Cyclodextrin Glucanotransferase Production by Alkaliphilic Bacteria Isolated from Paddy Fields Dimple S. Pardhi and Kiransinh N. Rajput* Department of Microbiology and Biotechnology, School of Science, Gujarat University, Ahmedabad-380009, Gujarat, INDIA Email: dimplespardhi@gmail.com
Introduction
Materials and Methods
▪ Cyclodextrin glucanotransferase (CGTase; EC 2.4.1.19) is an important extracellular enzyme, belongs to the ι-amylase glycoside hydrolase family which produces a non-reducing cyclic maltooligosaccharide compound cyclodextrin (CD) from utilizing starch through an intramolecular transglycosylation reaction called cyclization. It is also capable of coupling, disproportionation and weak hydrolyzing activity (Jemli et al., 2006). ▪ CDs are structurally classified as ι (cyclohexamilose cG6), β (cycloheptamilose cG7) and γ (cyclooctamilose cG8) based on the units linked by ι-1, 4, glycosidic bonds. The key difference between them is the apolar cavity size and the solubility in water (de Freitas et al., 2004). ▪ Generally CGTases produce a mixture of ι-, β- and γ-CDs in different ratios depending on the reaction conditions and the type of CGTase producing microorganisms. The maximum strains reported for CGTase production are belongs to the genus of alkaliphilic Bacillus sp. mainly for the production of βCD. CDs are widely used in pharmaceuticals, foods, textile and cosmetics and the reason is their capacity to form inclusion complexes with a variety of molecules to modify their physical and chemical properties which improves the action of the molecule (Menocci et al., 2008).
1. Screening of CGTase Producing Microorganisms: • Sample Collection: Total 35 soil samples were collected from rice fields of Gujarat and Dadra and Nagar Haveli. • Primary Screening: Horikoshi media (w/v; 1% soluble starch, 0.5% peptone, 0.5% yeast extract, 0.1% K2HPO4, 0.02% MgSO4.7H2O, 1% Na2CO3 (autoclave separately) and pH 10.0) supplemented with 0.03% (w/v) phenolphthalein and 0.01% (w/v) methyl orange was used for isolation and screening (Higuti et al., 2003). • The formation of yellowish halo zones around the colonies was considered as an initial indication of CGTase activity.
2. Production of CGTase: • Secondary Screening: The CGTase production of the selected isolates was carried out using Horikoshi basal medium. It was inoculated with 1% (v/v) inoculum having 1.0 O.D and incubated 37 ºC, 150 rpm for 48 h as shake flask culture. CGTase production was measured by phenolphthalein assay and absorbance was recorded at 550 nm (Rajput et al., 2016). • Standard calibration curve of β-CD (100 ¾g/ml) was prepared.
Background of the Study
3. Characterization of Selected Isolates:
â–Ş The present study deals with the screening of CGTase producing alkaliphilic bacteria and production of CGTase.
• Their cultural and morphological characteristics were observed using Horikoshi agar plate and Gram staining.
• Total 54 CGTase producing bacterial isolates were obtained from soil samples collected from rice fields of Gujarat and Dadra and Nagar Haveli using Horikoshi medium.
Results
Screening of Isolates Based on Zone of clearance: Spot diameter Isolate No.
Standard Curve of β-CD
Zone Diameter:Spot Diameter (mm)
0.12
0.08 0.06 0.04 0.02
Primary Screening of Isolate P3
0 0
Isolate No.
20
40
60
80
100
120
β-CD Concentration (¾g/ml)
Primary Screening of Isolate P3
Cultural Characters
2.67 2.29 3.01 2.30 2.87 2.74 2.20 2.44 2.0 2.57 2.55 2.18 2.27 2.25 2.13 2.20 2.32 2.03 2.07 2.15 2.09 2.34 2.22
Morphological Characters
P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P13 P16 P18 P19 P23 P25 P26 P28 P31 P34 P35 P37
OD at 550 nm
0.1
Phenolphthalein Assay of β-CD (100 ¾g/ml )
P1
P2
P3
P4
P5
P6
P7
P10
P26
P28
Size
Large
Large
Large
Large
Large
Large
Large
Large
Large
Large
Shape
Round
Round
Round
Round
Round
Round
Round
Round
Round
Round
Undulate
Entire
Entire
Entire
Entire
Entire
Entire
Entire
Entire
Entire
Elevation
Raised
Raised
Raised
Raised
Raised
Raised
Raised
Raised
Raised
Raised
Surface
Wavy
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Consistency
Moist
Moist
Moist
Moist
Moist
Moist
Moist
Moist
Moist
Moist
Opacity
Opaque
Opaque
Opaque
Opaque
Opaque
Opaque
Opaque
Opaque
Opaque
Opaque
Pigmentation
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Motility
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Gram’s nature
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Size
Long
Long
Long
Long
Long
Long
Long
Long
Long
Long
Shape
Rods
Rods
Rods
Rods
Rods
Rods
Rods
Rods
Rods
Rods
Arrangement
Pair
Single
Chain
Chain
Pair
Single
Chain
Pair
Single
Chain
Margin
Production of CGTase Isolate No.
Enzyme Activity (U/mL)
P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P13 P16 P18 P19 P23 P25 P26 P28 P31 P34 P35 P37
0.716 0.608 0.783 0.532 0.755 0.755 0.676 0.315 0.225 0.541 0.479 0.411 0.439 0.428 0.418 0.322 0.422 0.523 0.534 0.453 0.252 0.227 0.343
Conclusion CGTases are one of the most important economically and biotechnologically valuable enzymes. We have isolated total 54 CGTase producing bacterial strains from soil samples collected from rice fields. Amongst these, only 23 isolates were further screened based on their primary screening responses. The cultural and morphological characteristics revealed that all the isolates were Gram positive long rods, much similar to the Bacillus sp. Maximum CGTase activity of 0.783 U/ml was obtained by isolate P3 after 48 h of incubation at 37 ÂşC. These observations will help in the investigation of optimization studies of CGTase production by isolate P3 using one-factor-at-a-time and statistical methods in future.
References • • • • •
de Freitas, T. L., Monti, R. and Contiero, J. (2004). Production of CGTase by a Bacillus alkalophilic CGII strain isolated from wastewater of a manioc flour industry. Brazizilian Journal of Microbiology, 35: 255-260. Higuti, I. H., Grande, S. W., Sacco, R. and Nascimento, A. J. (2003). Isolation of alkalophilic CGTase-producing bacteria and characterization of cyclodextrin-glycosyltransferase. Brazilian Archives of Biology and Technology, Vol. 46, 2: 183-186. Menocci, V., Goulart, A. J., Adalberto, P. R., Tavano, O. L., Marques, D. P., Contiero, J. and Monti, R. (2008). Cyclodextrin glycosyltransferase production by new Bacillus sp. strains isolated from Brazilian soil. Brazilian Journal of Microbiology, 39:682-688. Jemli, S., Messaoud, E., Ayadi-Zouari, D., Naili, B., Khemakhem, B. and Bejar, S. (2006). A β-cyclodextrin glycosyltransferase from a newly isolated Paenibacillus pabuli US132 strain: Purification, properties and potential use in breadmaking. Biochemical Engineering Journal, 4373:1-7. Rajput, K. N., Patel, K. C. and Trivedi, U. B. (2016). �-Cyclodextrin production by cyclodextrin glucanotransferase from an alkaliphile Microbacterium terrae KNR 9 using different starch substrates. Biotechnology Research International, Vol. 2016.