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Recebido em 9/12/08; aceito em 27/5/09; publicado na web em 28/10/09 PRODUCTION, PURIFICATION AND PERFORMANCE IMPROVE OF CYCLODEXTRIN GLYCOSYL TRANSFERASES TO CYCLODEXTRINS PRODUCTION. Biospecific affinity chromatography was used to purify three cyclodextrin glycosyl transferases (CGTases) obtained from microorganisms isolated of soil. The cyclodextrins (CDs) production by CGTases was evaluated using starches from different sources. CDs were measured through the Complexation Theory and by HPLC. CGTase from Bacillus firmus strain 7B showed the best production (30 mmol/L of b-CD and 4.3 mmol/L of γ-CD), and its cultivation conditions were optimized. The maximum enzymatic activity was achieved using lung peptone, soluble starch and agitation speed of 160 rpm. Studied CGTases were shown quite interesting for the industrial production of CDs.Keywords: cyclodextrin; CGTase; biospecific affinity chromatography. INTRODUÇÃOCiclodextrina glicosiltransferase (CGTase, EC 2.4.1.19) é a única enzima capaz de converter amido e substâncias relacionadas em ciclodextrinas (CDs) através de uma reação de transglicosilação intramolecular (ciclização). Também catalisa reações de transglicosilação intermolecular, tais como, acoplamento e desproporcionamento. É uma enzima geralmente extracelular, com fraca atividade de hidrólise e produzida por vários tipos de microrganismos, como Thermoanaerobacterium, Klebsiella oxytoca e algumas espécies de Bacillus, como B. macerans, B. circulans, B. megaterium, B. firmus, B. stearothermophilus e B. lentus. 1As CDs são oligossacarídeos cíclicos contendo seis (a-CD), sete (b-CD), oito (γ-CD) ou mais unidades de glicopiranose unidas por ligações a-(1,4). A molécula de CD é descrita como um cone truncado, cujo interior é hidrofóbico e a superfície hidrofílica.2 As características estruturais das mesmas são conhecidas com base em investigações por cristalografia de raios-X, 3 entretanto, Britto e colaboradores 4 mostraram que o método da mecânica molecular é o mais indicado para determinação da estrutura geométrica das CDs. Devido esta estrutura, as CDs podem formar complexos de inclusão com várias moléculas orgânicas e inorgânicas, sendo que as propriedades físicas, químicas e/ou biológicas da molécula hóspede podem ser alteradas. As CDs são amplamente utilizadas nas indústrias de alimentos, cosméticos, farmacêutica e na agricultura, atuando como agentes estabilizantes de substâncias voláteis e/ou instáveis, emulsificantes, antioxidantes, na solubilização de compostos insolúveis em água, em sistemas de liberação controlada de drogas, na imobilização de compostos tóxicos para proteção ambiental. 3,[5][6][7] As iniciativas para aplicação industrial das CDs foram bastante tímidas até a década de 70, devido à baixa disponibilidade e preços inacessíveis. Contudo, o consumo anual de CDs no mundo está crescendo em altas proporções como consequência da intensa pesquisa neste campo no sentido de melhorar a produção, recuperação e purificação deste oligossacarídeo cíclico.3 Além disso, a busca por substratos ...

The preservation of Bacillus firmus strain 37 cells by lyophilization was evaluated and response surface methodology (RSM) was used to optimize the β-cyclodextrin (β-CD) production by cells immobilized on loofa sponge. Interactions were studied with the variables temperature, pH and dextrin concentration using a central composite design (CCD). Immobilization time influence on β-CD production was also investigated. B. firmus strain 37 cells remained viable after one year of storage, showing that the lyophilization is a suitable method for preservation of the microorganism. From the three-dimensional diagrams and contour plots, the best conditions for β-CD production were determined: temperature 60 °C, pH 8, and 18% dextrin. Considering that the amount of dextrin was high, a new assay was carried out, in which dextrin concentrations of 10, 15, and 18% were tested and the temperature of 60 °C and pH 8 were maintained. The results achieved showed very small differences and therefore, for economic reasons, the use of 10% dextrin is suggested. Increasing the immobilization time of cells immobilized on synthetic sponge the β-CD production decreased and did not change for cells immobilized on loofa sponge. The results of this research are important for microorganism preservation and essential in the optimization of the biosynthesis of CD.

This study aimed to improve the production of β-cyclodextrin (β-CD) by microbial cells immobilized on synthetic or loofa sponges both with and without the use of alginate or chitosan. The most suitable matrix for the immobilization of Bacillus firmus strain 7B was synthetic sponge and for Bacillus sphaericus strain 41 was loofa sponge. After 330 days of storage, the β-CD production by Bacillus firmus and Bacillus sphaericus remained at around 41% and 49%, respectively, of initial levels. After 24 days of immobilization on loofa sponge, Bacillus sphaericus strain 41 achieved an improved operational stability, reaching 86.6 mM β-CD after 20 days of production, compared to only 32.8 mM of β-CD produced by free Bacillus sphaericus strain 41 cells. The expected increase in β-CD production by immobilized cells of Bacillus firmus strain 7B on synthetic sponge for 4 days was not statistically different to that for cells immobilized for 24 days. The application of this process on an industrial scale using loofa sponge, an inexpensive and renewable matrix, will allow the stable production of β-CD.

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