Made available in DSpace on 2016-06-02T19:56:57Z (GMT). No. of bitstreams: 1 6472.pdf: 2357652 bytes, checksum: b48a2e541ddf343b775996e49d4e7f1f (MD5) Previous issue date: 2014-03-31 / Financiadora de Estudos e Projetos / Cyclodextrins (CDs) are cyclic oligosaccharides that show the ability to encapsulate a large number of organic molecules at the molecular level. Thus, they have large application in pharmaceutical, food, and cosmetics industries. CDs may have different sizes; however the most common are formed by 6, 7, and 8 glucose units, called α, β and γ-CD, respectively. These compounds are produced from starch by hydrolysis catalyzed by cyclodextrin glycosyltransferase ( CGTase - EC 2.4.1.19 ) enzyme. CGTase is commonly produced from bacteria of the genus Bacillus and Klebsiella pneumoniae. Due to the commercial importance of this enzyme and of the microorganisms producing CGTase whose enzyme is capable to produce only one CD, this work aimed to evaluate the CGTase production by the bacterium Paenibacillus sp. F37. The experiments aimed to assess the potential for enzyme production by Paenibacillus sp. F37 strain and also the characterization and classification of the enzyme. Analysis of enzymatic activity was accomplished by cyclization activity of the supernatant of the culture medium, which was monitored by the production rate of β-CD at 50°C, pH 8.0 using a solution of dextrin as substrate. Initially it was evaluated the effect of the pH (6.0 to 10.0) over the microorganism growth into solid Horikoshi medium without starch. It was observed that the microorganism grew better at pH 8.0, being this pH adopted for assays using liquid Horikoshi medium without starch. In this step it was established the following conditions to the bacterium growth: 10h, 37oC, and agitation of 120 rpm in an incubator. The CGTase production step was performed into Horikoshi medium containing 1% (m/v) soluble starch (enzyme inducer), and pH and production time were evaluated. Under standard conditions (37oC, 120 rpm, 16h, and culture medium prepared in 0.1 M tris-HCl buffer pH 9.0) the volumetric activity from the culture medium achieved about 800 U/L, corresponding to a productivity of 50 U/L.h-1. The characterization of the crude enzyme showed that the CGTase from Paenibacillus sp. F37 strain has maximum cyclization activity around 50oC and pH 6.0. Under these conditions the enzyme showed to have a half-life time around 2 h. At the growth conditions (37oC, pH 9.0), the enzyme show to have a half-life time around 3h, being almost inactivated around 5h. Thus, the enzyme production in a medium where pH starts at 9.0 and is decreased to around 6.0 during the growth showed to be a good strategy to prevent denaturation of the enzyme. The CD production into batch reactor and their characterization by colorimetric and chromatographic methods allowed classifying the enzyme as a β-CGTase (ratio α-:β-:γ-CD of 0:1:0.26). At the CD-production conditions (50oC, pH 8.0, 50 g/L of dextrin, 1.6 U/g of dextrin, and 92 h) it was achieved β-CD productivity around 100 mg/L.h. These results showed that Paenibacillus sp. F37 strain is a promising microorganism for producing CGTase. / Ciclodextrinas (CDs) são oligossacarídeos cíclicos de grande importância comercial e que apresentam amplo emprego industrial nos setores farmacêutico, alimentício, de cosmético, dentre outros, por terem a capacidade de encapsulamento ao nível molecular de um grande número de moléculas orgânicas. CDs podem apresentar diversos tamanhos, sendo as que têm 6, 7 ou 8 unidades de glicose, denominadas α, β e γ-CD, respectivamente, são as mais conhecidas. Estas são obtidas a partir da hidrólise do amido pela ação da enzima ciclodextrina glicosiltransferase (CGTase EC 2.4.1.19). A CGTase geralmente é produzida por bactérias do gênero Bacillus e Klebsiella pneumoniae. Levando em consideração a importância comercial desta enzima e a busca por microrganismos produtores de CGTase que produzam predominantemente uma CD de interesse, esse trabalho buscou avaliar a produção de CGTase a partir de Paenibacillus sp. F37. Os experimentos tiveram como objetivo a avaliação do potencial de produção da enzima por esse microrganismo, bem como a caracterização e a classificação da enzima. A análise de atividade enzimática foi realizada pela atividade de ciclização do sobrenadante do meio de cultivo, que foi monitorada pela taxa de produção de β−CD a 50°C, pH 8,0, usando uma solução de dextrina como substrato. Avaliou-se inicialmente a influência do pH (6,0 a 10,0) no crescimento do microrganismo em meio Horikoshi sólido, sem amido. Observou-se melhor crescimento em pH 8,0, definindo-se esse valor para ensaios de crescimento em meio Horikoshi líquido, sem amido. Nessa etapa definiram-se as condições de crescimento como 10 h, 37ºC e agitação de 120 rpm em câmara incubadora. A etapa de produção da CGTase foi realizada em meio Horikoshi com amido solúvel 1%, m/v (indutor da enzima), avaliando-se pH e tempo de cultivo. Sob condições padronizadas (37ºC, 120 rpm, 16 h e meio de produção preparado em tampão tris-HCl 0,1 M, pH 9,0) atingiu-se uma atividade volumétrica no meio de produção da ordem de 800 U/L, correspondendo a uma produtividade de aproximadamente 50 U/L.h-1. A caracterização da enzima bruta mostrou que a CGTase de Paenibacillus sp. F37 apresenta atividade máxima de ciclização em 50ºC, pH 6,0. Nessas condições a enzima apresentou meia-vida aproximada de 2 h. Nas condições de cultivo (37ºC, pH 9,0), a enzima apresentou baixa estabilidade, com meia-vida em torno de 3 h, sendo praticamente inativada em 5 h. Por isso, produzir a enzima em meio cujo pH inicial é 9,0 e esse reduz-se ao longo do cultivo para em torno de 6,0, mostrou-se uma boa estratégia para prevenir a desnaturação da enzima. A produção de CDs em reator batelada e a sua caracterização colorimétrica e por CLAE permitiram a classificação da enzima como uma β-CGTase (razão α-:β-:γ-CD de 0:1:0,26). Nas condições de produção de CDs (50ºC, pH 8,0, 50 g/L de dextrina, 1,6 U/g de dextrina e 92 h) atingiu-se uma produtividade de β-CD da ordem de 100 mg/L.h. Os resultados indicam, portanto, que Paenibacillus sp. F37 é um microrganismo promissor na produção de CGTase.

