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      Obtaining partial purified xylose reductase from Candida guilliermondii Translated title: Obtenção de xilose redutase de Candida guilliermondii parcialmente purificada

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          Abstract

          The enzymatic bioconversion of xylose into xylitol by xylose reductase (XR) is an alternative for chemical and microbiological processes. The partial purified XR was obtained by using the following three procedures: an agarose column, a membrane reactor or an Amicon Ultra-15 50K Centrifugal Filter device at yields of 40%, 7% and 67%, respectively.

          Translated abstract

          A bioconversão enzimática da xilose em xilitol pela xilose redutase (XR) é uma alternativa para as vias química e microbiológica. Avaliouse a purificação parcial da XR, utilizando os três seguintes procedimentos: uma coluna de agarose, um reator com membrana ou tubos de ultracentrifugação Amicon Ultra-15 50K, com rendimento de 40%, 7% ou 67%, respectivamente.

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          Most cited references13

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          Mutational analysis of the role of the conserved lysine-270 in the Pichia stipitis xylose reductase.

          Xylose reductase catalyzes the NAD(P)H-dependent reduction of xylose to xylitol and is essential for growth on xylose by yeasts. To understand the nature of coenzyme binding to the Pichia stipitis xylose reductase, we investigated the role of the strictly conserved Lys270 in the putative IPKS coenzyme binding motif by site-directed mutagenesis. The Lys270Met variant exhibited lower enzyme activity than the wild-type enzyme. The apparent affinity of the variant for NADPH was decreased 5-16-fold, depending on the substrate used, while the apparent affinity for NADH, measured using glyceraldehyde as the substrate, remained unchanged. This resulted in 4.3-fold higher affinity for NADH over NADPH using glyceraldehyde as the substrate. The variant also showed a 14-fold decrease in Km for xylose, but only small changes were observed in Km values for glyceraldehyde. The wild-type enzyme, but not the Lys270Met variant, was susceptible to modification by the Lys-specific pyridoxal 5'-phosphate. Results of our chemical modification and site-directed mutagenesis study indicated that Lys270 is involved in both NADPH and D-xylose binding in the P. stipitis xylose reductase.
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            Structure of xylose reductase bound to NAD+ and the basis for single and dual co-substrate specificity in family 2 aldo-keto reductases.

            The co-ordinates reported have been submitted to the Protein Data Bank under accession number 1MI3. Xylose reductase (XR; AKR2B5) is an unusual member of aldo-keto reductase superfamily, because it is one of the few able to efficiently utilize both NADPH and NADH as co-substrates in converting xylose into xylitol. In order to better understand the basis for this dual specificity, we have determined the crystal structure of XR from the yeast Candida tenuis in complex with NAD(+) to 1.80 A resolution (where 1 A=0.1 nm) with a crystallographic R -factor of 18.3%. A comparison of the NAD(+)- and the previously determined NADP(+)-bound forms of XR reveals that XR has the ability to change the conformation of two loops. To accommodate both the presence and absence of the 2'-phosphate, the enzyme is able to adopt different conformations for several different side chains on these loops, including Asn(276), which makes alternative hydrogen-bonding interactions with the adenosine ribose. Also critical is the presence of Glu(227) on a short rigid helix, which makes hydrogen bonds to both the 2'- and 3'-hydroxy groups of the adenosine ribose. In addition to changes in hydrogen-bonding of the adenosine, the ribose unmistakably adopts a 3'- endo conformation rather than the 2'- endo conformation seen in the NADP(+)-bound form. These results underscore the importance of tight adenosine binding for efficient use of either NADH or NADPH as a co-substrate in aldo-keto reductases. The dual specificity found in XR is also an important consideration in designing a high-flux xylose metabolic pathway, which may be improved with an enzyme specific for NADH.
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              Cloning and expression of Candida guilliermondii xylose reductase gene (xyl1) in Pichia pastoris.

              A xylose reductase gene (xyl1) of Candida guilliermondii ATCC 20118 was cloned and characterized. The open reading frame of xyl1 contained 954 nucleotides encoding a protein of 317 amino acids with a predicted molecular mass of 36 kDa. The derived amino acid sequence of C. guilliermondii xylose reductase was 70.4% homologous to that of Pichia stipitis. The gene was placed under the control of an alcohol oxidase promoter (AOX1) and integrated into the genome of a methylotrophic yeast, Pichia pastoris. Methanol induced the expression of the 36-kDa xylose reductase in both intracellular and secreted expression systems. The expressed enzyme preferentially utilized NADPH as a cofactor and was functional both in vitro and in vivo. The different cofactor specificity between P. pastoris and C. guilliermondii xylose reductases might be due to the difference in the numbers of histidine residues and their locations between the two proteins. The recombinant was able to ferment xylose, and the maximum xylitol accumulation (7.8 g/l) was observed when the organism was grown under aerobic conditions.
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                Author and article information

                Journal
                bjm
                Brazilian Journal of Microbiology
                Braz. J. Microbiol.
                Sociedade Brasileira de Microbiologia (São Paulo, SP, Brazil )
                1517-8382
                1678-4405
                September 2009
                : 40
                : 3
                : 631-635
                Affiliations
                [01] São Paulo SP orgnameUniversidade de São Paulo orgdiv1Escola de Farmácia orgdiv2Departamento Bioquímica e Tecnologia Farmacêutica Brasil
                [02] Lorena SP orgnameUniversidade de São Paulo orgdiv1Escola de Engenharia de Lorena orgdiv2Departamento de Biotecnologia Brasil
                Article
                S1517-83822009000300027 S1517-8382(09)04000327
                2b879151-a29e-41a5-b949-4eb531069c01

                This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

                History
                : 15 July 2008
                : 25 September 2008
                : 03 May 2009
                Page count
                Figures: 0, Tables: 0, Equations: 0, References: 13, Pages: 5
                Product

                SciELO Brazil

                Self URI: Full text available only in PDF format (EN)
                Categories
                Industrial Microbiology

                Purificação parcial,Partial purification,Xylitol,Xylose reductase,Xilitol,Xilose redutase

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