Bessy Cutiño-Avila

A thermostable exo-β-fructosidase immobilised through rational design

Thermotoga maritima exo-β-fructosidase (BfrA) secreted by a recombinant Pichia pastoris strain was optimally immobilised on Glyoxyl-Sepharose CL 4B using the Rational Design of Immobilised Derivatives (RDID) strategy. Covalent attachment of the N-glycosylated BfrA onto the activated support at pH 10 allowed total recovery of the loaded enzyme and its activity. The immobilisation process caused no variation in the catalytic properties of the enzyme and allowed further enhancement of the thermal stability. Complete inversion of cane sugar (2.04 M) in a batch stirred tank reactor at 60 °C was achieved with a productivity of 22.2 g of substrate hydrolysed/gram of biocatalyst/hour. Half-life of the immobilised enzyme of 5 days at 60 °C was determined in a continuously operated fixed-bed column reactor. Our results promote the applicability of the BfrA-immobilised biocatalyst for the complete hydrolysis of concentrated sucrose solutions under industrial conditions, especially at a high reaction temperature.

Rational design of immobilized lipases and phospholipases.

Immobilization of lipases and phospholipases on, mainly, water insoluble carriers, helps in their economic reuse and in the development of continuous bioprocesses. Design of efficient lipases and phospholipases-immobilized system is rather a difficult task. A lot of research work has been done in order to optimize immobilization techniques and procedures and to develop an efficient immobilized system. A new rational design of immobilized derivatives strategy (RDID) has been conceived in favor of the successful synthesis of optimal lipases and phospholipases-immobilized derivatives, aiming prediction of the immobilized derivatives functionality and the optimization of load studies. RDID begins with the knowledge of structural and functional features of synthesis components (protein and carrier), and the practical goal of immobilized product. RDID was implemented in software named RDID ( 1.0 ). The employment of RDID allows selecting the most appropriate way to prepare immobilized derivatives more efficient in enzymatic bioconversion processes and racemic mixture resolution.

Synthesis of Tetanus Toxoid-Sepharose CL 4B derivatives by Rational Design

Tetanus is an infectious disease caused by contamination of wounds from bacteria that live in the soil. Efforts for the development of vaccines against Clostridium tetani have focused primarily upon two antigens, tetanus toxoid (TTn) and capsular polysaccharides (CapP). Our interest is the production of polyclonal antibodies (PAb) in rabbit that efficiently recognize different serotypes of CapP. The aim of this work was the development of an efficient affinity chromatography support; with immobilize TTn on Sepharose CL 4B, that allows separate from a mixture anti-CapP (fraction not adsorbed to the support) and anti-TTn (fixed fraction) antibodies.

Computational Mathematic Model for the Immobilization of Cells and Proteins on Charged Solid Surfaces by Electrostatic Interactions

Non-covalent immobilization techniques such as ionic adsorption on ionic exchanger supports may be a good option because immobilization is very simple and produces very little work and time consumption and the suppErts may be reused after desorption and, in this way, reduce the final price and generate less residues. Rational Design of Immobilized Derivative (RDID) is a strategy that combines mathematical and bioinformatics tools for designing optimal immobilization processes. In this work are described new mathematical algorithms to optimize proteins/cells immobilization via electrostatic on ionic exchangers, these algorithms belongs to RDID strategy. Were estimated the support maximum loading capacity on the immobilization of cells (Scenedesmus obliquus), spores (Aspergillus niger), enzymatic pancreatic extract (pancreatin) and purified enzymes (pancreatic porcine lipase, PPL). At the same time, the most probable configurations of the immobilized derivative were predicted for PPL. RDID predictions were highly accurate when comparing with experimental results.

Rational Design Strategy as a Novel Immobilization Methodology Applied to Lipases and Phospholipases

Immobilization of lipases and phospholipases, mainly on water-insoluble carriers, helps in their economic reusing and in the development of continuous bioprocesses. Design of efficient lipase and phospholipase-immobilized systems is rather a difficult task. A lot of research work has been done in order to optimize immobilization techniques and procedures and to develop efficient immobilized systems. We conceived a new strategy for the rational design of immobilized derivatives (RDID) in favor of the successful synthesis of optimal lipase and phospholipase-immobilized derivatives, aiming the prediction of the immobilized derivatives functionality and the optimization of load studies. The RDID strategy begins with the knowledge of structural and functional features of synthesis components (protein and carrier) and the practical goal of the immobilized product. The RDID strategy was implemented in a software named RDID1.0. The employment of RDID allows selecting the most appropriate way to prepare immobilized derivatives more efficient in enzymatic bioconversion processes and racemic mixture resolution.

Rational design and synthesis of affinity matrices based on proteases immobilized onto cellulose membranes

ABSTRACT Discovery of new protease inhibitors may result in potential therapeutic agents or useful biotechnological tools. Obtainment of these molecules from natural sources requires simple, economic, and highly efficient purification protocols. The aim of this work was the obtainment of affinity matrices by the covalent immobilization of dipeptidyl peptidase IV (DPP-IV) and papain onto cellulose membranes, previously activated with formyl (FCM) or glyoxyl groups (GCM). GCM showed the highest activation grade (10.2 µmol aldehyde/cm2). We implemented our strategy for the rational design of immobilized derivatives (RDID) to optimize the immobilization. pH 9.0 was the optimum for the immobilization through the terminal α-NH2, configuration predicted as catalytically competent. However, our data suggest that protein immobilization may occur via clusters of few reactive groups. DPP-IV−GCM showed the highest maximal immobilized protein load (2.1 µg/cm2), immobilization percentage (91%), and probability of multipoint covalent attachment. The four enzyme-support systems were able to bind at least 80% of the reversible competitive inhibitors bacitracin/cystatin, compared with the available active sites in the immobilized derivatives. Our results show the potentialities of the synthesized matrices for affinity purification of protease inhibitors and confirm the robustness of the RDID strategy to optimize protein immobilization processes with further practical applications.