京都大学 / 新制・論文博士 / 博士(農学) / 乙第13567号 / 論農博第2913号 / 新制||農||1101(附属図書館) / (主査)教授 小川, 順, 教授 阪井, 康能, 教授 栗原, 達夫 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

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.

Cyclodextrin glucanotransferase (EC 2.4.1.19) from the alkaliphilic Bacillus sp. G-825-6 converts starch mainly to c-cyclodextrin (CD8). A combination of error-prone PCR and DNA shuffling was used to obtain variants of this enzyme with higher product specificity for CD8 and a broad pH activity range. The variant S54 with seven amino acid substitutions showed a 1.2-fold increase in CD8-synthesizing activity and the product ratio of CD7:CD8 was shifted to 1:7 compared to 1:3 of the wild-type enzyme. Nine amino acid substitutions of the cyclodextrin glucanotransferase were performed to generate the variant S35 active in a pH range 4.0–10.0. Compared to the wild-type enzyme which is inactive below pH 6.0, S35 retained 70% of its CD8-synthesizing activity at pH 4.0.

Cyclodextrin glucanotransferase (EC 2.4.1.19) from the alkaliphilic Bacillus sp. G-825-6 converts starch mainly to c-cyclodextrin (CD8). A combination of error-prone PCR and DNA shuffling was used to obtain variants of this enzyme with higher product specificity for CD8 and a broad pH activity range. The variant S54 with seven amino acid substitutions showed a 1.2-fold increase in CD8-synthesizing activity and the product ratio of CD7:CD8 was shifted to 1:7 compared to 1:3 of the wild-type enzyme. Nine amino acid substitutions of the cyclodextrin glucanotransferase were performed to generate the variant S35 active in a pH range 4.0–10.0. Compared to the wild-type enzyme which is inactive below pH 6.0, S35 retained 70% of its CD8-synthesizing activity at pH 4.0.

Background: Cyclodextrin glucanotransferases (CGTases) catalyze the synthesis of cyclodextrins, cyclic oligosaccharides composed of glucose monomers that find applications in the pharmaceutical, food, and cosmetic industries. An economic application of these industrially important enzymes requires their efficient production and recovery. In this study, the effect of Sec-type signal peptides on the recombinant expression of a CGTase derived from Bacillus sp. G825-6 was investigated in Escherichia coli BL21(DE3) using a codon-adapted gene. In addition, a novel purification method for the CGTase using starch adsorption was developed. Results: Expression vectors encoding N-terminal PelB, DacD, and the native Bacillus sp. G825-6 CGTase signal peptides (SP) were constructed for the recombinant CGTase. With the DacD SP derived from E. coli, a 3.9- and 3.1-fold increase in total enzyme activity was obtained compared to using the PelB and the native CGTase SP, respectively. DacD enabled a 7.3-fold increase of activity in the extracellular fraction after induction for 24 h compared to the native CGTase SP. After induction for 48 h, 75% of the total activity was detected in the extracellular fraction. By a batch wise adsorption to starch, the extracellular produced CGTase could be purified to homogeneity with a yield of 46.5% and a specific activity of 1637 U/mg. Conclusions: The signal peptide DacD promoted the high-level heterologous extracellular expression of a recombinant CGTase from Bacillus sp. G825-6 with a pET20b(+) vector in E. coli BL21(DE3). A protocol based on starch adsorption enabled a fast and efficient purification of the recombinant enzyme.

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.