Economic model for obtaining cyclodextrins from commercial cgtase

A repetitive batch process was employed followed by membrane ultrafiltration system to produce low-cost cyclodextrins (CDs) using commercial enzymes Toruzyme® cyclomaltodextrin glucanotransferase (CGTase) and its kinetic parameters were determined. The ultrafiltration system enabled the removalof inhibitory products from the reaction medium, allowing the enzyme to be recovered for reuse. A 10 kDa membrane was used to separate the different CDs produced by the CGTase. The substrates evaluated were maltodextrin, corn starch and cassava starch at 5, 10 and 15% (w/V), in the presence and absence of 10% (V/V) ethanol. After reaction for 132 h, 10% (w/V) cassava starch in the presence of ethanol provided the best results with 32.1 mg/mL of ß-CD. Maximum production occurred after 72 h of reaction, with a yield of 87.4% of ß-CD and an α-CD, ß-CD and γ-CD production ratio of 1:1:0.08 g, respectively. When eight repetitive batches of 72 h followed by ultrafiltration and crystallization of ß-CD were performed, 2.1 g of precipitate was obtained with a purity of 67.6% ß-CD. The supernatant from the crystallization process was lyophilized and resulted in 35.3% α-CD. The developed model can be used industrially for the production of low cost CDs from easily obtained raw material

High level expression of α-CGTase and optimize biotransformation conditions of AA-2 G / 中国生化药物杂志

Objective To construct a prokaryotic expression vector in BL21 to secretorily expressα-Cyclodextrin Glycosyltransferase(α-CGTase). Methods α-CGT gene was amplified from Bacillus macerens genome by PCR.pET26b and α-CGT gene were connected after digested with Nco I, Xho I respectivly, and then transformed into Escherichia coli BL21 strain.α-CGTase was expressed in fermentation culture medium and AA-2G was prepared by using α-CGTase, VC and starch.Results α-CGTase was expressed secretorily and the enzyme activity was up to 120 U/mL.AA-2G was prepared by the biotransformation of VC and starch using α-CGTase which proved to be correct by HPLC.Conclusion AA-2G was prepared by using self-madeα-CGTase, after optimized the preparation conditions the yield of AA-2G was 17.46 g/L, and the conversion rate reached 58.2%(mg/mg).

Secretion of cyclodextrin glucanotransferase in E. coli using Bacillus subtilis lipase signal peptide and optimization of culture medium.

The cyclodextrin glycosyltransferase (CGTase) of Paenibacillus pabuli US132 was fused to the secretive lipase signal peptide of B. subtilis. This leads to an efficient secretion of the recombinant enzyme into the culture medium of E. coli as an active and soluble form contrasting with the native construction leading to a periplasmic production. In order to enhance the yield of CGTase production, an experimental design methodology was applied for the optimization of the culture composition. Hence, the media components were submitted to preliminary screening using a Plakett-Burman design. The concentrations of the major operating ones were then optimized to enhance the secretion of CGTase using response surface methodology. The findings revealed that concentrations of 0.5% potato starch, 3% yeast extract, 3% tryptone, 1.5% casein hydrolysate, 0.5% NaCl, 0.2% KH2PO4, and 0.02% MgSO4 were the optimal conditions for CGTase production. The experimental value (9.43 U/ml) obtained for CGTase activity was very close to the predicted value (9.27 U/ml).

Cyclodextrin glycosyltransferase production by new Bacillus sp. strains isolated from brazilian soil / Produção de ciclodextrina glicosiltransferase por novas cepas de Bacillus sp. isoladas de solo brasileiro

