Purificação e caracterização bioquímica da CGTase produzida por Paenibacillus campinasensis H69-3
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Data
2018-12-18
Autores
Sanches, Raisa Déli de Oliveira [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
Uma ciclomaltodextrina glucanotransferase (E.C. 2.4.1.19, CGTase) produzida pelo microrganismo Paenibacillus campinasensis linhagem H69-3, foi obtida por fermentação submersa a 45 °C, e a CGTase purificada por cromatografia de afinidade bioespecífica em resina Sepharose 6B ativada com β-CD. A enzima foi purificada até 23 vezes, obtendo um rendimento de 11,0%, e a atividade específica de 32,6 U mg-1 ao final da etapa de purificação. A massa molecular da enzima purificada foi estimada em 72 kDa por eletroforese desnaturante em gel de poliacrilamida na presença de dodecil-sulfato de sódio. O pH ótimo para a atividade enzimática foi de 6,5 e a enzima foi estável na faixa de pH de 6,5 a 8,0. A temperatura ótima foi de 65 °C em pH 6,5, e foi térmicamente estável até 55 °C na ausência de substrato durante 1 h de incubação e estável até 65 °C na presença de Ca2+ 5 mmol L-1. A atividade enzimática aumentou na presença de Ba2+, Mg2+, Ca2+, Ni2+, K+, Zn2+ e foi inibida na presenca dos íons metálicos Cu2+, Hg2+, Ag+, Li2+, Al3+ e NH42+ e por β‑, γ‑CD, DMSO, Triton, SDS e NaN3. Utilizando maltodextrina como substrato a CGTase apresentou 11,0±0,5 µmol min·mg e 0,30 ± 0,07 mg mL‑1, Vmáx e Km, respectivamente. Avaliaram-se os parâmetros termodinâmicos da CGTase, que demonstrou mais de 11 horas a 65 ºC como tempo de meia vida, e elevada resistência estrutural à desnaturação térmica quando avaliados os resultados de entropia, energia de ativação e entalpia.
A cyclomaltodextrin glucanotransferase (E.C. 2.4.1.19, CGTase) produced by 4 microorganism Paenibacillus campinasensis strain H69-3 was obtained by submerged fermentation at 45 °C and CGTase purified by biospecific affinity chromatography on β-CD activated Sepharose 6B resin. The enzyme was purified up to 23 times, yielding 11.0% yield, and the specific activity of 32.6 U mg-1 at the end of the purification step. The molecular weight of the purified enzyme was estimated at 72 kDa by denaturing polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The optimum pH for the enzymatic activity was 6.5 and the enzyme was stable in the pH range of 6.5 to 8.0. The optimum temperature was 65 °C at pH 6.5 and was thermally stable at 55 °C in the absence of substrate for 1 h incubation and stable at 65 ° C in the presence of Ca2+ 5 mmol L -1. The enzyme activity increased in the presence of Ba2+, Mg2+, Ca2+, Ni2+, K+, Zn2+ and was inhibited in the presence of Cu2+, Hg2+, Ag+, Li2+, Al3+ and NH42+ ions and by β, γ -CD, DMSO, Triton, SDS and NaN3. Using maltodextrin as substrate CGTase presented 11.0 ± 0.5 μmol min · mg and 0.30 ± 0.07 mg mL-1, Vmax and Km, respectively. The thermodynamic parameters of CGTase, which demonstrated more than 11 hours at 65 ºC as a half-life, and high structural resistance to thermal denaturation were evaluated when the results of entropy, activation energy and enthalpy were evaluated.
A cyclomaltodextrin glucanotransferase (E.C. 2.4.1.19, CGTase) produced by 4 microorganism Paenibacillus campinasensis strain H69-3 was obtained by submerged fermentation at 45 °C and CGTase purified by biospecific affinity chromatography on β-CD activated Sepharose 6B resin. The enzyme was purified up to 23 times, yielding 11.0% yield, and the specific activity of 32.6 U mg-1 at the end of the purification step. The molecular weight of the purified enzyme was estimated at 72 kDa by denaturing polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The optimum pH for the enzymatic activity was 6.5 and the enzyme was stable in the pH range of 6.5 to 8.0. The optimum temperature was 65 °C at pH 6.5 and was thermally stable at 55 °C in the absence of substrate for 1 h incubation and stable at 65 ° C in the presence of Ca2+ 5 mmol L -1. The enzyme activity increased in the presence of Ba2+, Mg2+, Ca2+, Ni2+, K+, Zn2+ and was inhibited in the presence of Cu2+, Hg2+, Ag+, Li2+, Al3+ and NH42+ ions and by β, γ -CD, DMSO, Triton, SDS and NaN3. Using maltodextrin as substrate CGTase presented 11.0 ± 0.5 μmol min · mg and 0.30 ± 0.07 mg mL-1, Vmax and Km, respectively. The thermodynamic parameters of CGTase, which demonstrated more than 11 hours at 65 ºC as a half-life, and high structural resistance to thermal denaturation were evaluated when the results of entropy, activation energy and enthalpy were evaluated.
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Ciclomaltodextrina glucanotransferase, CGTase, Paenibacillus campinasensis, Ciclodextrinas, Cromatografia de afinidade, Cyclomaltodextrin glucanotransferase, Cyclodextrins, Affinity chromatography