Cloning, purification, and biochemical characterization of an esterase from Aspergillus nidulans

A large accumulation of agro-industrial waste from different segments is generated daily and is often not properly managed. There are now other fronts in research to give a destination to these residues; these studies are generally aimed at obtaining new and better enzymes and the formulation of enzymatic cocktails that contain (for example, cellulases and hemicellulases) responsible for the degradation of lignocellulosic material. The plant cell wall is mainly composed of cellulose, hemicellulose, and lignin, forming a complex structure. Xylan is one of the main constituents of hemicellulose. To degrade this structure, enzymatic hydrolysis must occur synergistically with xylanolytic enzymes, such as endo-beta-1,4-xylanases, -xylosidases, and acetyl xylan esterase (AXE). In the current work, we reported the purification and biochemical characterization of an acetyl xylan esterase (AxeCE3) from Aspergillus nidulans. The axeCE3 gene was cloned into the pEXPYR vector and transformed into A. nidulans A773 for protein expression. The enzyme AxeCE3 was purified and characterized for its biochemical properties. AxeCE3 showed activity over a wide range of pH (3.0-9.0) and temperature (30-70 °C), with maximum activity at 55 °C, pH 7.0. Regarding the stability at temperature, AxeCE3 showed values above 90% of residual activity after 24 h of incubation at 45 and 50 °C. In relation to stability at pH, AxeCE3 maintained more than 90% of its residual activity after being incubated at 25 °C for 24 h between the pH range 3.0 to 9.0. It was also verified the effect of possible inhibitors (ethylenediamine tetraacetic acid (EDTA), Furfural, and 5- Hydroxymethylfurfural (5-HMF)) on the enzyme activity. AxeCE3 maintained 88% of relative activity at 5 mM EDTA, 43% and 82% at 50 mM furfural and 5-HMF, respectively. The results showed that AxeCE3 has interesting properties to use in the development in the formulation of enzymatic cocktails for the hydrolysis of lignocellulosic residues.The work was supported by the following: FAPESP (São Paulo Research Foundation, grants: 2014/50884 and 2018/07522-6) and National Institute of Science and Technology of Bioethanol, INCT, CNPq (grant: 465319/2014-9) and process 301963/2017-7. Research scholarships were granted to RCA and DA by FAPESP (Grant No: 2020/00081-4 and No: 2020/15510-8), to GSA by CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Finance Code 001).info:eu-repo/semantics/publishedVersio

Development of new eco-friendly supports for immobilization of enzymes based on cellulose residues

The pulp and paper industry generates a high volume of solid wastes that are usually burned to obtain energy, directed to landfills, or incinerated. Among the wastes generated in this process is the paper sludge, a residue rich in cellulose with low lignin content making it a useful raw material to produce high-value products such as cheap immobilization supports. In the current work, paper sludge was activated using different functional groups (amino, epoxy, and aldehyde). The xylanase GH10 from Malbranchea pulchella was used as a model enzyme for the immobilization assays. The enzyme was efficiently immobilized through reversible immobilization on aminated support monoaminoethyl-N-ethyl (MANAE) and polyethyleneimine (PEI), achieving yields of more than 90 %. Furthermore, the yield and activity of the biocatalyst immobilized with paper sludge using groups glyoxyl and epoxy (irreversible immobilization) were higher than the enzyme immobilized on agarose supports. The biocatalyst immobilized on paper sludge-epoxy presented the best results, reaching 12.54 U.g-1 of support. Therefore, the use of paper sludge, such as backbone of different immobilization supports, was an efficient method and promising approach for the immobilization of enzymes such as xylanase. More studies are necessary to optimize the displayed potential for future applications as tests in other enzymes of different characteristics and their behavior with bifunctional reagents as the glutaraldehyde. Furthermore, the valorization of these residues in a biorefinery context holds great socio-economical relevance for Portugal and Brazil. Thus, our perspectives are the development of a hybrid biocatalyst using magnetics nanoparticles and paper sludge.info:eu-repo/semantics/publishedVersio

