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Malays. Appl. Biol.



of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia


of Science and Technology, School of Chemical Sciences and Food Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia


of Biotechnology Engineering, Faculty of Engineering, International Islamic University of Malaysia, Jalan Gombak, 53100 Kuala Lumpur, Malaysia and Chemical Technology Centre, SIRIM Berhad, P.O. Box 7035, Section 2, 40911 Shah Alam, Selangor, Malaysia of Chemical Engineering and Natural Resources Engineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia



Cyclodextrin glycosyltransferases (CGTases) are extracellular bacterial enzymes that generate cyclodextrin from starch. Screening, isolation and characterisation of CGTases are extensively carried out due to its importance in industrial biotechnology. Conventionally, identification of CGTase-producing bacteria involves the use of solid media containing phenolphthalein-methyl orange as indicators by colour changes. The formation of CGTase does not require a specific inducer as the presence of starch is essentially sufficient. In the present study, modification of the conventional method was developed by substituting soluble starch with five other types of starches, namely, corn, rice, tapioca, glutinous starch and sago. The improved method enables simultaneous isolation and screening for CGTase-producing bacteria and identification of the most suitable carbon source (starch) for enzyme production. The diameter of the clearing zone formation around the bacterial colony in the starch-containing medium is used to gauger the hydrolytic efficiency of the bacteria. Out of the 250 soil bacterial samples screened, strain MK 6 was identified as the most prolific CGTase producer. This isolated strain was capable of hydrolysing sago starch best (and least in glutinous starch). The modified method using simple substrate substitution and the phenolphthalein colour reaction studied here was thus found to be useful for the rapid and qualitative method for preliminary screening of CGTase-producing bacteria.

Siklodekstrin glikosiltransferase (CGTases) adalah enzim bakteria ekstraselular yang bertanggungjawab dalam penghasilan siklodekstrin daripada kanji. Penyaringan, pemencilan dan pencirian yang ekstensif dijalankan ke atas CGTase berdasarkan kepentingannya dalam bidang bioteknologi industri. Kebiasaanya, penentuan bakteria penghasil CGTase melibatkan penggunaan media pepejal yang mengandungi phenolphthalein-methyl orange sebagai penanda bagi perubahan warna yang berlaku. Pembentukan CGTase tidak memerlukan pengalak spesifik kerana kehadiran kanji di dalam sistem adalah mencukupi. Di dalam kajian ini, pengubahsuaian ke atas kaedah sedia ada telah dilakukan melalui penggantian kanji terlarut dengan lima jenis kanji terpilih seperti kanji jagung, beras, keledek, pulut dan sagu. Pengubahsuaian kaedah ini membolehkan penyaringan dan pemencilan bakteria penghasil CGTase dapat dilakukan dengan mudah dan penentuan sumber karbon yang paling sesuai dapat dikenalpasti bagi penghasilan

* To whom correspondence should be addressed.


enzim. Pembentukan kawasan zon cerah di keliling koloni bakteria pada media yang mengandungi kanji dapat digunakan sebagai pengukur keberkesanan hidrolisis oleh bakteria. Daripada 250 sampel bakteria yang disaring, strain MK 6 telah dikenalpasti dan dipilih sebagai penghasil CGTase yang berpotensi. Strain yang dipencilkan ini berupaya menghidrolisis kanji sagu dengan baik dan diakhiri dengan kanji pulut. Kaedah terubahsuai ini menggunakan substrat ringkas dan tindakbalas perubahan warna fenolftalein didapati berguna untuk kaedah yang cepat dan kualitatif dalam proses penyaringan awal bakteria penghasil CGTase.

Key words: Rapid screening method, CGTase, Bacteria, Carbon, Starch

INTRODUCTION Cyclodextrin glycosyltransferase (EC (CGTase) or [1, 4 - -D-glucopyranosyl]transferase is an extracellular enzyme, which degrades starches into cyclodextrin (CDs) molecules via cyclisation reaction. Cyclisation happens when a linear oligosaccharide (starch) chain is cleaved and the new reducing end sugar is transferred to the non-reducing end sugar of the same chain. Therefore cyclodextrins are cyclic oligosaccharides consisting of 6-12 units of glucose joined by the -1, 4-linkages. CGTases also catalyses two intermolecular transglycosylation reactions: coupling, in which a cyclodextrin ring is cleaved and transferred to an acceptor maltooligosaccharide substrate and disproportionation, in which a linear maltooligosaccharide is cleaved and the new reducing end sugar is transferred to an acceptor maltooligosaccharide substrate. Besides these reactions, the enzyme has a weak hydrolysing activity (Penninga et al., 1995; Bart et al., 2000). Cyclodextrins with 6, 7 and 8 glucose units are most common and also known as -, - and cyclodextrin, respectively. Bacillus species constitute the major contributor of industrially important enzymes (Starnes, 1990). The wide range of enzyme application, e.g. in the detergent, pulp and paper industry prompted the isolation of strains from a variety of alkaline environments as a source of enzymes with suitable activities. Major producers of CGTase also belong to the Bacillus sp. In fact, the industrial production of CGTase was made attractive only when alkalophilic Bacillus was introduced as a production organism (Priest, 1977). However, production by other species such as Klebsiella, Micrococcus, Brevibacterium, Thermoactinomyces, Aspergillus and thermophillic Archaea has also been reported (Rita and Rajni, 2002). Various types of starch substrates may be used for CGTase production; including potato, corn and rice starch. The present study aims to investigate the potential of sago starch as an

