Avaliação da produção de ciclodextrina glicosiltransferase (CGTase) por fermentação submersa de Paenibacillus sp.

Abstract

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.