Biology, Medicine, & Natural Product Chemistry  ISSN 2089-6514 (paper) 

Volume 13, Number 1, April 2024 | Pages: 193-197 | DOI: 10.14421/biomedich.2024.131.193-197 ISSN 2540-9328 (online) 
 

 

 

 

Isolation of β-Cyclodextrin Gycosyl Transferase (β-CGTase) 

Producing Bacteria from Potato Plantation Soil 

 
Helman Kurniadi1,*, Desi Sagita2, Barmi Hartesi3 

1,2Pharmacy Department; Adiwangsa Jambi University, Jl. R. Wijaya, The Hok, Jambi Sel., Jambi 36125, Indonesia. 
3Faculty of Pharmacy, Jenderal Achmad Yani University, Jl. Ters Jend Sudirman, Cimahi, Jawa Barat 40633, Indonesia. 

 

 

Corresponding author* 
helmankurniadi@gmail.com 

 

 

 

 

Abstract 

 

Cyclodextrin glycosyltransferase (CGTase) is an extracellular enzyme produced by several microorganisms, particularly bacteria, that 

converts starch into cyclodextrin (CD). CD has various applications in fields such as food, textiles, pharmaceuticals, chemistry, and 

biotechnology. The objective of this study was to isolate β-CGTase-producing bacteria from soil in a potato plantation located in Kerinci, 

and to conduct their characterization. Screening was conducted using Horikoshi agar media with phenolphthalein and methyl orange color 

indicators. The results indicated that one isolate, coded CK-2, produced the optimum β-CGTase enzyme when incubated for 30 hours at 

37 ºC and pH 10. The CGTase enzyme converted starch to CD when incubated for 60 minutes at 37 ºC and pH 7. The CK-2 isolate was 

identified as monobacilli Gram-positive bacteria, having spores, positive in the catalase test, and motile. The study concluded that the 

bacteria producing β-CGTase enzyme are found in the soil of potato plantations in Kerinci, which is believed to originate from the genus 

Bacillus sp. 

 

Keywords: CK-2 isolate; cyclodextrin (CD); cyclodextrin glycosyltransferase (CGTase); soil. 

 

Abbreviations: CD: cyclodextrin; CGTase: cyclodextrin glycosyltransferase. 

 

 

INTRODUCTION 
 

Cyclodextrin glycosyl transferase, which is classified 

with CGTase number E.C 2.4.1.19, is a bacterial enzyme 

belonging to the R-amylase family that has the ability to 

convert starch into cyclodextrin (Jeang et al., 2005). The 

CGTase enzyme can synthesize starch to form three main 

cyclodextrin compounds, namely α-CD, β-CD, and γ-CD 

(Kesaulya, 2015). This enzyme converts starch to 

cyclodextrin (CD) through the transglycosylation and 

starch hydrolysis pathway. The resulting CD is a glucose 

residue compound bound by α-1,4 glycosidic to form 

cyclic oligosaccharides (Kamble & Gupte, 2015). 

Cyclodextrin is a cup-shaped molecule with a cavity 

in the middle that is hydrophobic on the inside and 

hydrophilic on the outside. The specialty of cyclodextrins 

lies in their ring structure, which has the ability to 

enclose guest molecules in the cyclodextrin cavity. This 

can be applied in several ways, including modifying the 

physicochemical properties of molecules such as 

stability, solubility, bioavailability, conjugate 

preparation, and the bonding of several polymers 

(Duchene, 2011; Jansook et al, 2018; Poulson et al. 

2022). 

The formation of cyclodextrins must be in accordance 

with the environmental conditions and reactions needed 

for growth and reproduction so that some CGTase-

producing bacteria can form three cyclodextrin 

molecules, namely α-CD with 6 glucose molecules, β-

CD with 7 glucose molecules, and γ-CD with 8 glucose 

molecules (Kesaulya, 2015). Due to the difference in the 

number of molecules that make up each cyclodextrin, the 

main difference between them is the size of the nonpolar 

cavity and their solubility in water (Koo et al., 2017; 

Braga, 2019). 

