Rafael C Rodrigues

• PhD
• Professor (Full) at Federal University of Rio Grande do Sul

The chitosan‐genipin complex has been extensively studied and applied in various fields, attracting increasing attention due to its unique characteristics and properties. This complex enables the development of novel materials for applications in biomedical engineering, biotechnology, and medicine. Chitosan is a natural biopolymer, and genipin is a...

This study investigated the impact of a support matrix and active group on the support to the nutritional properties of orange juice after juice clarification. Pectinase was immobilized on chitosan and aminated silica supports, activated with genipin or glutaraldehyde, and applied for juice clarification. The effects on various juice properties, in...

This study aimed to modify the porosity of chitosan beads using Na2CO3 as a porogen agent and to crosslink them with genipin for the immobilization of β-galactosidase from Aspergillus oryzae. Immobilization was performed under four different pH conditions (4.5, 6.0, 7.5, and 9.0), resulting in biocatalysts named B4, B6, B7, and B9, respectively. Th...

Beer production is the largest among alcoholic beverages. Its production process is complex and demands several steps. Lager beers commonly present an off-flavor of butter that is due to the presence of diacetyl, and to avoid such a problem, a long period of maturation (3–5 weeks) is required. Another way is the application of (α-acetolactate decar...

In this work, we studied the covalent immobilization of α‐acetolactate decarboxylase (ALDC) on glutaraldehyde activated chitosan beads for application in beers to prevent off‐flavor. The immobilized preparation was characterized regarding yield, efficiency, activity recovery, thermal and pH stabilities, and operational stability in beer. The concen...

Most of the studies regarding phenolic compounds (PC) have been focused only on one fraction of PC, named extractable phenolic compounds (EPC). As the name suggests, EPC can be directly extracted from the food matrix by using an appropriate solvent. Otherwise, non-extractable phenolic compounds (NEPC) remain in the food matrix after the conventiona...

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 ana...

Feijoa fruit (Acca Sellowiana) has a short postharvest period, thus, processing is usually an alternative. The objective of this work was to evaluate the efficiency of three different commercial enzyme preparations in the extraction of feijoa juice in the yield and quality parameters. The juices with Pectinex®, Lallzyme® and Rohapect® were evaluate...

The use of enzymes in industrial processes requires the improvement of their features in many instances. Enzyme immobilization, a requirement to facilitate the recovery and reuse of these water-soluble catalysts, is one of the tools that researchers may utilize to improve many of their properties. This review is focused on how enzyme immobilization...

In this study, the extraction of oil from seeds of castor (Ricinus communis) was intended using aqueous enzymatic extraction (AEE). After evaluating several enzymes (cellulase, pectinase, hemicellulase and Viscozyme L), the enzymatic cocktail Viscozyme L gave the best oil extraction yield (64 %) at pH 4 and 50 • C, and it was chosen for the optimiz...

In this study, aqueous enzymatic extraction (AEE) was evaluated during the process of obtaining oil from mamey sapote seed (OMSS). Viscozyme L enzyme complex was used at pH 4 and 50 °C during the optimization of the extraction process by central composite design and response surface methodology. Optimal conditions were: 3.5% (w/w) of enzyme (regard...

The oxidation activity of laccase on a broad range of substrates has attracted great interest in the development of technologies for industrial and environmental applications. In this work, a crude laccase from Marasmiellus palmivorus was immobilized onto chitosan-coated magnetic particles. The support was developed and characterized by X-ray diffr...

The effect of the degree of deacetylation (DD) on the characteristics of the support matrix formed through chitosan (CH) and genipin (GE) crosslinking was investigated by using CH with DD of 83, 94 and 96%. The rheological properties of CH dispersions were determined in order to study the influence of DD on the CH viscosity, gel network structures...

The enzymatic hydrolysis of residual cellulose casings from sausage processing was assessed. A central composite design (CCD) was performed to determine the best enzyme and substrate concentrations to release reducing sugars (RS) using the enzymatic complex Celluclast. The optimal response found in the CCD was a substrate concentration of 34.1 mg/m...

This review is focused on the use of glucose oxidase, mainly in the production of D-gluconic acid, a reactive of undoubtable interest in different industrial areas. The enzyme has been used in many instances as a model reaction to study the problems of oxygen supply in bioreactors. One of the main topics in this review is the problem of the generat...

The increasing relevance of cascade reactions in biocatalysis has sparked a great interest for enzyme co-immobilization. Enzyme co-immobilization allows access to some kinetic advantages that in some instances are necessary to get the desired product, avoiding side-reactions. However, the kinetic effect is very relevant mainly at the initial reacti...

