Cyclodextrin glucanotransferase from alkalophilic bacteria of Taiwan.

Nomoto, Masao; Shew, Dey-Chyi; Chen, Shew-Jie; Yen, Tai-Min; Liao, Chao-Wei; Yang, Chin‐Ping

doi:10.1271/bbb1961.48.1337

Cited by 9 publications

(3 citation statements)

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“…As características que interferem na atividade enzimática foram avaliadas pela capacidade de formação de ciclodextrinas CD-TCE (NOMOTO et al, 1984). No estudo do efeito da temperatura os valores permaneceram entre 20 e 80°C.…”

Section: Características Da Enzimaunclassified

Isolamento De Microrganismos Alcalofílicos Produtores Da Enzima Ciclodextrina Glicosil-Transferase (Cgtase)

Mendonça¹,

Sabioni²

2004

B. do CEPPA

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Uma cultura de bactéria alcalofílica, bastonete Gram+, com alta capacidade de produção da enzima ciclodextrina glicosiltransferase (CGTase) foi isolada de amostras de solo da cidade Ouro Preto, Minas Gerais (Brasil). A produção máxima da enzima ocorreu a 37°C após 72 horas de incubação, sob agitação de 120 ciclos por minuto, em meio alcalofílico (pH 10,3) contendo 2% de amido solúvel, 0,5% de peptona, 0,5% de extrato de levedura, 0,1% de K2HPO4, 0,02% de MgSO4.7H2O e 1% de Na2CO3. A temperatura ótima de atividade hidrolítica da enzima situou-se entre 55 e 60°C. Na ausência de substrato, a CGTase mostrou-se 100% estável até 60°C por 30 min. A adição de 10 mmol/L de cloreto de cálcio aumentou sua termo-resistência, resultando em maior produção de ciclodextrinas. O pH ótimo em tampão borato 50 mmol/L foi 8,0. A atividade e a estabilidade em altas temperaturas indicam potencialidade industrial para essa CGTase. Assim, estudos complementares devem ser realizados para identificar a espécie microbiana, purificar a enzima, avaliar o efeito de inibidores enzimáticos, determinar o tipo de ciclodextrina produzida em maior proporção e a taxa de conversão do amido em ciclodextrinas. ISOLATION OF ALKALOPHILIC MICROORGANIMS PRODUCERS OF THE ENZYME CYCLODEXTRIN GLYCOSYLTRANSFERASE (CGTase) Abstract A culture of an alkalophilic bacterium, rod shaped Gram positive, with high production capacity of the enzyme CGTase was isolated from soil samples of the city of Ouro Preto, Minas Gerais state (Brazil). Maximum enzyme production occurred at 37º C after 72 h of incubation, under agitation of 120 cycles per minute, in alkalophilic medium (pH 10.3) containing 2% soluble starch, 0.5 % peptone, 0.1 % K2HPO4, 0.02 % MgSO4.7H2O and 1% Na2CO3. The optimum temperature of the enzyme hydrolytic activity was between 55 and 60º C. In the absence of substrate, the CGTase showed stability of 100% until 60º C per 30 min. The addition of 10 mmol/L of calcium chloride enhanced its thermal resistance, resulting in a higher production of cyclodextrins. The optimum pH in borate buffer 50 mmol/L was 8.0. The activity and the stability in high temperatures indicate industrial potential for this CGTase. Like this, complementary studies must be realized to identify the microbial species, to purify the enzyme, to evaluate the effect of enzymatic inhibitors, to determine the type of cyclodextrins produced in greater proportion and the conversion rate of starch in cyclodextrins.

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Section: Características Da Enzimaunclassified

Isolamento De Microrganismos Alcalofílicos Produtores Da Enzima Ciclodextrina Glicosil-Transferase (Cgtase)

Mendonça¹,

Sabioni²

2004

B. do CEPPA

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“…(Akimura, et al, 199 1;Kato and lorikoshi, 1986;Kitahata et al, 1974;Kitahata and Okada, 1982;Kobayashi ef al., 1978;Lee et al, 1992;Makela et al, 1988;Nakamura and Horikoshi, 1976;Nomoto et al, 1984;Pongsawasdi and Yakisawasdi, 1987;Yagi, et al, 1986;Yu et al, 1989) and some other microorganisms (Bender, 1977;Lee, et al, 1992;Norman and J4rgensen, 1992;Yagi et al, 1980). The CGTase is an important industrial enzyme because CDs have already been used widely for food pharmaceutical, and chemical industries (Nakamoto, 1985;S&mid, 1989;Szejtli, 1989).…”

Section: Introductionmentioning

confidence: 99%

Purification and characterization of a ?-cyclodextrin glucosyltransferase from an alkalophilic Bacillus sp.