Research conducted by Higuti et al. (2003) concluded 

that there are 75 strains of CGTase-producing bacteria 

that exist in various types of soil. Based on the summary 

of Qi & Zimmermann (2005), CGTase-producing 

bacterial isolates are currently known to produce the 

most β-CD. Miftahurrohmah & Moordiani (2014) found 

that the CGTase-producing JS-1 isolate from soil in 

Jatinangor was a Gram-positive bacterial isolate that was 

bacillus-shaped, motile, facultative anaerobic, and had a 

positive catalase test, in line with research by 
Rostinawati & Lestari (2017) showing that CGTase was 

produced by the bacteria Bacillus sp. The aim of this 

research is to obtain isolates of bacteria producing the 

enzyme β-CGTase from potato plantation soil in Kerinci 

Manuscript received: 03 March, 2024. Revision accepted: 20 May, 2024. Published: 04 June, 2024. 

https://doi.org/10.14421/biomedich.2024.131.193-197


 

 
 

194 Biology, Medicine, & Natural Product Chemistry 13 (1), 2024: 193-197 

 

 

and identify isolates producing β-CGTase based on 

biochemical characteristics. 

 

 

MATERIALS AND METHODS 
 

This research was conducted at the Pharmaceutical 

Microbiology Laboratory, Harapan Ibu Jambi College of 

Health Sciences from April 2019 to July 2019. The 

samples examined in this research were soil samples 

taken from the potato plantations, specifically the soil 

closest to the potato tubers. A 1-gram sample was taken 

at one point and diluted into 9 mL of sterile distilled 

water to create a dilution of 10-8. Then, 100 µl of 

suspension from dilutions 10-2 to 10-8 was poured into 

solid Horikoshi medium and incubated for 24 hours at 37 

ºC. 

The presence of a yellow hollow zone around the 

growing colony indicated the ability of the bacteria to 

produce the enzyme β-CGTase. The colonies were then 

identified using macroscopic, microscopic, and 

biochemical tests, and inoculated into liquid Horikoshi 

media to measure CGTase activity. CGTase activity was 

measured using spectrophotometry, which involved 

observing the absorbance of the color indicator bound by 

cyclodextrin to create a curve of cyclodextrin 

concentration against the incubation time of the CGTase 

enzyme.  

Absorbance was measured by inserting a color 

indicator compound into the cyclodextrin to form an 

inclusion. The measurement is carried out on the color 

indicator captured by the cyclodextrin, which was 

produced from the CGTase enzyme. The more 

cyclodextrin present, the more compound was captured, 

resulting in a smaller absorbance value of the color 

indicator. The percent decrease in absorbance of the 

color indicator was then correlated to the CGTase 

incubation time using the following formula: 

 

Percent reduction in phenolphthalein (%) 

 

U/mL=  
R0−R

R
× 100% 

 

R0 : Control absorbance (Time 0)  

R  : Sample absorbance 

 

 

RESULTS AND DISCUSSION 
 

Result 

Screening of Isolates Producing the β-CGTase Enzyme  

The isolate successfully obtained from potato plantation 

soil, which exhibited β-CGTase activity, was derived 

from a soil dilution of 10-2. A yellow hollow zone was 

observed on the β-Horikoshi media, and this isolate was 

designated as CK-2. The CGTase activity secreted by the 

CK-2 isolate in the β-Horikoshi medium is illustrated in 

Figure 1. 

 

 

 

   
 

Figure 1. Horikoshi agar as control (A), Horikoshi agar after CK-2 inoculation (B) 

 

 

Isolate Identification 

Macroscopically, the colonies of the CGTase-producing 

isolate that grew on β-Horikoshi agar had a round shape 

with a whitish-yellow color, flat edges, and a flat 

elevation. Microscopically, identification and 

characterization were carried out through several tests:  

 

Biochemical Test  

Biochemical tests were conducted using IMVIC media, 

including SIM media, Veges paskeuer, methyl red, 
Simon citrate & Triple Sugar Iron Agar, and sugar 

media. The results of the isolate identification can be 

seen in Table 1. 

 

Table 1. Results of identification of CK-2 isolates. 
 