Lipases are among the most utilized enzymes in biocatalysis. In many instances, the main reason for their use is their high specificity or selectivity. However, when full modification of a multifunctional and heterogeneous substrate is pursued, enzyme selectivity and specificity become a problem. This is the case of hydrolysis of oils and fats to p...

The objective of this research was to incorporate an ingredient obtained from spent cellulose casings in beef emulsion modeling systems. The test ingredient (residual sausage casing, RSC) was procured from cellulose sausage casings following thermal processing of the sausages. The casings were cleaned of contaminants before a combination of enzymat...

In this study, a commercial enzyme cocktail designed for fruit juice clarification was immobilized on chitosan beads activated with glutaraldehyde and used for setting-up continuous packed-bed and fluidized-bed reactors. Firstly, the enzyme cocktail concentration in the immobilization process was tested to get maximum biocatalyst activity in the re...

We investigated the immobilization of a tetrameric Kluyveromyces lactis β-galactosidase (EC: 3.2.1.23) (KL-Gal) on Immobead 150 using different support modification strategies. Immobead support was modified using an acid solution of H2SO4:HNO3 (3:1) (Immobead-Ac) or 5 % (v/v) glutaraldehyde (Immobead-Glu). Its unmodified form (Immobead) was also te...

Transglutaminases (TGases) are a class of transferases widely used in the food and biotechnology industries. In this work, we describe the production of recombinant Bacillus amyloliquefaciens TGase in Escherichia coli, obtaining the protein in its soluble and active form. In order to reduce TGase activity inside host cells and consequently its toxi...

The incomplete anthocyanin extraction during the industrial processes turns grape pomace into an inexpensive source of phenolic compounds. The effects of temperature and enzyme preparation percentage (% E/S) on the anthocyanin recovery from grape pomaces of eight grape varieties were evaluated by their physicochemical characteristics and phenolic c...

The objective of this study was to characterize dietary fiber from residual cellulose sausage casings using a combination of enzymatic hydrolysis and high-speed homogenization. To modify structure and function of the cellulose, residual cellulose casings (40 or 50 mg mL⁻¹) were subjected to enzymatic hydrolysis followed by high-speed homogenization...

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 usin...

Pectin lyase (from Rohapect 10 L) was immobilized on glutaraldehyde supports at low ionic strength at pH 5, 6.5 or 8 and later incubated at pH 8 for 48 h. The activity recovery of the biocatalysts versus pectin was quite low, under 10% for all of the immobilized biocatalyst at 20 °C. However, a high stabilization was found when the enzyme was immob...

Magnetic-chitosan particles were prepared following three different protocols enabling the preparation of particles with different sizes - nano (Nano-CMag, Micro (Micro-CMag) and Macro (Macro-CMag) - and used for pectinase immobilization and clarification of grape, apple and orange juices. The particle size had a great effect in the kinetic paramet...

Rohapect 10L is an enzyme cocktail commercialized for juice clarification. Here, we characterized the activity and stability of five enzymatic activities present in this cocktail: total pectinase (PE), polygalacturonase (PG), pectin lyase (PL), pectin methyl esterase (PME), and total cellulase (CE) activities. All these enzyme activities have the m...

Polygalacturonase (PG) from Aspergillus niger was immobilized using glyoxyl, vinylsulfone or glutaraldehyde-activated supports. The use of supports pre-activated with glutaraldehyde presented the best results. The immobilization of PG on glutaraldehyde-supports was studied under different conditions: at pH 5 for 24 h; at pH 5, 6.5 or 8 for 3 h and...

In this work, we studied the effect of some variables in the reaction between genipin and chitosan, to prepare a support for enzyme immobilization. Reaction temperature, pH, time and genipin concentration were evaluated for support activation and β-galactosidase from Aspergillus oryzae was used as the enzyme model. The immobilized enzyme was charac...

We applied ultrasonic technology for the transesterification of soybean oil catalyzed by a mixture of lipases (combi-lipase: 75 % Novozym 435; 10 % Lipozyme TL-IM; and 15 % Lipozyme RM-IM), verifying the effects of ultrasonic amplitude, pulse conditions, the ethanol:oil molar ratio, enzyme concentration, and the influence of solvent (tert-butanol o...

Lecitase Ultra is a chimera produced by the fusion of the genes of the lipase from Thermomyces lanuginosus and the phospholipase A1 from Fusarium oxysporum. The enzyme was first designed for the enzymatic degumming of oils, as that problem was not fully resolved before. It is commercialized only as an enzyme solution by Novo Nordisk A/S. This revie...

