Short communication Improvement in inulinase production by simultaneous action of physical and chemical mutagenesis in Penicillium purpurogenum Arun Dev Sharma, Prabhjot Kaur Gill*, Sukhdev Singh Bhullar and Prabhjeet Singh Department of Biotechnology, Guru Nanak Dev University, Amritsar 143005, Punjab, India *Author for correspondence: E-mail: goldi77700@yahoo.com Received 25 October 2004; accepted 24 November 2004 Keywords: Fructanase, inulinase, I/S ratio, mutants, Penicillium purpurogenum Summary An extracellular inulinase (fructanase)-producing strain of Penicillium purpurogenum was isolated from the rhizosphere soil of chicory. Conidia of this selected strain were subjected to simultaneous treatment with NTG–UV (N-methyl-N 0 -nitro-N-nitrosoguanidine and ultraviolet radiation) and EtBr–UV (Ethidium bromide–ultraviolet radiation). After mutagenesis, colonies were screened and among them a few were selected to carry out the inulinase study, which showed a significantly higher inulinase activity with higher I/S (inulin/sucrose) ratio in all the selected colonies, indicating enhancement of inulinase production after mutagenic treatments in all the selected mutants. Introduction Fructose is one of the sweetest natural sugars and is emerging as an alternative sweetener to sucrose in the food and pharmaceutical industries. The importance of fructose in human nutrition has increased significantly due to their favorable functionalities, such as use as a sweetening agent in food and drinks (Vandamme & Derycke 1983), utilization by diabetics (Vandamme & Derycke 1983) and as an aid in digestion by increasing iron absorption in children (Fleming et al. 1979). Fructose can be produced from starch by enzymatic methods involving a-amylase, amyloglucosidase and glucose isomerase resulting in the production of prod- ucts consisting of oligosaccharides (8%), fructose (45%) and glucose (50%) (Kochhar et al. 1997). Separation of fructose from this high fructose corn syrup is costly and thus makes this method uneconomical. Fructose can also be obtained by acid hydrolysis of the inulin, but fructose is easily degraded at low pHs and the process gives rise to coloring of the inulin hydrolysate and by- product formation in the form of difructose anhydrides (Vandamme & Derycke 1983; Barthomeuf et al. 1991). Another source includes microorganisms, some of which secrete high amounts of exoinulinase (fructanase, EC 3.2.1.80), which can cleave fructose from inulin (a polymer of fructose with terminal glucose), by a single step catalyzed reaction with yields up to 95% of fructose (Kaur et al. 1992). Therefore, keeping in mind the potential applications of inulinases in various processes, the present study was undertaken to enhance by mutagenesis the inulinase production potential of a fungal isolate Penicillium purpurogenum (Sharma et al. 1998). Methods Microorganism and inoculum preparation The strain of Penicillium purpurogenum used in the present study was isolated from rhizosphere soil of chicory (Sharma et al. 1998) and deposited with the Microbial Type Culture Collection (MTCC No. 3009) at the Institute of Microbial Technology, Chandigarh (India). The stock was maintained on Czapek–Dox Agar (CDA) at 4 °C and, whenever required, fresh culturing was done on CDA slants. The composition of CDA medium was as follows: (inulin/sucrose 1%; NaNO 3 0.3%; K 2 HPO 4 0.1%; MgSO 4 Á 7H 2 O 0.05%; KCl 0.05%; FeSO 4 Æ 7H 2 O 0.001% and agar 2.0%). The inoculum was preparated by adding physiological saline solution (0.9% (w/v) NaCl) containing 1% (v/v) Tween- 80 to a 4-day old slant culture. The spore suspension was obtained by scratching the mycelial pad with the help of a sterile inoculating needle. Spores were counted by using a haemocytometer. Mutagenesis The induction of mutants was carried out by a simulta- neous treatment of a conidia suspension by N-methyl-N 0 - nitro-N-nitrosoguanidine–UV (NTG–UV) and ethidium bromide–UV (EtBr–UV) according to the protocol of World Journal of Microbiology & Biotechnology (2005) 21: 929–932 Ó Springer 2005 DOI 10.1007/s11274-004-6723-y