Structure of Tagatose-1,6-bisphosphate Aldolase. Insight into chiral discrimination, mechanism, and specificity of class II aldolases

Tagatose-1,6-bisphosphate aldolase (TBPA) is a tetrameric class II aldolase that catalyzes the reversible condensation of dihydroxyacetone phosphate with glyceraldehyde 3-phosphate to produce tagatose 1,6-bisphosphate. The high resolution (1.45 Å) crystal structure of the Escherichia coli enzyme, encoded by the agaY gene, complexed with phosphoglycolohydroxamate (PGH) has been determined. Two subunits comprise the asymmetric unit, and a crystallographic 2-fold axis generates the functional tetramer. A complex network of hydrogen bonds position side chains in the active site that is occupied by two cations. An unusual Na(+) binding site is created using a interaction with Tyr(183) in addition to five oxygen ligands. The catalytic Zn(2+) is five-coordinate using three histidine nitrogens and two PGH oxygens. Comparisons of TBPA with the related fructose-1,6-bisphosphate aldolase (FBPA) identifies common features with implications for the mechanism. Because the major product of the condensation catalyzed by the enzymes differs in the chirality at a single position, models of FBPA and TBPA with their cognate bisphosphate products provide insight into chiral discrimination by these aldolases. The TBPA active site is more open on one side than FBPA, and this contributes to a less specific enzyme. The availability of more space and a wider range of aldehyde partners used by TBPA together with the highly specific nature of FBPA suggest that TBPA might be a preferred enzyme to modify for use in biotransformation chemistry

A multipurpose immobilized biocatalyst with pectinase, xylanase and cellulase activities

<p>Abstract</p> <p>Background</p> <p>The use of immobilized enzymes for catalyzing various biotransformations is now a widely used approach. In recent years, cross-linked enzyme aggregates (CLEAs) have emerged as a novel and versatile biocatalyst design. The present work deals with the preparation of a CLEA from a commercial preparation, Pectinex™ Ultra SP-L, which contains pectinase, xylanase and cellulase activities. The CLEA obtained could be used for any of the enzyme activities. The CLEA was characterized in terms of kinetic parameters, thermal stability and reusability in the context of all the three enzyme activities.</p> <p>Results</p> <p>Complete precipitation of the three enzyme activities was obtained with n-propanol. When resulting precipitates were subjected to cross-linking with 5 mM glutaraldehyde, the three activities initially present (pectinase, xylanase and cellulase) were completely retained after cross-linking. The V_max/K_mvalues were increased from 11, 75 and 16 to 14, 80 and 19 in case of pectinase, xylanase and cellulase activities respectively. The thermal stability was studied at 50°C, 60°C and 70°C for pectinase, xylanase and cellulase respectively. Half-lives were improved from 17, 22 and 32 minutes to 180, 82 and 91 minutes for pectinase, xylanase and cellulase respectively. All three of the enzymes in CLEA could be reused three times without any loss of activity.</p> <p>Conclusion</p> <p>A single multipurpose biocatalyst has been designed which can be used for carrying out three different and independent reactions; 1) hydrolysis of pectin, 2) hydrolysis of xylan and 3) hydrolysis of cellulose. The preparation is more stable at higher temperatures as compared to the free enzymes.</p

Purification and In Situ Immobilization of Papain with Aqueous Two-Phase System

Papain was purified from spray-dried Carica papaya latex using aqueous two-phase system (ATPS). Then it was recovered from PEG phase by in situ immobilization or preparing cross-linked enzyme aggregates (CLEAs). The Plackett-Burman design and the central composite design (CCD) together with the response surface methodology (RSM) were used to optimize the APTS processes. The highly purified papain (96–100%) was achieved under the optimized conditions: 40% (w/w) 15 mg/ml enzyme solution, 14.33–17.65% (w/w) PEG 6000, 14.27–14.42% (w/w) NaH2PO4/K2HPO4 and pH 5.77–6.30 at 20°C. An in situ enzyme immobilization approach, carried out by directly dispersing aminated supports and chitosan beads into the PEG phase, was investigated to recover papain, in which a high immobilization yield (>90%) and activity recovery (>40%) was obtained. Moreover, CLEAs were successfully used in recovering papain from PEG phase with a hydrolytic activity hundreds times higher than the carrier-bound immobilized papain

Didactic Strategies Stop The Development Of Critical Thinking In University Students

La educación debe evolucionar para afrontar los retos que plantea el futuro, en la actualidad vivimos en un mundo globalizado que comparte grandes cantidades de información en tiempo real, el problema radica en que no toda la información es fiable, por consiguiente, es necesario generar estrategias efectivas en las aulas, buscando que el estudiante genere independencia de pensamiento para que desarrolle criterios propios de pensamiento crítico, lo anterior permite que el estudiante adquiera adquirir las destrezas para generar habilidades de pensamiento superior que lo lleven a la independencia intelectual.Education must evolve to meet the challenges of the future, we live in a globalized world that shares large amounts of information in real time, the problem is that not all information is reliable, therefore it is necessary to generate strategies Effective in the classroom, seeking the student to generate independence of thought to develop criteria critical thinking, the previous allows the student to acquire the skills to generate higher thinking skills that lead to intellectual independence

One-point covalent Immobilization of enzymes on glyoxyl agarose with minimal physico-chemical modification: Immobilized “native enzymes”

The immobilization of soluble enzymes inside the porous structure of a preexisting support is one of the most interesting techniques to prepare heterogeneous biocatalysts. The main cause of inactivation of these biocatalysts is the distortion of the tridimensional structure of the immobilized enzymes. In some cases, immobilization of enzymes on preexisting supports can be used in order to improve its functional properties: stabilization by multipoint covalent immobilization, hyper-activation, and stabilization of lipases by interfacial adsorption on hydrophobic supports, etc. In other cases, the properties of the enzyme can be modified by additional interactions of the enzyme surface with the support surface: hydrophobic or electrostatic interactions. In all cases, it would be very interesting to evaluate the intrinsic tridimensional stability of native industrial enzymes. Under drastic experimental conditions, soluble enzymes may undergo undesirable aggregations, and the tridimensional stability of one enzyme is more accurately evaluated by using immobilized native enzymes. That is, immobilized derivatives associated to a minimal chemical modification of the enzyme surface placed in the proximity of a fully hydrophilic and inert support surfaces. In this chapter, the immobilization of enzymes with minimal physicochemical modification on glyoxyl agarose supports is proposed. At pH 8.5, the unique reactive amino group on the enzyme surface is the N-terminus. At the end of the immobilization, mild borohydride reduction, the primary amino terminus is simply converted into a secondary amino group, with similar physical properties, and aldehyde groups on the supports are converted into fully inert hydroxyl groups. The preparation of immobilized derivatives of penicillin G acylase (PGA) with identical properties (activity and stability) that one of the soluble enzyme is reported: preparation of immobilized native PGA.Peer reviewe