Cyclodextrin Glucanotransferase From Bacillus Sp. Sk13.002 And Its Application On Rutin Transglycosylation

Using a rapid screening method combined with enzymatic transglycosylation reaction of rutin, a bacterial strain SK13.002 producing cyclodextrin glucanotransferase (CGTase) with high efficiency in rutin transglycosylation was isolated from soil sample. Identification of this strain including 16S rDNA sequence, morphological, biochemical and physiological characteristic analyses has also been carried out.16S rDNA sequence analysis indicated that 16S rDNA gene composed of 1476 base pairs. 16S rDNA sequence blasting against RDPII database showed that the SK13.002 strain 16S rDNA shared 98.5% identity with 16S rDNA of Bacillus amyloliquefaciens NBRC 15535, and 97.5% identity with that of Bacillus vallismortis DSM11031. Phylogenetic trees were generated by MEGA version 4.0 using 16S rDNA sequences of strain SK13.002 and 10 related Bacillus species. The gene sequences obtained in this study were deposited to the NCBI GenBank database under accession number GU570959.Morphological, biochemical and physiological characteristic analyses showed that this strain was a Gram-positive rod-shaped, sporulating bacterium with ellipsoidal spores. It was positive for starch hydrolysis and could reduce nitrate to nitrite. Combined with 16S rDNA sequence analysis, this strain was identified as Bacillus sp. SK13.002.The optimization of the composition of fermentation media and fermentation conditions for CGTase production has been investigated. The results indicated that the highest CGTase production could be obtained in medium containing (g/L): soluble starch 17.91, soy peptone 10.08, yeast extract 5.45, K2HPO4·12H2O 1.0, MgSO4·7H2O 0.2 and Na2CO3 8.0. And the optimal fermentation conditions are as follows: inoculation volume (3%), temperature (37°C), rotation speed of shaker (200 rpm) and cultivation time (96 h).Two CGTase isozymes produced by Bacillus sp. SK13.002 were purified to homogeneity using DEAE Sepharose Fast Flow anion exchange chromatography and Superdex 75 gel filtration chromatography to show molecular weights of 67.5 and 46.8 kDa on SDS-PAGE respectively, which were confirmed by LC-MS analysis. Purification protocol resulted in increased CGTase specific activity from 0.49 U/mg for the crude enzyme to 9.47 U/mg and 8.83 U/mg for the individual isozymes, respectively, with a purification fold of 37.3 and 19.1% yield recovery.The optimal temperature of starch hydrolysis for CGTase-1 and CGTase-2 are 65 and 60 oC respectively; CGTase-1 and CGTase-2 both have good thermal stability: under 60°C condition for 2 h, 80% enzyme activity are still remained; under 65°C condition for 2 h, 42 and 58% activities of CGTase-1 and CGTase-2 can still be remained. The optimal pH of starch hydrolysis for CGTase-1 and CGTase-2 are 8.0 and 6.5 respectively. Actually, within pH from 6.0 to 10.0, the activity can reach as high as 93%. CGTase-1 and CGTase-2 both have excellent pH stability: under pH 3.0 conditions for 2 h, the remained enzymatic activities are 96.3 and 85.2% respectively; while under pH 11.0 conditions for 2 h, the remained activities are still 95.4 and 93.1% respectively. Under pH from 4.0 to 10.0 conditions, remained enzymatic activities of CGTase-1 and CGTase-2 are over 96%. Only Ca ²⁺ stimulated slightly the enzymatic activity of CGTase-2, whereas Fe³⁺,Cu²⁺ and Zn²⁺ can result in inhibition of enzymatic activities for both isozymes among whichCu²⁺ and Zn²⁺ have the strongest inhibition. Mg²⁺,Fe²⁺,Mn²⁺,Na+ and K+ have almost no negative effect on enzymatic activities for both CGTase-1 and CGTase-2; Co²⁺ and Ni+ have no inhibition of enzymatic activities for CGTase-2, whereas they have to some extent inhibition for CGTase-1. In summery, the results of characteristics of CGTase indicated that compared to other CGTases, CGTase produced by Bacillus sp. SK13.002 has its unique characteristics in terms of enzymatic conditions and catalyzing capacity.Rutin is a bioflavonoid and has been used as a nutritive element, pigment or an antioxidant in food, cosmetic and pharmaceutical industry. However, its application has been limited by its insolubility and instability in water. To improve its physicochemical properties, transglycosylation of rutin using Bacillus sp. SK13.002 CGTase has been investigated. The optimization of enzymatic reaction conditions of the transglycosylation of rutin has been investigated. The results indicated that under the optimized enzymatic reaction condition (pH 5.5, temperature 35°C, the amount of enzyme 20 U/mL reaction system, reaction duration 24 h), the conversation rate of rutin can reach as high as 65.7%. Furthermore, the process of enzymatic reaction has also been studied. The results of rutin transglycosylation also revealed that Bacillus sp. SK13.002 CGTase possesses its unique characteristics and catalyzing capacity.Microwave irradiation has been utilized in rutin transglycosylation. And the results showed that microwave irradiation with specific power could increase dramatically the speed of rutin transglycosylation. As a result, the duration of rutin transglycosylation had been curtailed dramatically. Under the optimized Microwave-assisted reaction conditions of 40°C and 60 W of power (2, 450 KHz), the rutin transglycosylation could finish within only several minutes. Compare to conventional rutin transglycosylation, the speed of MAR of rutin transglycosylation increased dozens of times while the similar conversion rate of rutin could be obtained. Furthermore, the process of MAR rutin transglycosylation has been investigated and the difference between MAR and conventional rutin transglycosylation and the possible mechanism have also been investigated.