Introduction

Cyclodextrins (CDs) are cyclic oligosaccharides with a variable number of glucose units joined by α linkages -1.4, the most common features of which are 6, 7 and 8 glucose units with diameters of about 5.2 Å (α -CD), 6.6 Å (β-CD) and 8.4 Å (γ-CD) [1] and sufficiently hydrophilic externally to make CDs water soluble [2]. CDs solubilize and modify drugs, foods, cosmetics, etc. due to the formation of inclusion complexes [3]. Cyclodextrin glycosyltransferase (CGTase) EC 2.4.1.19 (α-1,4-glucan 4-glycosyltransferase) is a monomeric enzyme with a molecular weight of around 75 kDa [4]. CGTase has an amino acid sequence similar to that of the enzyme α-amylase and therefore belongs to the amylase family. This enzyme catalyzes the conversion of starch into CDs through a reversible intramolecular transglycosylation reaction, cyclization, hydrolysis and coupling to the starch [5]. CDs production involves starch treatment with CGTase produced by bacteria [6]. The selection of carbon sources is a critical factor in the fermentation process for CGTase production. The wrong carbon source may cause catabolite repression of the enzyme production and reduce the growth rate of the microorganism [7]. Sorghum is a starchrich grain grown widely in Brazil. The increase in sorghum crops is due the capacity of the plant to adapt to conditions of water deficiency and recycle nutrients in the soil. Nitrogen fixation in sorghum crops has been addressed in recent studies [8]. According to the National Sorghum Producers, sorghum has a low production cost – at least 20 percent less than other major grain crops. Thus, the use of sorghum as a source for downstream products has an economic benefit. The sorghum has been used as a carbon source for products as ethanol [9] and Lactic acid except for CGTase production are not yet known [10]. Sorghum use as a carbon source in the media may lower the cost of CGTase production. The aims of the present study were to evaluate the use of sorghum as a substrate for the production of CGTase using the microorganism Bacillus circulans and optimize its concentration and other production variables, such as temperature and initial pH, in an experimental design.

Materials and Methods

Analysis of starch in Sorghum The starch in sorghum was calculated as the difference between total sugars and reducing sugars: Sorghum starch = total sugars – reducing sugars. For the analysis of total sugars, 2 g of sorghum were transferred to a 100-mL Erlenmeyer flask; 1 ml of sodium hydroxide 10% (v/v) was added and autoclaved (1 atm at 120°C) for 1 hour. At room temperature, 10 ml of hydrochloric acid were added and autoclaved again (1 atm at 120°C) for 30. At 25°C, the excess hydrochloride acid was neutralized with sodium hydroxide 40% (v/v), transferred to a 500-mL volumetric flask and the volume was completed with distilled water. The resultant solution was stirred and filtered with dry filter paper (90 mm). Two hundred fifty µL of filtered solution were added to 250 µL of 3,5-dinitro salicyclic acid (DNS), boiled for 5 min. and cooled. Absorbance of the final solution was read using a spectrophotometer at 540 nm. The standard curve was made with glucose P.A. For the analysis of reducing sugars, 2 g of sorghum was transferred to a 500-mL volumetric flask; the volume was completed with distilled water, stirred and filtered with dry filter paper (90 mm). Two hundred fifty µL of the solution volume were added to 250 µL of DNS, boiled for 5 min. and cooled in an ice bath. Absorbance of the final solution was read using a spectrophotometer at 540 nm. The standard curve was made with glucose P.A.