STUDIES ON β-CYCLODEXTRIN PRODUCTION FROM CGTase PRODUCING BACTERIA AND ITS EFFECT ON DRUG SOLUBILITY Original Article SUKHMINDERJIT KAUR*, SIMRANJEET KAUR, KIRANDEEP KAUR Department of Biotechnology Shaheed Udham Singh College of Research and Technology, Tangori, Mohali. Email: sukhminderphd_6@yahoo.co.in Received: 03 Aug 2014 Revised and Accepted: 05 Sep 2014 ABSTRACT Objective: To produce cyclodextrin (CD) from Cyclodextrin glycosyltransferase (CGTase) producing bacteria isolated from various samples and to study its effect on drug solubility. Methods: CGTase producing bacteria were isolated from various samples viz. Soil, rotten potatoes, and stale corn flour dough using Horikoshi - phenolphthalein agar. All the isolates were screened for CGTase activity. Isolate showing highest CGTase activity was characterized morphologically and biochemically. CD was produced using CGTase which was further used for drug solubility studies. Results: A total of fifty samples were studied of which 20 bacterial isolates showed the presence of halo around them. Upon further screening, the culture supernatant of 12 isolates showed cyclization activity, of which CD 18 strain produced highest amount of CGTase. Morphological and biochemical characterization revealed that the isolate belonged to Bacillus sp. The isolate showed maximum growth, reducing sugars 0.724 mg/ml and maximum enzymatic activity for dextrinisation as well as cyclisation viz. 9.45 U/ml and 7.41 U/ml respectively after 48 hrs of incubation. Thus, Bacillus sp. CD 18 was used to produce CGTase, which was further used for the production of CD. CD increased the solubility of acclofenac and paracetamol. Conclusion: Bacteria are regarded as important sources of CGTases. New strains might suit better for industrial production of CGTase after optimizing the various cultures and process parameters. Keywords: CGTase, Cyclodextrins, Bacillus sp. INTRODUCTION Cyclodextrin glycosyltransferase (CGTase), EC 2.4.1.19 is an important enzyme that can convert starch to cyclodextrins (CDs) as product [1]. CDs are oligosaccharides consisting of 6 to 12 glucose units, the three most common forms of CDs are α-CD, β-CD and γ-CD consisting of six, seven and eight glucose molecules respectively joined by α- 1, 4 glucoside linkages. They are also known as cycloamyloses, cyclomaltoses and Schardinger dextrins respectively having molecular mass of 70 to 75 kDa and shaped into a conical doughnut shape [2]. Of the three kinds of CDs, β-CD is of the most practical use because its inclusion complexes are easily prepared and stable, Moreover β-CD is easily separated from reaction mixtures because of its low solubility in water. CDs produced by the action of CGTase on starch and related compounds are cyclic, non hygroscopic and crystalline substances. They are conical doughnut shaped molecules with their OH groups facing outside making them water soluble while the interior cavity is relatively apolar and hydrophobic so they can easily form an inclusion complex with many organic hydrophobic substances [3]. Therefore, CDs are becoming increasingly popular in various industries like food industry, cosmetic industry, pharmaceutical field as stabilizers for flavoring agents, to reduce unpleasant odor, taste as stabilizers of chemically labile compounds, to obtain prolonged action, to decrease local irritation and to reduce unpleasant odors [4, 5]. In pharmaceuticals CDs have mainly been used as complexing agents to increase the aqueous solubility of poorly water-soluble drugs and to increase their bioavailability and stability. In addition, CDs have been used to reduce or prevent gastrointestinal or ocular irritation, reduce or eliminate unpleasant smells or tastes, prevent drug-drug or drug-additive interactions, or even to convert oils and liquid drugs into microcrystalline or amorphous powders [6]. Due to the above mentioned novel properties the industrial demand for β-CD has increased many folds within a few past years. The isolation and screening of microorganisms from natural sources have always been the most powerful means for obtaining useful and genetically stable strain for industrially important products. In view of the above lacunae, the present study was planned to produce β- CD from CGTase producing bacteria isolated from various samples and to study its effect on drug solubility. MATERIALS AND METHODS Isolation of CGTase producing microorganisms Fifty samples from different sources such as soil, rotten potatoes and stale corn flour dough were used for the isolation of CGTase producing bacteria. The samples were collected in sterile bags and immediately transferred to biotechnology laboratory of Shaheed Udham Singh College of Research and Technology, Tangori, Mohali (Punjab) to isolate CGTase producing microorganisms for further processing. Each sample was suspended in normal saline, serially diluted and inoculated on Horikoshi - phenolphthalein agar containing (w/v) 1.0% soluble starch, 0.5% yeast extract, 0.5% peptone, 0.1% KH2 PO4 , 0.02% MgSO4 ×7H2 O, 0.02% phenolphthalein, 1.0% Na 2 CO3 and 1.5% agar [7]. The plates were incubated at 37 ° Screening of isolates for CGTase activity C and were observed daily for 1 week. Bacterial colonies with yellowish clearance zone around them were selected as β-CGTase producing isolate and were sub-cultured on Horikoshi - phenolphthalein agar plates for purification. The presence of halos as a result of bacterial growth on the plates containing phenolphthalein suggests the presence of β-CGTase producing bacteria [8]. The purified isolates were stored on nutrient agar slants at 4° C and were screened for CGTase activity. Flasks containing 50 ml of sterile Horikoshi liquid production media containing 1.5% soluble starch, 0.4% yeast extract, 0.15g of KH2 PO4 , MgSO4 .7H2 O, FeSO4 .7H2 O, 0.12g of 20% KCl, and 0.9% NaCl [7] were inoculated with each isolate. Each flask was then incubated at 37°C in a rotatory shaker at 120 rpm. Samples from each flask were withdrawn at specific intervals of 24 hrs, 48 hrs and 72 hrs to analyze bacterial growth, amount of reducing sugars produced, dextrinising activity and cyclisation activity. International Journal of Pharmacy and Pharmaceutical Sciences ISSN- 0975-1491 Vol 6, Issue 10, 2014 Innovare Academic Sciences