Yan

Lin

1995

Biotechnol Tech

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“…Agitated cultivation proceeded for up to five days at 37°C. Then cells were separated in a centrifuge, and CGTase activity for production of ~ and y-CD was determined in the supernatant by colorimetric methods [6], and by the method of successive dilution s of the enzyme and characterisation 0 f CD produced by precipitation with TCE-trichloroethylene [7].The CGTase enzyme was obtained from the supernatant by precipitation with ammonium sulfate, and further purified by bioespecific affinity chromatography according to Laszlo et al[8] using either ~-CD or y-CD as affinants [3].Purified CGTase was concentrated by ultrafiltration, washed with Tris-HCl buffer five times to remove molecules with MW smaller than 30,000, and used thereafter for CD production. These tests, run at 50°C, used as substrate a 10% solution of Dextrin 10 Fluka, Tris-HCI buffer pH 8.0, O.ol M and 5mM CaCho Enzyme concentration in the reaction medium was set around I mgIL.…”

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confidence: 99%

Screening For Gamma-Cgtase

Matioli

Zanin

Guimarães

et al. 1996

Proceedings of the Eighth International Symposium on Cyclodextrins

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CGTase positive alkalophilic microorganisms were selected by parallel application of two screening techniques based on colorimetric distinction of the colonies at the cultivation plate level. One of the techniques uses Congo red and xylene cyanole dyes which form a clear halo only in the presence of y-cyclodextrin (y-CD), whereas the other uses phenolphthalein that is specific for f3-cyclodextrin (f3-CD). The CGTase produced by strains selected up to now yields, however, mainly f3-CD. The production of f3-CD by the CGTase of strain 37 is, nevertheless, very interesting since f3-cyclodextrin concentration reached 16 mM at 50°C, pH 8.0, 10% w/v initial substrate concentration. INTRODUCTIONThe potential application by the pharmaceutical industry of large drug molecules encapsulated by y-cyclodextrin (y-CD) has driven research interest into: (i) better ways of separating y-CD from a mixture of cyclodextrins normally produced by action of CGTase enzymes upon starch [1]; (ii) increase the production ofy-CD by complexation during these reactions [2]; or (iii) search for a microorganism whose CGTase kinetically favours y-CD production at the beginning of the reaction [3]. In this work the third option was adopted. 2. MATERIALS AND METHODSSelection of alkalopbilic microorganisms that produce CGTase enzyme was accomplished using the Horikoshi II plate screening medium [4]: soluble starch (1 %), polypetone (0.5%), yeast extract (0.5%), KZHP04 (0.1 %), MgS04 (0.02%), NaZC03(1 %), agar (1.5%), final pH 10.3, modified by inclusion of the following dyes: Congo red (0.01 %) and Xylene cyanole FF (0.001 %). These dyes were used by Hamaker and Tao [5] with Luria-Bertani neutral media (pH 7.0) containing 1% starch, to detect y-CD production by recombinant E. coli. The presence of y-CD is revealed by a clear halo around the colonies. A drop of solution containing either of each cyclodextrin (a, ~ or y) has shown that also for modified Horikoshi medium, only y-CD produces a clear halo. Samples of soils used for cultivation of wheat, corn, potato and cassava, were suspended in distilled water; screening plates were inoculated with these suspensions and incubated for 24 h at 37°C.To determine the CGTase activity of positive microorganisms, these were cultivated in a liquid medium composed of the same substances aforementioned with exception of the dyes and agar. Agitated cultivation proceeded for up to five days at 37°C. Then cells were separated in a centrifuge, and CGTase activity for production of ~ and y-CD was determined in the supernatant by colorimetric methods [6], and by the method of successive dilution s of the enzyme and characterisation 0 f CD produced by precipitation with TCE-trichloroethylene [7].The CGTase enzyme was obtained from the supernatant by precipitation with ammonium sulfate, and further purified by bioespecific affinity chromatography according to Laszlo et al.[8] using either ~-CD or y-CD as affinants [3].Purified CGTase was concentrated by ultrafiltration, washed with Tris-HCl buffer five times to...

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Cyclodextrin glucanotransferase from alkalophilic bacteria of Taiwan.

Cited by 9 publications

References 0 publications

Isolamento De Microrganismos Alcalofílicos Produtores Da Enzima Ciclodextrina Glicosil-Transferase (Cgtase)

Isolamento De Microrganismos Alcalofílicos Produtores Da Enzima Ciclodextrina Glicosil-Transferase (Cgtase)

Purification and characterization of a ?-cyclodextrin glucosyltransferase from an alkalophilic Bacillus sp.

Screening For Gamma-Cgtase

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