Novozym 435 (N435) is a commercially available immobilized lipase produced by Novozymes. It is based on the immobilization via interfacial activation of the lipase B from Candida antarctica on a resin, Lewatit VP OC 1600. This resin is a macroporous support formed by poly(methyl methacrylate) crosslinked with divinylbenzene. N435 is perhaps the mos...

Lipases are the most widely used enzymes in biocatalysis, and the most utilized method for enzyme immobilization is using hydrophobic supports at low ionic strength. This method allows the one step immobilization, purification, stabilization, and hyperactivation of lipases, and that is the main cause of their popularity. This review focuses on thes...

The β-glucosidase activities of nine commercial enzymatic 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 assayed β-glucosidases. Celluclast® 1.5 L showed the hi...

Three β-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 immo...

This work describes the use of an ultrasound system for the enzymatic transesterification of oils using combi-lipases as biocatalyst. The reactions were carried out evaluating the individual use of waste oil and fresh soybean oil, and the immobilized lipases CALB, TLL, and RML were used as biocatalysts. It was performed in a mixture design of three...

Opuntia monacantha mucilage was investigated for its physicochemical characteristics and functional properties. The mucilage extraction yield was 12% (DW) and it consisted of 80.12% carbohydrates, 15.14% ashes, 3.55% proteins, and 1.19% lipids. Monosaccharide profiling demonstrated a predominantly presence of galactose, glucuronic acid, and arabino...

For optimization of biochemical processes in food and pharmaceutical industries, the evaluation of enzyme inactivation kinetic models is necessary to allow their adequate use. Kinetic studies of thermal inactivation of β-galactosidase from Aspergillus oryzae were conducted in order to critically evaluate mathematical equations presented in the lite...

BACKGROUND This study investigated the use of core–shell biocatalysts, synthesized using recombinant lipase B from Candida antarctica (CALB), for the production of isopropyl palmitate and the optimization of this biocatalytic route. RESULTS Initially, three distinct core–shell biocatalysts, were evaluated for the production of isopropyl palmitate...

We describe the development of cross-linked enzyme aggregates (CLEA) of dextransucrase of Leuconostoc mesenteroides B-512 F. Treatments of enzyme preparation using dextranase were evaluated varying incubation time to remove the dextran layer involving the enzyme molecule, turning it suitable for immobilization. Results showed 21 days of treatment a...

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...

This work describes the continuous synthesis of ethyl esters via enzymatic catalysis on a packed-bed continuous reactor, using mixtures of immobilized lipases (combi-lipases) of Candida antarctica (CALB), Thermomyces lanuginosus (TLL), and Rhizomucor miehei (RML). The influence of the addition of glass beads to the reactor bed, evaluation of the us...

In the present study, we prepared two different magnetic biocatalysts of pectinase and cellulase: carrier-free magnetic CLEAs (CLEA-MP*) and immobilization on glutaraldehyde-activated magnetite (Enz-Glu-MP*). The biocatalysts were compared to their magnetic properties, immobilization parameters, stability and grape juice clarification. Enz-Glu-MP*...

This review discusses the possible roles of polyethylenimine (PEI) in the design of improved immobilized biocatalysts from diverse perspectives. This includes their use to activate supports and immobilize enzymes via ion exchange, as well as to improve immobilized enzymes by coating with PEI. PEI is a polymer containing primary, secondary and terti...

We carried out the physico-chemical characterization of Immobead 150, a hydrophobic support for the immobilization of lipases. Thermomyces lanuginosus lipase (TLL) was immobilized on Immobead 150 by multipoint covalent attachment (ImmTLL) and the morphological, textural, structural, thermal, and physico-chemical properties of the support, before an...

This study reports the immobilization of a β-CGTase on glutaraldehyde pre-activated silica and its use to production of cyclodextrins in batch and continuous reactions. We were able to modulate the cyclodextrin production (α-, β- and γ-CD) by immobilization and changing the reaction conditions. In batch reactions, the immobilized enzyme reached to...

In this work the effects of ultrasound (US), mechanical stirring (MS) and nine commercial enzyme preparations (EP) were investigated on Vitis vinifera Cabernet Sauvignon must extraction (GME). Yield, quality parameters and bioactive compounds were analyzed for individual and combined extraction techniques. The EP Zimopec PX5® presented the best res...

Ultrasound effects were investigated on pectinase (PE), xylanase (XLN) and cellulase (CE) activities at different pH, temperatures, and by sonication pre-treatment, comparing the reaction at ultrasound bath (US) and at a mechanical stirring (MS). In general, US increased the activity of the enzymes by 5% for PE, 30 % for XLN and 25% for CE compared...