alternative substrate for CGTase production. The low cost and the high yield associated with sago starch production makes it additionally attractive as an alternative substrate. In South East Asia alone, it is estimated that 60 Mtons of starch is produced from sago palms annually (Wang et al., 1996). Many methods are available for the screening of CGTase producing microorganisms. These methods may either employ detection on solid culture media or/and on liquid culture media. In both situations, the basis for detection is by the reduction of the colour intensity of phenolphthalein under alkaline conditions. In 1989, Park et al. developed a rapid screening method by incorporating phenolphthalein and methyl orange to Horikoshis medium II. Clear yellowish hollow zones will be observed on bacterial strains having CGTase activity. In modified Horikoshis medium II, soluble starch was used as the main carbon source. However, the preferences of different types of Bacillus sp towards different types of starch have to be taken into consideration for enhancing the CGTase production. Rapid screening methodology enables simultaneous identification of CGTase producing microorganism and the most suitable carbon source for its growth. This screening procedure helps in identifying the best carbon source for CGTase production In the present investigation, a qualitative rapid screening procedure was developed by modifying a screening method proposed by Park et al. (1989).

MATERIALS AND METHODS Chemicals All chemicals were of analytical grade, unless stated otherwise. Soluble starch and phenolphthalein was purchased from Merck. Rice starch, corn starch, tapioca starch and sago starch were of industrial grade. Yeast extract and peptone were purchased from BDH.


Culture Conditions Isolated strains were grown in 20 ml Horikoshis medium II (Park et al., 1989) contained the following solutions after autoclaving. Solution 1: Starch 1.0% (w/v), peptone 0.5% (w/v), yeast extracts 0.5 % (w/v), K 2 HPO 4 0.1% (w/v), MgSO 4 0.02% (w/v). Solution 2: Na2CO3 1.0% (w/v). The culture was incubated at 37 oC, agitated at 200 rpm for 18 hours. Cells were harvested by centrifugation at 5000 rpm for 5 minutes and washed once with normal saline solution (0.85% w/v NaCl) and were then suspended in normal saline solution to give an optical density reading of 0.5 at 660 nm, using a UV-spectrophotometer. Screening and Isolation of the Bacterial Strains from Soil Soil samples were collected from locations including sago plantation(s) in Johore, a several herbs plantation (s) in Sarawak and a market place at Selangor. Criteria for soil selection were mainly based on soil pH (pH 5-pH 6). Collected soil samples were suspended in sterile saline and the solid particles were allowed to settle. Plating was done at high pH (up to pH 10) to discourage growth of fungal species. Screening for cyclodextrin producers was done according to Park et al. (1989). In the primary selection, soil samples were diluted and streaked onto the isolation media. The plates were incubated at 37 oC for 24-48 hours. Bacterial colonies with yellowish clearance zones were selected and streaked onto Horikoshi-Phenolphthalein (PHP) plate several times until pure colonies were obtained. Suspensions of vegetative cells were grown in Horikoshi medium II for 24 hours at 37 oC, mixed with sterilised glycerol (20%) and kept in 1 ml aliquots at -80oC until further used. Modified Horikoshi-Phenolphthalein (PHP) Method The best CGTase producer from the above screening method was further used to study the most suitable carbon source. Soluble starch in the method proposed by Park et al. (1989) will be substituted with local starches from sago starch, tapioca, corn and rice. Culture broth of the isolated bacteria strain will be incubated in Horikoshi medium II, without agar at 37C for 24 hours. The bacterial stock strain was examined on the agar medium containing the phenolphthalein methyl orange and different type of starches. For colonies grown on plates, a 24 hours incubation at 37C was sufficient for the appearance of colonies with halo zones. Bacterial colonies with the widest yellowish clear zones were selected for further optimisation processes.

CGTase Assay Method CGTase activity was determined by using phenolphthalein assay (Kaneko et al., 1987). Reaction mixture containing 1 ml of 40 mg of soluble starch in 100 mM phosphate buffer (pH 6.0) and 0.1 ml enzyme solution. The mixture was incubated at 60C for 10 minutes in a water bath. Subsequently, 0.5 ml of 0.02% (w/v) phenolphthalein in 5 mM Na2CO3 solution was then added to the reaction mixture and mix well. After 15 minutes, the reduction in colour intensity was measured at 550 nm. Blanks lacking the CGTase were analysed simultaneously with each batch of samples. As a standard, the soluble starch and enzyme were replaced by 0.5 mg of CD and 0.1 ml of water, respectively. A calibration curve was made using -CD in 100 mM phosphate buffer at pH 6.0. One unit of enzyme activity was defined as the amount of enzyme that formed 1mol -CD per min under the conditions defined above.