Identification Results 

Sulfur Indol Motility (-),(-),(+) 

Methyl Red (+) 

Veges Paskeuer (-) 

Triple Sugar Iron Agar K/K 

Simon Citrate (-) 

Glukosa (-) 

Dextrosa (-) 

Mannitol (-) 

Sucrosa (-) 

Lactosa (-) 

Gram (+) 

Spora (+) 

Katalase (+) 



 

 
 

 Kurniadi et al. – Isolation of β-Cyclodextrin Gycosyl Transferase (β-CGTase)  195 
 
 

Gram Staining and Endospores 

In Gram staining, rod-shaped bacteria are purple in color, 

indicating that they were Gram-positive. The bacteria 

had subterminal spores because they had a green dot on 

1/4 of the cell. The bacteria also produce the enzyme 

catalase. 

 

 
 
Figure 2. Result of Gram staining under light microscope with 

magnification 1000x. 

 

Characterization of the incubation time of enzymes 

on CGTase activity 

The characterization of enzyme incubation time was 

conducted to determine the optimal time for the enzyme 

to catalyze starch and form CD. According to the results 

of the characterization, as shown in Figure 3 (A), the 60-

minute incubation time exhibited the lowest 

phenolphthalein absorbance, indicating that this is the 

optimal time for CGTase activity in producing CD. 

 

Preparation of a Growth Curve for Isolate CK-2 

In the growth curve, the results of the OD600 

measurement shown in Figure 3 (B) indicate that the 

isolate experienced a log phase starting from the 12th 

hour, followed by a stationary phase at the 24th hour. 

Subsequently, the death phase was observed from the 

30th to the 72nd hour of incubation. 

 

Characterization of Isolates in β-CD 

Production The best time for an isolate to produce the β-

CGTase enzyme was seen based on incubation of the 

CK-2 isolate at a temperature of 37 ºC. Isolate CK-2 

secreted the CGTase enzyme maximally after 30 hours of 

incubation. In Figure 3 (C), it can be seen that the 

phenolphthalein absorbance was the smallest at 30 hours 

of incubation, followed by the 36th hour of incubation. 

Figure 3 (D) illustrates that isolate CK-2 secreted the 

maximum amount of CGTase at the 30-hour mark, as 

indicated by a percent decrease in phenolphthalein 

absorbance reaching 98.39%. 

 

         

        
Figure 3. (A) Decreased phenolphthalein absorbance corresponds to CD formation. (B) Growth curve of isolate CK-2. (C) Phenolphthalein absorbance 

over incubation time. (D) The percentage decrease in phenolphthalein absorbance is directly proportional to β-CD formation. 
 

  

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196 Biology, Medicine, & Natural Product Chemistry 13 (1), 2024: 193-197 

 

 

Discussion 

Screening and identification of isolates 

An isolate of bacteria producing the CGTase enzyme has 

been successfully isolated from potato plantation soil in 

the Kerinci area. This isolate exhibits a yellow hollow 

zone on Horikoshi media. The formation of this yellow 

hollow zone is attributed to the isolate's capability to 

produce the enzyme β-CGTase, which converts starch 

into the compound β-CD. Consequently, this compound 

binds to phenolphthalein, causing the disappearance of 

the reddish-purple color of phenolphthalein around the 

colony. 

Based on the results of biochemical identification of 

bacteria, it is suspected that the CK-2 isolate comes from 

the genus Bacillus (Kiranmayee Rao & M. Lakshmi, 

2007). This identification has several advantages, namely 

that it is easy to carry out and costs less. However, it has 

the disadvantage of a lower level of accuracy due to 

several types of species that exhibit nearly identical 

biochemical metabolic results. Therefore, further 

molecular identification of the CK-2 isolate is necessary. 

This aligns with several local studies that have identified 

Bacillus as CGTase-producing bacteria, including 

Bacillus sp BK-1 (Yunianto et al., 2000), Bacillus sp 

BGG-1 (Perbadi, 2001), Bacillus sp LT1B and PT2B 

(Sagita, 2011), as well as Bacillus sp BV-3 (Vionita, 

2017). 

 

Characterization of enzyme incubation time on CGTase 
activity 

This characterization was carried out in order to obtain 

the optimum incubation time for the enzyme that reacts 

with starch to produce β-CD. Figure 3 (A) shows that the 

best time for the enzyme to react with starch to produce 

CD is at 60 minutes of incubation. In the test carried out 

by Rostinawati et al (2016), the incubation time for 

CGTase with starch was 30 minutes but with the starch 

chain breaking first by gelatinization. This research also 

did not carry out enzyme purification so that a mixture of 

substances originating from the media still remained. It is 

possible that the incubation time was longer in this study 

due to the need for additional time for the reaction to 

break the starch structure and the disruption of the bond 

between the enzyme and starch by residual substances 

from the media. 

