Purification and characterization of cyclodextrin β-glucanotransferase from novel alkalophilic bacilli

Abstract

The discovery of novel bacterial cyclodextrin glucanotransferase (CGTase) enzyme could provide advantages in terms of its production and relative activity. In this study, eight bacterial strains isolated from soils of a biodiversity-rich vegetation in Egypt based on their hydrolyzing activity of starch, were screened for CGTase activity, where the most active strain was identified as Bacillus lehensis. Optimization process revealed that the using of rice starch (25%) and a mixture of peptone/yeast extract (1%) at pH 10.5 and 37 °C for 24 h improved the bacterial growth and enzyme activity. The bacterial CGTase was successively purified by acetone precipitation, gel filtration chromatography in a Sephadex G-100 column and ion exchange chromatography in a DEAE-cellulose column. The specific activity of the CGTase was increased approximately 274-fold, from 0.21 U/mg protein in crude broth to 57.7 U/mg protein after applying the DEAE-cellulose column chromatography. SDS-PAGE showed that the purified CGTase was homogeneous with a molecular weight of 74.1 kDa. Characterization of the enzyme exhibited optimum pH and temperature of 7 and 60 °C, respectively. CGTase relative activity was strongly inhibited by Mg(2+), Zn(2+), Al(3+) and K(+), while it was slightly enhanced by 5 and 9% with Cu(2+) and Fe(2+) metal ions, respectively.

Production and characterization of β‐cyclodextrin glucanotransferase fromBacillus sp. ND1

“…The purification studies of CGTase from Bacillus sp. ND1 was similar to the studies reported in Bacillus lehensis with overall 7.7% enzyme yield and 57.7 U mg −1 specific activity . The bacterium Bacillus firmus has been reported to have an exceptional amount of yield of 80% with 23.1‐fold purification .…”

Purification and biochemical characterization of a cyclodextrin glycosyltransferase from Geobacillus thermoglucosidans CHB1

“…These results differ from those found for the Thermococcus sp. CL1 CGTase, which is strongly inhibited by NiCl (5 mM) , and Bacillus lehensis CGTase, which is significantly inhibited by MgSO 4 (1 mM) . Ca 2+ at a concentration of 1 mM can slightly enhance the activity of the enzyme.…”

“…Cyclodextrin glycosyltransferase (EC 2.4.1.19; CGTase) is an important member of the α‐amylase family that can catalyze four different reactions, specifically the cyclization, coupling, disproportionation, and hydrolysis of starch . The most common application of CGTase is in the production of three types of cyclodextrin ( i.e ., α‐CD, β‐CD, and γ‐CD) from starch or dextrin . Cyclodextrins consist of six to eight glucose molecules that form an annular structure by linking in a head‐to‐tail manner with no reducing end.…”

“…CGTases can be produced by various microorganisms. Bacteria belonging to the genus Bacillus , such as Bacillus licheniformis , B. circulans , B. macerans , B. agaradhaerens , B. cereus , B. lehensis , and B. sphaericus , are the major strains that produce CGTase . Other bacteria, such as Paenibacillus sp.…”

“…, also produce CGTases. Starch gelatinization is important for cyclodextrin production in industrial process , and because the starch gelatinization step is usually performed at temperatures higher 50°C , thermophilic CGTases are more attractive for cyclodextrin production.…”