RESULTS AND DISCUSSION Isolation and Characterisation of Microorganism Soil samples were screened for -cyclodextrin producer. A plate assay was used for the detection of CGTase activity consisting of soluble starch as substrate and phenolphthalein as the indicator. Secretion of the CGTase enzyme was detected by formation of clearance zone around the colonies. A total of 250 isolates have been successfully isolated in this study. The diameter of the isolates ranged from 0.5 to 3.5 cm following a 24 hour incubation at 37C. The highest producer of CGTase enzyme is the isolate MK 6 with clearance zone diameter of 5.3 cm. The isolated strain MK 6 was further characterised using the modified phenolphthalein method. Park et al. (1989) reported that phenolphthalein was transformed into a colourless dianion within the cavity of -cyclodextrin. Therefore CGTase activity was determined on the basis of the reduction of the colour intensity of phenolphthalein under alkaline conditions. However in the conventional method, detection may only be done using soluble starch as the substrate. In the present study, the simultaneous determination of CGTase producing strains and the best carbon source using different starches was successfully achieved. The preference ability of the strain to excrete CGTase enzyme in different carbon source can be evaluated using this method. The diameter of the clearance zone, signifying the amounts of CGTase excreted varies depending on the starches used. The choice of starch used in this experiment


pertaining to its economic feasibility. Culture broth of strain, MK 6 after 24 hours of incubation on modified PHP method was found to exhibit CGTase activity by the formation of clearing zone. Strain MK 6 showed observable usage of starches of industrial grade through formation of halo zone; in decreasing order as shown below: Sago Starch (5.3 cm) > Tapioca Starch (4.9 cm) > Corn starch (4.2 cm) > Rice starch (3.7 cm) > Green peas starch (2.5 cm) > Glutinous starch (1.5 cm) The modified PHP method is only a qualitative indicative measure of excretion of CGTase enzyme. Therefore further quantitative measurement method, such as the study for reduction of phenolphthalein using spectrophotometric analysis was studied. Different Type of Starches on the Effect of CGTase Production The selection of the types of starch used in this experiment was based on the result obtained from the modified PHP method. Since the formation of CGTase did not require the presence of specific inducer, therefore production of the enzyme depends mostly on the substrate used. (Kabaivanova et al., 1999). It was observed that the bacteria can grow well on most of the starches used. Sago starch, tapioca starch and corn starch are the most suitable sources for enzyme synthesis. Nogrady et al. (1995) found that CGTase was the only starch degrading enzyme in Bacillus macerans which hydrolysed the -1, 4-glucosidic linkages of starch and therefore they suggested that CGTase was thought to play a primary role in the degradation of both amylase and amylopectin. Simple sugars however such as glucose, maltose and even hydrolysed sago starch did not produce significant result as summarised in Table 1. The results obtained were contradicted as reported for B. stearothermophillus, where glucose was found to be the most suitable substrate (Stefanova et al.,

1999). However in earlier report by Jin-Bong et al. (1990), soluble starch was more suitable for B. stearothermophillus. Xylose and glucose were best for B. cereus (Jamuna et al., 1993), in different cases production was enhance with addition of starch (Thatai et al., 1999). Sreenivasan et al. (1991) reported maximum CGTase activity when tapioca was used as carbon source for B. macerans. The differences in enzyme activity obtained in media with various starches may be related to their physical structure, a trait that seems to be discriminated by the Bacillus species (Adriana et al., 2002). The preference for starch rather than simple sugar can be explained from genetic approach. The amino-acid sequences of CGTases from bacilli show 60-80% identity. Although the similarity between the sequences of CGTases and -amylase is limited (< 30%), four highly conserved amino-acid regions have been discovered (Pekka et al., 1995). From the cgt gene of Bacillus circulans strain 251 which had been cloned and sequenced, the crystal structure of the CGTase protein has been determined at 2.0 resolutions. The protein consists of a single polypeptide chain of 686 amino acid residues; as in other known CGTase structures, five domains (A-E) can be recognized. The three N-terminal domains (A-C) have structural similarity with the three -amylase domains. Domain E contains a raw starch binding motif. This domain was shown to be essential for degradation of raw starch (Penninga et al., 1996). Therefore starches exhibit the role of inducer in production of CGTase enzyme.

CONCLUSION Strain MK 6 had been successfully isolated from local soil using a phenolphthalein solid medium. The modified phenolphthalein assay has high reproducibility and significant operational advantages. The effect of different carbon sources on CGTase production showed that enzyme

Table 1. Comparisons of yield for CGTase production using different type of starches Starch Sago Soluble starch (Potato) Tapioca Maltose Glucose Hydrolysed sago starch Relative Activity (%) 12.30 01.16 11.67 04.15 03.49 08.80 CGTase Activity (U/ml) 0.458 0.033 0.422 0.127 0.150 0.100 Increment from soluble starch 42.50 38.90 09.40 11.70 06.67


production was highest when sago starch was used as carbon source. Use of hydrolysed starch and simple sugars gave low yield of CGTase activity.

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