A ciclodextrina glucosiltransferase (CGTase) é uma enzima industrialmente muito importante, capaz de converter o amido em ciclodextrinas (CDs). As CDs são capazes de formar complexos de inclusão com uma grande gama de compostos orgânicos e inorgânicos, podendo mudar suas propriedades químicas e físicas, propriedades estas que lhes confere extensiva aplicabilidade na indústria de alimentos, farmacêutica, química, cosmética e agrícola. Atualmente, diversas CGTases já foram isoladas e caracterizadas a partir de vários microrganismos, principalmente Bacillus, Paenibacillus, Pseudomonas, Klebsiella, Xanthomonas, Thermococcus, Vibrio, Geobacillus e Thermoanaerobacterium. Neste trabalho, demonstramos o primeiro relato envolvendo a clonagem e expressão heteróloga da CGTase de Stenotrophomonas maltophilia, microrganismo isolado do solo brasileiro. O gene codificador da CGTase de S. maltophilia, foi amplificado com êxito através da técnica de PCR, clonado no vetor pET-23a(+) e expresso em Escherichia coli BL21(DE3). As células recombinantes necessitaram de aproximadamente 4 horas de cultivo em meio Luria Bertani (LB) após a adição de 0,1 mM de IPTG para a expressão elevada da proteína alvo. Porém, a CGTase recombinante foi expressa sob forma de corpos de inclusão permanecendo na fração insolúvel, sendo necessário utilizar protocolos para solubilização, incluindo diferentes concentrações de uréia, mas a precipitação não foi eficaz. Embora tenha sido observada uma expressão elevada da proteína com cerca de 60 kDa em SDS-PAGE 12%, que correspondeu ao tamanho esperado da proteína, a forma ativa da enzima não foi obtida. Uma análise bioinformática foi realizada, onde foi observou-se uma proteína conhecida como uma importante possível facilitadora transmembrana (PMFTP – putative Major Facilitator Transmembrane Protein) que ancora o gene cgt. Proteína esta que pode ser utilizada em novos estudos a fim de desenvolver um novo e mais eficaz sistema para expressão da CGTase, podendo facilitar a sua expressão extracelular. Assim, mais estudos são necessários para desenvolver um sistema de co-expressão de rCGTase::PMFTP em E. coli e obter mais informações desta proteína de Stenotrophomonas maltophilia. / Cyclodextrin glucosyltransferase (CGTase) is an industrially important enzyme, capable to convert starch into cyclodextrins (CDs). The CDs are able to form inclusion complexes with a wide range of organic and inorganic compounds, which can change their chemical and physical properties, that gives them extensive applicability in the food, pharmaceutical, chemical, cosmetics and agricultural. Currently, many CGTases have been isolated and characterized from various microorganisms, particularly Bacillus, Paenibacillus, Pseudomonas, Klebsiella, Xanthomonas, Thermococcus, Vibrio, Geobacillus and Thermoanaerobacterium. This work demonstrates the first report involving cloning and expression of heterologous CGTase from Stenotrophomonas maltophilia, microorganism isolated from Brazilian soil. The gene encoding the S. maltophilia CGTase, was successfully amplified by PCR, cloned into the vector pET-23a (+) and expressed in Escherichia coli BL21(DE3). Recombinant cells required about 4 h of cultivation in Luria Bertani (LB) after addition of 0.1 mM IPTG for high expression of the target protein. However, the CGTase was expressed recombinant form of inclusion bodies remaining in the insoluble fraction, being necessary use protocols for solubilization, including different concentrations of urea, but the precipitation was not effective. Although it was observed a high expression of the protein about 60 kDa on SDS-PAGE 12%, corresponding to the expected size of the protein, but the active form of the enzyme was not obtained. Bioinformatic analysis was performed and observed a Putative Major Facilitator Transmembrane Protein (PMFTP) harboring the cgt gene. This protein can be used in further studies to develop a new and more effective system for expression of the CGTase, which may facilitate its extracellular expression. Thus, more studies are needed to develop a system of co-expression of rCGTase::PMFTP in E. coli and acquire more information about this protein of Stenotrophomonas maltophilia.