Three strains of Bacillus sp. (BACRP, BACNC-1 and BACAR) were isolated from soil adhered to cassava husk. CGTase specific activity for the three isolated strains was higher when cultivated at 40¨¬C. Potato starch, cassava starch, maltodextrin and glucose were used as carbon source and growth temperatures varied from 25 to 55¨¬C. The three isolates presented higher CGTase specific activity when cultivated with potato starch at 40¨¬C. Isolated BACRP and BACAR presented specific activity of 4.0x10-3 and 2.2x10-3 U/mg prot at pH 7.0, respectively, when cultivated in mediums added with NaCl 2 percent; at pH 10,0 their activities were of 3.4x10-3 and 3.0x10-3 U/mg prot, respectively, in the same concentration of NaCl. On the other hand, the isolated BACNC-1 presented activity specific of 2.4x10-3 U/mg prot when cultivated at pH 7.0 added of NaCl 1 percent, and at pH 10.0 the specific activity was of 3.4x10-3 U/mg prot without NaCl addition. This work also showed the presence of cyclodextrins formed during fermentation process and that precipitation with acetone or lyophilization followed by dialysis was efficient at removing CDs (cyclodextrins), thus, eliminating interference in the activity assays. The enzyme produced by the BACAR strain was partially purified and ¥â-CD was liberated as a reaction product.

Três linhagens de Bacillus sp (BACRP, BACNC- 1 e BACAR) foram isoladas a partir de solo aderido em casca de mandioca. Foram utilizados amido de batata, amido de mandioca, maltodextrina e glicose como fonte de carbono, e temperaturas de crescimento de 25-55¨¬C, sendo que os três isolados apresentaram maior atividade específica de CGTase quando cultivados com amido de batata a 40¨¬C. Em pH 7,0 os isolados BACRP e BACAR apresentaram atividade específica de 4,0x 10-3 e 2,2x10-3 U/mg prot, respectivamente, quando cultivados em meios acrescidos de 2 por cento de NaCl; em pH 10,0 suas atividades foram de 3,4x10-3 e 3,0x10-3 U/mg prot na mesma concentração de NaCl. Por outro lado, o isolado de BACNC-1 apresentou atividade específica 2,4x10-3 U/mg prot quando cultivado em pH 7,0 acrescido de 1 por cento de NaCl, e em pH 10,0 sua atividade específica foi de 3,4x10-3 U/mg prot sem adição de NaCl. Também foi demonstrada neste trabalho que ciclodextrinas são formadas durante o processo fermentativo, e que a precipitação com acetona ou liofilização seguida de diálise foram eficientes na remoção destas CDs, eliminando sua interferência nos ensaios enzimáticos. A enzima produzida pela cepa BACAR foi purificada parcialmente liberando b-CD como produto da reação.

Metodologia de seleção de cepas para produção etodologia da ciclodextrina glicosiltransferase e para purificação da enzima / Strains selection methodology for cyclodextrin glycosyltransferase production and enzyme purification

As ciclodextrinas (CDs) são maltooligossacarídeos, produzidas a partir do amido, pela enzima ciclodextrina glicosiltransferase (CGTase). Esta pesquisa teve por objetivo estabelecer metodologias de seleção de cepas para produção de CGTase e para purificação da enzima. Os microrganismos foram selecionados a partir de 53 análises de solos de cultura de amido, em placas contendo meio de cultivo específico, para seleção de cepas produtoras de CGTase. As enzimas foram obtidas com cultivo destes microrganismos em meio líquido. As atividades enzimáticas das CGTases foram determinadas pelos métodos espectrofotométricos e precipitação com tricloroetileno. A cepa isolada do solo de aveia foi a que apresentou maior atividade [0,1864 µmol de alfa-CD (min mL)-1]. Esta cepa foi utilizada para a produção da enzima em escala laboratorial e purificação em cromatografia de afinidade bioespecífica. A cepa selecionada nesta pesquisa abre novas perspectivas para produção de enzima e CDs em escala industrial. Palavras-chave: ciclodextrina, glicosiltransferase, CGTase.

Cyclodextrins (CDs) are maltooligosaccharides produced from starch by cyclodextrin glycosyltransferase (CGTase) enzyme. This research aimed at establishing method for strains selection for CGTase production and enzyme purification. The microorganisms were selected from 53 analyses of starch cultures soils on plates containing specific culture medium for strains selection that produce CGTase. The enzymes were obtained by culturing these microorganisms in liquid medium. The enzyme activity was determined with photospectrometric methods and precipitation with trichloroethylene. The strain isolated from oat soil was the one that showed the highest activity [0.1864 µmol of alpha-CD (min mL)-1]. This strain was used for enzyme production in laboratory scale and purification by biospecific affinity chromatography. The strain selected in this research opens new perspectives for enzymes production and CDs in industrial scale.