Abstract
Cyclodextrin glycosyltransferase (CGTase) is an enzyme that produces cyclodextrins (CDs) from starch and related carbohydrates, producing a mixture of α-, β-, and γ-CDs in different amounts. CGTase production, mainly by Bacillus sp., depends on fermentation conditions such as pH, temperature, concentration of nutrients, carbon and nitrogen sources, among others. Bacillus megaterium CGTase produces those three types of CDs, however, β-CD should prevail. Although, waxy corn starch (CS) is used industrially to obtain CGTase and CDs because of its high amylopectin content, alternative sources such as amaranth starch (AS) could be used to accomplish those purposes. AS has high susceptibility to the amylolytic activity of CGTase because of its 80% amylopectin content. Therefore, the aim of this work was evaluate the AS as carbon source for CGTase production by B. megaterium in a submerged fermentation. Afterwards, the CGTase was purified partially and its activity to synthesize α-, β-, and γ-CDs was evaluated using 1% AS as substrate. B. megaterium produced a 66 kDa CGTase (Topt = 50°C; pHopt = 8.0), from the early exponential growth phase which lasted 36 h. The maximum CGTase specific activity (106.62 ± 8.33 U/mg protein) was obtained after 36 h of culture. CGTase obtained with a Km = 0.152 mM and a Vmax = 13.4 μM/min yielded 40.47% total CDs using AS which was roughly twice as much as that of corn starch (CS; 24.48%). High costs to produce CDs in the pharmaceutical and food industries might be reduced by using AS because of its higher α-, β- and γ-CDs production (12.81, 17.94, and 9.92%, respectively) in a shorter time than that needed for CS.
Highlights
Cyclodextrins (CDs) are synthesized from starch and related carbohydrates such as amylose, amylopectin and maltooligosaccharide by cyclodextrin glycosyltransferase (CGTase, E.C.2.4.1.19) which is a bacterial extracellular enzyme (Ahmed and El-Refai, 2010)
Lag phase was practically negligible; the total growth time of B. megaterium was of 4 days (Figure 1A), the exponential phase was shorter when amaranth starch (AS) was used as substrate (36 h) compared to that observed with corn starch (CS), which lasted 48 h
The shape of the curve of extracelular proteins was identical with that of Cyclodextrin glycosyltransferase (CGTase) specific activity. This activity obtained during the submerged fermentation (SmF), when AS was used at any time of the exponential phase, was roughly 25% greater than that observed when CS was utilized (Figure 1B)
Summary
Cyclodextrins (CDs) are synthesized from starch and related carbohydrates such as amylose, amylopectin and maltooligosaccharide by cyclodextrin glycosyltransferase (CGTase, E.C.2.4.1.19) which is a bacterial extracellular enzyme (Ahmed and El-Refai, 2010). The formation of the inclusion complexes modifies the physical and chemical properties of the host molecule, mostly in terms of water solubility. In this sense, CDs are important ingredients as molecular encapsulators for applications in food, cosmetic, and pharmaceutical industries (Sivakumar and Shakilabanu, 2013). A comparative analysis of more than 30 currently known CD containing pharmaceutical formulations shows that β-CD is the most commonly employed The reason for this lies in the ease of its production and subsequent low price (more than 10,000 tons produced annually with an average bulk price of approximately 5 USD per kg). A universal solution to this problem was found in the substitution of multiple β-CD hydroxyls on both rims of the molecule resulting in a notably improved aqueous solubility (Kurkov and Loftsson, 2013)
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