Ciclodextrina glicosiltransferase (CGTase, EC 2.4.1.19) é a enzima capaz de converter o amido e seus açúcares relacionados em ciclodextrinas (CDs) através da reação de ciclização. As CDs têm inúmeras aplicações na indústria farmacêutica, cosmética e de alimentos, devido à sua capacidade de encapsular moléculas hidrofóbicas dentro de sua cavidade. A CGTase de Thermoanaerobacter sp. é capaz de converter o amido em CDs sob condições de processo industrial, em temperaturas elevadas. A produção de CDs em escala industrial é feita, geralmente, em processos de batelada, nos quais é utilizada a enzima livre diretamente. No entanto, a imobilização da CGTase tem sido testada, com o propósito de permitir seu uso contínua e repetidamente, de modo a prevenir sua solubilização e promover uma forma molecular mais estável. Neste trabalho, buscouse imobilizá-la em microesferas de sílica-polietilenoglicol (sílica-PEG). O suporte foi silanizado com 3- aminopropiltrimetoxisilano (APTMS) e ativado com glutaraldeído para gerar condições de imobilização de enzimas, que foi realizada a 6ºC e pH 6, durante 15h. O rendimento de imobilização e a atividade recuperada foram 83% e 73%, respectivamente. Os resultados foram comparados com estudos anteriores sobre a imobilização covalente de CGTase. As propriedades enzimáticas da CGTase imobilizada foram investigadas e comparadas com as da enzima solúvel. CGTases solúveis e imobilizadas apresentaram valores similares de pH ótimo. Por outro lado, a temperatura ótima foi de 100ºC e 80ºC para as formas solúvel e imobilizada da enzima, respectivamente. Em comparação com a CGTase solúvel, a forma imobilizada apresentou maior Km (constante de Michaelis), menor Vmax (velocidade máxima de reação), a estabilidade de armazenamento diminuiu cerca de 15% e apenas um ligeiro decréscimo foi observado quando a estabilidade térmica estava sob avaliação. A estabilidade operacional foi medida em repetidos processos de batelada e a enzima imobilizada reteve cerca de 60% da atividade catalítica inicial, após 15 ciclos. / Cyclodextrin glicosyltransferase (CGTase, EC 2.4.1.19) is the enzyme able to convert starch and related sugars into cyclodextrins (CDs) via cyclization reaction. Cyclodextrins have numerous applications in the pharmaceutical, cosmetics, and food industry, because of their capacity to encapsulate hydrophobic molecules within their cavity. The CGTase from the Thermoanaerobacter sp. is able to degrade starch into CDs under industrial conditions (high temperature). For the industrial scale production of CDs, conventional batch production methods, which utilize soluble CGTase directly, have been mainly adopted. However the immobilization of CGTase has been pursued with the purpose of allow its reuse continuously and repeatedly by avoiding enzyme solubilization and promoting a more stable molecule form. In this research, Thermoanaerobacter CGTase was immobilized on silica-polyethyleneglycol (silica-PEG) microspheres. The support was silanized with 3- aminopropyltrimethoxysilane (APTMS) and activated with glutaraldehyde to generate conditions for enzyme immobilization, which was carried out at 6ºC and pH 6, during 15h. The immobilization yield and recovery activity was around 83% and 73%, respectively. Results were compared with previous studies on covalent immobilization of CGTase. The enzymatic properties of immobilized CGTase were investigated and compared with those of the soluble enzyme. Soluble and immobilized CGTases showed similar values for optimum pH. On the other hand, the optimum temperature was 100ºC and 80ºC for the soluble and immobilized forms, respectively. In comparison with the soluble CGTase, the immobilized form exhibited higher Km (Michaelis constant), lower Vmax (maximal reaction rate), the storage stability was decreased about 15% and just a slight decrease was observed when thermal stability was under evaluation. The operational stability was evaluated in repeated batch process and the immobilized enzyme retained about 60% of the initial catalytic activity after 15 cycles.

A ciclodextrina glucosiltransferase (CGTase) é uma enzima industrialmente muito importante, capaz de converter o amido em ciclodextrinas (CDs). As CDs são capazes de formar complexos de inclusão com uma grande gama de compostos orgânicos e inorgânicos, podendo mudar suas propriedades químicas e físicas, propriedades estas que lhes confere extensiva aplicabilidade na indústria de alimentos, farmacêutica, química, cosmética e agrícola. Atualmente, diversas CGTases já foram isoladas e caracterizadas a partir de vários microrganismos, principalmente Bacillus, Paenibacillus, Pseudomonas, Klebsiella, Xanthomonas, Thermococcus, Vibrio, Geobacillus e Thermoanaerobacterium. Neste trabalho, demonstramos o primeiro relato envolvendo a clonagem e expressão heteróloga da CGTase de Stenotrophomonas maltophilia, microrganismo isolado do solo brasileiro. O gene codificador da CGTase de S. maltophilia, foi amplificado com êxito através da técnica de PCR, clonado no vetor pET-23a(+) e expresso em Escherichia coli BL21(DE3). As células recombinantes necessitaram de aproximadamente 4 horas de cultivo em meio Luria Bertani (LB) após a adição de 0,1 mM de IPTG para a expressão elevada da proteína alvo. Porém, a CGTase recombinante foi expressa sob forma de corpos de inclusão permanecendo na fração insolúvel, sendo necessário utilizar protocolos para solubilização, incluindo diferentes concentrações de uréia, mas a precipitação não foi eficaz. Embora tenha sido observada uma expressão elevada da proteína com cerca de 60 kDa em SDS-PAGE 12%, que correspondeu ao tamanho esperado da proteína, a forma ativa da enzima não foi obtida. Uma análise bioinformática foi