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-β-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.The authors gratefully acknowledge FAPESP (São Paulo Research Foundation, grants No: 2018/07522-6) and FCT (POCI-01-0145-FEDER-032206)—transnational cooperation project EcoTech, and National Institute of Science and Technology of Bioethanol, INCT, CNPq 465319/2014-9/FAPESP n ◦ 2014/50884- 5) for financial support. Research scholarships were granted to RCA, DA, and JCSS by FAPESP (Grant No: 2020/00081-4, 2020/15510-8, and 2019/21989-7, respectively), to CCVD and VEP by CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Finance Code 001).info:eu-repo/semantics/publishedVersio

Immobilization of beta-glucosidases on agarose supports activated with different functional groups

As β-glicosidases hidrolisam ligações β-glicosídicas a partir da extremidade não redutora de oligossacarídeos e glicosídeos conjugados, e possuem largo espectro de aplicações biotecnológicas como, na estabilização e enriquecimento de bebidas a exemplo dos sucos e vinhos. Porém, estes processos são realizados em um meio com alta concentração de glicose, uma molécula com grande efeito inibitório das β- glicosidases. Deste modo, o presente trabalho visou desenvolver biocatalisadores imobilizados com atividade de β-glicosidase, capazes de serem aplicados em bebidas à base de uva. A imobilização desta enzima é uma ferramenta extremamente relevante para ultrapassar os desafios da sua aplicação industrial através da melhoria das suas características, como, um melhor controle operacional, e menor oneração. Inicialmente, as atividades de β-glicosidase de nove preparações enzimáticas comerciais foram comparadas avaliando-se os efeitos do pH, estabilidade térmica, parâmetros cinéticos e tolerância à glicose. O valor do pH do meio de reação influenciou fortemente as atividades, estabilidades, e inibição à glicose das β- glicosidases testadas. As preparações de Pectinex Ultra SP-L, Pectinex Utra Clear e Celluclast® 1,5 L, e o extrato enzimático de Aspergillus niger URM 6642 foram selecionadas com base em sua atividade de β-glicosidase e tolerância à glicose. Em seguida, a imobilização das enzimas selecionadas foram testadas em 5 diferentes suportes: MANAE, MANAE-glutaraldeído, glioxil-agarose, divinilsulfona-agarose e epóxi-agarose. A imobilização das β-glicosidases de Pectinex Ultra SP-L, Pectinex Utra Clear e de A. niger URM 6642 no suporte MANAE-agarose foram realizadas em pH 5, 7 e 9 para permitir diferentes orientações das moléculas da enzima na superfície do suporte, e, posteriormente, também modificadas com glutaraldeído. Os resultados demonstraram que o protocolo ideal para imobilizações de β-glicosidases, usando a o glutaraldeído, deve ser testado individualmente e adaptado para cada tipo de enzima. A imobilização das β-glicosidases de Pectinex Ultra SP-L e Celluclast® 1.5L em DVS-agarose também foram testadas nos pHs 5, 7, 9 e 10. Os resultados demonstraram que o pH de imobilização ocasionou forte influência nos rendimentos de imobilização, e afetou a estabilidade das enzimas e a tolerância à glicose. As enzimas também foram estudadas quanto a descoloração do suco de uva e do vinho tinto. Os resultados deste trabalho demonstraram que os biocatalisadores estudados possuem características promissoras, e, portanto, de grande importância para indústria.β-glucosidases hydrolyze β-glycosidic bonds from nonreducing terminal of oligosaccharides and conjugated glycosides, and have wide range of biotechnological applications such as, in the stabilization and enrichment drinks like juices and wines. However, these processes are carried out in a reaction medium with a high concentration of glucose, a molecule with high inhibitory effect on β-glucosidases. Thus, the present work aimed to develop immobilized biocatalysts with β-glucosidase activity subject to application in grape-based beverages. The immobilization of this enzyme is an extremely relevant tool to overcome the challenges of its industrial application through the improvement of its characteristics, such as higher operational control and lower cost. Initially, the β-glucosidase activities of nine commercial enzyme preparations were compared by evaluating the effects of pH, thermal stability, kinetic parameters, and glucose tolerance. The pH value of the reaction medium strongly influenced the activities, stabilities, and glucose inhibition of the β-glucosidases tested. The Preparations of Pectinex Ultra SP-L, Pectinex Utra Clear, Celluclast® 1.5 L, and a strain of Aspergillus niger URM 6642 were selected based on their β-glucosidase activity and glucose tolerance. Then, the immobilization of selected enzymes were tested on 5 different supports: MANAE, MANAE-glutaraldehyde, glyoxyl-agarose, divinylsulfone-agarose and epoxy-agarose. The immobilization of β-glucosidases from Pectinex Ultra SP-L, Pectinex Utra Clear and A. niger URM 6642 on the MANAEagarose support were carried out at pH 5, 7 and 9 to allow different orientations of the enzyme molecules on the support surface, and later also modified with glutaraldehyde. Results showed that optimal protocol for β-glucosidases immobilizations using the glutaraldehyde chemistry must be individually tested and tailored to each type of enzyme. The immobilization of β-glucosidases from Pectinex Ultra SP-L and Celluclast® 1.5L in DVS-agarose were also tested at pHs 5, 7, 9 and 10. The esults showed that the immobilization pH had a high influence on the immobilization yields. The change in immobilization pH also affected enzyme stability and glucose tolerance. Enzymes were also studied for discoloration of grape juice and red wine. The results of this work showed that the studied biocatalysts have promising characteristics, and therefore, of great importance for industry