 

▪ Making an Isolate Growth Curve 
In the growth curve of the CK-2 isolate, it is known that 

from the initial time of incubation to the 12th incubation 

hour there is a lag phase, namely the time when the 

isolate adapts and is then able to reproduce. This is 

different from research conducted by Kamble & Gupte 

(2014) which is known to be the lag phase of Bacillus. 

producing CGTase at 0 hours to 8 hours of incubation, 

this difference is probably caused because the CK-2 

isolate takes longer to adapt to the pH 10 liquid 

Horikoshi medium, while the isolate used by Kamble & 

Gupte (2014) is an isolate that has been proper pH 

optimization for growth. 

Next, the CK-2 isolate experienced a log phase 

starting from the 12th hour of incubation to the 24th hour 

of incubation, indicating that the CK-2 isolate 

reproduced maximally during that incubation hour. In 

line with the difference in the lag phase with the research 

of Kamble & Gupte (2014), there was also a difference in 

the log phase. In this study, the log phase was obtained 

for 22 hours of incubation, namely from the 8th hour to 

the 30th hour of incubation. This can be caused by 

adjustments to the nutrient concentration that they make, 

such as optimizing the concentration of peptone, yeast 

extract and starch so that the possibility of bacterial 

growth can last longer. Meanwhile, the stationary phase 

in this study started at the 24th incubation hour until 

before the 30th incubation hour, this shows a state of 

balance between the growth rate and death rate of 

bacteria at that time and is in line with the reduction in 

nutrients in the media, so the number of deaths continues 

to increase and the growth rate continues to decrease, this 

occurs at the 30th to 72nd incubation hours. This phase is 

also different from the research of Kamble & Gupte 

(2014) which concluded that the stationary phase from 

their growth curve measurements started from the 30th 

incubation hour to the 176th incubation hour, while the 

death phase is known to occur later, this difference is 

probably caused by the addition of nutrients. In their 

media, the starch used was tapioca starch, the addition of 

0.2% Ammonium dihydrogen Phosphate and a difference 

in Magnesium Sulfate concentration of 0.12%. 

 

▪ Characterization of Isolates in β-CD Production 

The characterization of isolates in the production of β-

CD, as depicted in Figure 3 (C), reveals that the 30th 

hour of incubation exhibits the lowest phenolphthalein 

absorbance value. This observation suggests that this 

specific incubation time is optimal for bacteria to 

produce β-CD, resulting in a significant 98.39% increase 

in β-CD formation, illustrated in Figure 3 (D). 

Furthermore, the 30th hour of incubation marks the 

conclusion of the stationary phase, indicating a 

correlation between β-CGTase production and this phase. 

Specifically, the formation of β-CGTase escalates 

concomitantly with the conclusion of the stationary 

phase. 

 

 

CONCLUSIONS 
 

Bacteria producing β-CGTase, named isolate CK-2, have 

been found. Based on biochemical identification, the CK-

2 isolate is a gram positive bacterium in the form of a 

bacillus, facultative anaerobe, motile and positive in the 

catalase test. This isolate is thought to be a bacterium 

from the genus Bacillus. The CGTase enzyme secreted 

by the CK-2 isolate has optimum cyclization activity in 



 

 
 

 Kurniadi et al. – Isolation of β-Cyclodextrin Gycosyl Transferase (β-CGTase)  197 
 
 

incubation time 60 minutes at 37 ºC. This bacterium is 

one of the CGTase-producing bacteria which can 

produce the maximum CGTase enzyme at the 30th hour 

of incubation at a temperature of 37 ºC. 

 

Authors’ Contributions: Helman Kurniadi, Desi Sagita, 

and Barmi Hartesi designed the research, carried out 

laboratory work, and analyzed the data. Soil sampling 

was carried out by Helman Kurniadi. 

 

Competing Interests: Regarding competing interests, it is 

important to note that there was no conflict of interest 

observed in this study. 

 

Funding: It is worth mentioning that no party provided 

cost support for this research. Instead, the study was 

carried out at the researchers' own expense, 

demonstrating their dedication and commitment to 

advancing scientific knowledge in their field. 

 

 

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