realizada, onde foi observou-se uma proteína conhecida como uma importante possível facilitadora transmembrana (PMFTP – putative Major Facilitator Transmembrane Protein) que ancora o gene cgt. Proteína esta que pode ser utilizada em novos estudos a fim de desenvolver um novo e mais eficaz sistema para expressão da CGTase, podendo facilitar a sua expressão extracelular. Assim, mais estudos são necessários para desenvolver um sistema de co-expressão de rCGTase::PMFTP em E. coli e obter mais informações desta proteína de Stenotrophomonas maltophilia. / Cyclodextrin glucosyltransferase (CGTase) is an industrially important enzyme, capable to convert starch into cyclodextrins (CDs). The CDs are able to form inclusion complexes with a wide range of organic and inorganic compounds, which can change their chemical and physical properties, that gives them extensive applicability in the food, pharmaceutical, chemical, cosmetics and agricultural. Currently, many CGTases have been isolated and characterized from various microorganisms, particularly Bacillus, Paenibacillus, Pseudomonas, Klebsiella, Xanthomonas, Thermococcus, Vibrio, Geobacillus and Thermoanaerobacterium. This work demonstrates the first report involving cloning and expression of heterologous CGTase from Stenotrophomonas maltophilia, microorganism isolated from Brazilian soil. The gene encoding the S. maltophilia CGTase, was successfully amplified by PCR, cloned into the vector pET-23a (+) and expressed in Escherichia coli BL21(DE3). Recombinant cells required about 4 h of cultivation in Luria Bertani (LB) after addition of 0.1 mM IPTG for high expression of the target protein. However, the CGTase was expressed recombinant form of inclusion bodies remaining in the insoluble fraction, being necessary use protocols for solubilization, including different concentrations of urea, but the precipitation was not effective. Although it was observed a high expression of the protein about 60 kDa on SDS-PAGE 12%, corresponding to the expected size of the protein, but the active form of the enzyme was not obtained. Bioinformatic analysis was performed and observed a Putative Major Facilitator Transmembrane Protein (PMFTP) harboring the cgt gene. This protein can be used in further studies to develop a new and more effective system for expression of the CGTase, which may facilitate its extracellular expression. Thus, more studies are needed to develop a system of co-expression of rCGTase::PMFTP in E. coli and acquire more information about this protein of Stenotrophomonas maltophilia.

Cyclodextrins (CD) are cyclic oligosaccharides composed of six to more than sixty glucose units. Large-ring cyclodextrins (LR-CD) are novel CD comprised of more than eight glucose units with cavity structures and sizes different from that of commercially available CD₆ – CD₈. LR-CD may offer unique molecular recognition properties and can be produced biocatalytically from starch using cyclodextrin glucanotransferase (CGTase, E.C. 2.4.1.19) in a short reaction time. LR-CD were isolated from glucose, CD₆ – CD₈ and other compounds by complexation of CD₆ – CD₈ as well as precipitation techniques. The yield of LR-CD (degree of polymerization from 9 to 21) was optimized using central composite design. Addition of polar organic solvents to the synthesis resulted in higher yields of LR-CD. LR-CD composed of 9 to 21 glucose units were successfully separated using reversed-phase of ODS-AQ chromatography and normal-phase of polyamine II chromatography. Maintaining optimized reaction conditions aided in a high yield of CD₉; it could be separated with reasonable yield using a single step of polyamine II chromatography. A co-grinding method helped to obtain higher solubilization levels of glibenclamide, vitamin A acetate and vitamin D₃ in CD₁₃, CD₁₀ and CD₁₁, respectively when compared to other CD. Vitamin K₁ was solubilized in distilled water with CD₆ – CD₁₃ using a co-precipitation method. When compared with other CD, CD₉ was seen to be the best solubilizer. The analysis of complexes using ESI MS showed spironolactone and glibenclamide complexed with CD₉ and CD₁₃, respectively.

Cyclodextrins (CD) are cyclic oligosaccharides composed of six to more than sixty glucose units. Large-ring cyclodextrins (LR-CD) are novel CD comprised of more than eight glucose units with cavity structures and sizes different from that of commercially available CD₆ – CD₈. LR-CD may offer unique molecular recognition properties and can be produced biocatalytically from starch using cyclodextrin glucanotransferase (CGTase, E.C. 2.4.1.19) in a short reaction time. LR-CD were isolated from glucose, CD₆ – CD₈ and other compounds by complexation of CD₆ – CD₈ as well as precipitation techniques. The yield of LR-CD (degree of polymerization from 9 to 21) was optimized using central composite design. Addition of polar organic solvents to the synthesis resulted in higher yields of LR-CD. LR-CD composed of 9 to 21 glucose units were successfully separated using reversed-phase of ODS-AQ chromatography and normal-phase of polyamine II chromatography. Maintaining optimized reaction conditions aided in a high yield of CD₉; it could be separated with reasonable yield using a single step of polyamine II chromatography. A co-grinding method helped to obtain higher solubilization levels of glibenclamide, vitamin A acetate and vitamin D₃ in CD₁₃, CD₁₀ and CD₁₁, respectively when compared to other CD. Vitamin K₁ was solubilized in distilled water with CD₆ – CD₁₃ using a co-precipitation method. When compared with other CD, CD₉ was seen to be the best solubilizer. The analysis of complexes using ESI MS showed spironolactone and glibenclamide complexed with CD₉ and CD₁₃, respectively.