Preparation of immobilized/stabilized biocatalysts of β-glucosidases from different sources : importance of the support active groups and the immobilization protocol

β-Glucosidases from two different commercial preparations, Pectinex Ultra SP-L andCelluclast 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 byglyoxyl, and epoxide as reagent groups. The best immobilization results were observedusing higher pH values on DVS-agarose, and for Celluclast 1.5L, good results were alsoobtained using the glyoxil-agarose immobilization. The biocatalyst obtained using PectinexUltra SP-L showed the highestthermal stability, at 65 C, and an operational stability of67% of activity after 10 reuses cycles when immobilized on DVS-agarose immobilized atpH 10 and blocked with ethylenediamine. The β-glucosidase from Celluclast 1.5L pro-duced best results when immobilized on DVS-agarose immobilized at pH 9 and blockedwith glycine, reaching 7.76-fold higher thermal stability compared to its free form andmaintaining 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

Preparation of immobilized/stabilized biocatalysts of β‐glucosidases from different sources: Importance of the support active groups and the immobilization protocol

β-Glucosidases from two different commercial preparations, Pectinex Ultra SP-L andCelluclast 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 byglyoxyl, and epoxide as reagent groups. The best immobilization results were observedusing higher pH values on DVS-agarose, and for Celluclast 1.5L, good results were alsoobtained using the glyoxil-agarose immobilization. The biocatalyst obtained using PectinexUltra SP-L showed the highestthermal stability, at 65 C, and an operational stability of67% of activity after 10 reuses cycles when immobilized on DVS-agarose immobilized atpH 10 and blocked with ethylenediamine. The β-glucosidase from Celluclast 1.5L pro-duced best results when immobilized on DVS-agarose immobilized at pH 9 and blockedwith glycine, reaching 7.76-fold higher thermal stability compared to its free form andmaintaining 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

Tuning Almond Lipase Features by Using Different Immobilization Supports

The lipase from Prunus dulcis almonds has been immobilized for the first time. For this purpose, two different supports, an octadecyl methacrylate particulate support, and aminated agarose (monoaminoethyl-N-aminoethyl) have been utilized. Both immobilized biocatalysts show improved enzyme stability, but great changes in enzyme specificity were detected. The enzyme immobilized via ion exchange maintained its activity intact versus p-nitrophenyl butyrate, while the enzyme immobilized on the hydrophobic support fully lost its activity versus this substrate, which was confirmed to be due to substrate adsorption on the support. However, this biocatalyst was much more active versus triacetin (more than 10-fold), R- or S- methyl mandelate at pH 7. At pH 9, a strong effect of using phosphate or bicarbonate as reaction buffers was detected. Using bicarbonate, the interfacially immobilized enzyme presented no activity versus R-isomer, but it was very active versus the S-isomer and triacetin. Using a phosphate buffer during the reaction, all compounds were recognized as substrates. The enzyme immobilized via ion exchange was significantly more active using phosphate; in fact, using bicarbonate, the enzyme was inactive versus both methyl mandelate isomers. This paper shows for the first time a great interaction between the effects of the immobilization protocol and buffer used during reaction on the enantiospecificity of lipases

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 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