This work describes the preparation of combi-CLEAs of pectinases using feather meal as novel proteic feeder. Initially, four solvents were tested as precipitant agents, and ethanol was chosen as the best solvent. Subsequently, a 2³ factorial design was carried out to define the optimal conditions for combi-CLEAs preparation, which were set as 110 m...

In this study, two different approaches were assessed in order to direct the immobilization of a cyclodextrin glycosyltransferase on functionalized silica support, one by amino groups using glutaraldehyde activation (Si-NH-G-CGTase) and other by disulfide bond through the Cys on the enzyme surface (Si-SH-CGTase). The efficiency of the immobilizatio...

A new bioprocess for the synthesis of lactosucrose was studied using a covalently immobilized β-galactosidase on macrospheres of chitosan. The effects of temperature and pH on the production of lactosucrose and other oligosaccharides were evaluated. At 30 °C and pH 7.0, the maximum concentration of lactosucrose reached to 79 g L⁻¹. The change of th...

Enzymatic synthesis of fatty acid esters from macaúba (Acrocomia aculeata) acid oil was studied in a solvent-free system using a fermented and dry babassu (Orbygnia oleifera) cake with lipase activity from Rhizomucor miehei, named solid enzymatic preparation (SEP). The reactions were carried out under the influence of ultrasound irradiation and usi...

In this work, we evaluated the effects of nine commercial enzymes—fungal (AMY-F), bacterial (AMY-B), and maltogenic (AMY-M) α-amylases, fungal (XYL-F) and bacterial (XYL-B) xylanases, glucose oxidase (GOX), laccase (LAC), lipase (LIP), and transglutaminase (TG) on the rheological properties of common wheat flour. Falling Number (FN), farinography,...

Dextransucrase from Leuconostoc mesenteroides B-512F was covalently immobilized on glutaraldehyde-actived chitosan particles. The best initial protein loading (400 mg/g of dried support) showed 197 U/g of catalytic activity. The optimal reaction pH and temperature of this new biocatalyst were determined to be 4.5 and 20 °C, respectively. Regarding...

In this work, the synergistic effects of the combination of Pectinex Ultra Clear (PUC), presenting high pectinase activity, and Lallzyme Beta (LB), presenting cellulase activity were tested for grape juice extraction from Vitis labrusca L. variety Concord. The variables time, temperature, enzyme concentration and PUC/LB ratio were analyzed with the...

In this work, the combined effects of nine commercial enzymes were studied on the rheological properties of dough wheat flour. Fungal (AMY-F), bacterial (AMY-B), and maltogenic (AMY-M) α-amylases; fungal (XYL-F) and bacterial (XYL-B) xylanases; glucose oxidase (GOX); laccase (LAC); lipase (LIP); and transglutaminase (TG) were tested in two types of...

Glycoside hydrolases (GH) are enzymes capable to hydrolyze the glycosidic bond between two carbohydrates or even between a carbohydrate and a non-carbohydrate moiety. Because of the increasing interest for industrial applications of these enzymes, the immobilization of GH has become an important development in order to improve its activity, stabili...

Thermomyces lanuginosus lipase (TLL) was covalently immobilized on Immobead 150 (ImmTLL) via epoxy groups of the support and the lysine of the enzyme to be used in the synthesis of butyl butyrate. The optimization of the esterification reaction parameters was carried out by central composite design and response surface methodology, having product y...

Chemical modification of enzymes and immobilization used to be considered as separate ways to improve enzyme properties. This review shows how the coupled use of both tools may greatly improve the final biocatalyst performance. Chemical modification of a previously immobilized enzyme is far simpler and easier to control than the modification of the...

Combined cross-linked enzyme aggregates (combi-CLEAs) are a novel prospect for immobilization of a mixture of enzymes and the present study addresses the preparation, characterization and application of pectinases–cellulases combi-CLEAs for grape juice clarification. Initially, 8 enzymatic preparations were tested for turbidity reduction in grape j...

In this work, the effects of eight different enzymatic preparations were compared in terms of the extraction of bioactive compounds as well as the yield and quality of grape juice from Vitis labrusca L. variety Concord. Juices were prepared by enzymatic treatment and the used preparations were characterized through five enzymatic activities: total...

Abstract In order to develop safer processes for the food industry, we prepared a chitosan support with the naturally occurring crosslinking reagent, genipin, for enzyme. As application model, it was tested the immobilization of β-D-galactosidase from Aspergillus oryzae. Chitosan particles were obtained by precipitation followed by adsorption of th...

