ß-glucosidase from Celluclast® 1.5L produced best results when immobilized on DVS-agarose immobilized at pH 9 and blocked with glycine, reaching 7.76-fold higher thermal stability compared to its free form and maintaining 76% of its activity after 10 successive cycles. The new biocatalysts obtained by these protocols showed reduction of glucose inhibition of enzymes, demonstrating the influence of immobilization protocols, pH, and blocking agent.

Immobilization and stabilization of different ß-glucosidases using the glutaraldehyde chemistry: Optimal protocol depends on the enzyme.

Three ß-glucosidases (Pectinex Ultra SP-L, Pectinex Ultra Clear and homemade preparation from Aspergillus niger) were immobilized using different strategies: ionic adsorption on aminated (MANAE)-agarose beads at pH 5, 7, and 9, followed by biocatalysts modification with glutaraldehyde, or on glutaraldehyde pre-activated supports. The pH of the immobilization was altered to allow different enzyme molecule orientations on the support surface. The biocatalysts from Pectinex Ultra SP-L showed the highest thermal and operational stabilities when immobilized on MANAE-agarose-glutaraldehyde at pH 7. The ß-glucosidase from Pectinex Ultra Clear and from A. niger produced best results when immobilized on MANAE-agarose beads at pH 5 and 7, respectively, which was later treated with glutaraldehyde. The best immobilization results using pre-activated supports were observed for the enzyme present in Pectinex Ultra SP-L, to which the highest thermal stabilities were obtained. Remarkably, the enzyme from A. niger, immobilized on MANAE-agarose at pH 9 and subsequently treated with glutaraldehyde, produced the highest stabilization (approximately 560 times more stable than soluble enzyme at 60 °C). Results showed that optimal protocol for ß-glucosidases immobilizations using the glutaraldehyde chemistry must be individually tested and tailored to each type of enzyme.