Immobilization and purification of enzymes are usual requirements for their industrial use. Both purification and immobilization have a common factor: they use a solid activated support. Using a support for enzyme purification means having mild conditions for enzyme release and a selective enzyme–support interaction is interesting. When using a sup...

Supports based on poly-styrene-divinylbenzene (PSD) are commercially available since a long time ago. However, they are not commonly used as enzyme immobilization matrices. The main reason for this lies in the negative effect of the very hydrophobic surface on enzyme stability that produces the instantaneous enzyme inactivation in many instances. H...

In this review, we detail the efforts performed to couple the purification and the immobilization of industrial enzymes in a single step. The use of antibodies, the development of specific domains with affinity for some specific supports will be revised. Moreover, we will discuss the use of domains that increase the affinity for standard matrices (...

Lipases are being extensively researched for the production of biodiesel as a "silver bullet" in order to avoid the drawbacks of the traditional alkaline transesterification. In this review, we analyzed the main factors involved in the enzymatic synthesis of biodiesel, focusing in the choice of the immobilization protocol, and the parameters involv...

In this work it was evaluated for the first time, the ester synthesis catalyzed by the phospholipase Lecitase-Ultra immobilized styrene-divinylbenzene beads (MCI-Lecitase), comparing the mechanical stirring and the ultrasonic energy. It was studied the specificity of the enzyme using carboxylic acids from C4 to C18, as well as the effects of alcoho...

In this work, β-fructofuranosidase and β-fructosyltransferase were covalently immobilized on chitosan spheres, using glutaraldehyde as a coupling agent, in order to produce invert sugar and fructooligosaccharides (FOS). Maxinvert L was used to make β-fructofuranosidase biocatalyst yielding 7000 HU/g. A partial purified β-fructosyltransferase from V...

Although reactions of transesterification are generally catalyzed by one specific lipase preparation, the concept of “combi-lipase” could be better explored for the production of biodiesel, since oils are heterogeneous substrates. In this research, we tested this concept by evaluating the enzymatic transesterification of olive and palm oils, two di...

Improvement of the features of an enzyme is in many instances a pre-requisite for the industrial implementation of these exceedingly interesting biocatalysts. To reach this goal, the researcher may utilize different tools. For example, amination of the enzyme surface produces an alteration of the isoelectric point of the protein along with its chem...

It is well established that the performance of lipase B from Candida antarctica (CALB) as catalyst for esterification reactions may be improved by the use of ultrasound technology or by its immobilization on styrene-divinylbenzene beads (MCI-CALB). The present research evaluated the synthesis of butyl acetate using MCI-CALB under ultrasonic energy,...

A commercial and very hydrophobic styrene-divinylbenzene matrix, MCI GEL® CHP20P, has been compared to octyl-Sepharose® beads as support to immobilize three different enzymes: lipases from Thermomyces lanuginosus (TLL) and from Rhizomucor miehie (RML) and Lecitase® Ultra, a commercial artificial phospholipase. The immobilization mechanism on both s...

Glutaraldehyde is one of the most widely used reagents in the design of biocatalysts. It is a powerful crosslinker, able to react with itself, with the advantages that this may bring forth. In this review, we intend to give a general vision of its potential and the precautions that must be taken when using this effective reagent. First, the chemist...

In this work, the combined use of ultrasound energy and molecular sieves was investigated for the synthesis of ethyl butyrate, ester with mango and banana notes, catalyzed by the immobilized lipase from Thermomyces lanuginosus (Lipozyme TL-IM). Initially, the best concentrations of biocatalysts (35%) and butyric acid (0.7M) were tested using ultras...

The enzymatic synthesis of fructooligosaccharides (FOS) was carried out using a partially purified β-fructofuranosidase from the commercial enzyme preparation Viscozyme L. Partial purification of β-fructofuranosidase from Viscozyme L was done by batch adsorption using ion-exchange resin DEAE-Sepharose, showing a 6-fold increase in specific activity...

Lipase from Staphylococcus warneri EX17 (SWL) was purified and immobilized via interfacial adsorption using the hydrophobic supports Octyl-sepharose, Immobead 150, and MCI GEL CHP20P. The purity of the obtained immobilized biocatalysts, Octyl-SWL, Immobead-SWL, and MCI-SWL, was evaluated by SDS-PAGE and their thermal and solvent stability were test...

The concept of the combi-lipase biocatalyst has been proposed. It is based on the combination of different lipases as biocatalysts in reactions using heterogeneous substrates. The hydrolysis of soybean oil was evaluated as a model substrate, and Novozym 435 (CALB), Lipozyme TL-IM (TLL), and Lipozyme RM-IM (RML) were used as biocatalysts. Results sh...