Production Of Pullulanase Using Novel Organic Substrates

Chaitanya Velhal, Mrudula Sant, Surojit Das, Chandrashekhar Kulkarni
Mitcon Biotechnology and Pharmaceutical Center, Shivajinagar, Pune – 411005.


Production of pullulanase was carried out from I. batatas, P. vulgaris, E. crassipes and P. stratiotes using A. pullulans. The fermentation reactions were carried out in minimal basal media at 28°C. E. crassipes and P. stratiotes as substrates produced more pullulanase than I. batatas and P. vulgaris. The highest enzyme production from Pistia stratiotes was of 9.3 U/ml after 72 hours of incubation at 28°C and Eichhornia crassipes showed most enzyme production of 4.65 U/ml after 48 hours of incubation at 28°C. The two novel substrates, E. crassipes and P. stratiotes, could successfully be used for the production of pullulanase and are a promising resource for large-scale production of the same.

Keywords: Eichhornia crassipes, Pistia stratiotes, Ipomoea batatas, Phaseolus vulgaris, pullulanase, Aureobasidium, Solid state fermentation


The roles of enzymes in many processes is no new to us as their use can been dated back to the history of ancient Greece when the Greeks used enzymes from microorganisms for applications such as in baking, brewing, alcohol production and cheese making [1]. In modern age they often find themselves useful in food, beverage and pharmaceutical and chemical industries [2].The growing demand for industrially
important enzymes is demanding the usage of cheap resources to fulfil these needs. One such industrially important enzyme is pullulanase also known asα-dextrin 6- glucanohydrolase [3] that is responsible for the hydrolysis of pullulan and amylopectin to produce maltosetriose units [4], [5] Various types of pullulanases are found in nature. Pullulanase type I hydrolyses α-(1,6) glycosidic linkages in pullulan and branched polysaccharides to give maltosetriose units [6], [7]. Pullulanase type II is used extensively because of its ability to hydrolyse both α-(1,6) and α-(1,4) glucosidic linkages in amylopectin [3] which is one of the main components of starch[8], [9], [10]. Pullulanase hydrolase type 1 acts on the α- (1,4)glycosidic linkages in pullulan to give panose [11], [12], [13]. Pullulanase hydrolase type II hydrolyses α-(1,4) bonds in pullulan to give isopanose and pullulanase hydrolase type III acts on both α-(1,4) and α-(1,6) glycosidic linkages of pullulan and starch to give out various end products such as panose, maltose and maltotriose[14], [15]. Pullulanases have a wide variety of applications as it acts as a starch de-branching enzyme [5]. Some of its applications include the production of high-glucose syrup, high-maltose corn syrup that is mainly used in the food processing industry and high-fructose corn syrup that is used as a carbon source in production media.

Pullulanases are also used for the production of ‘resistant starch’ in starch processing industries, detergent industry, baking industry and for the production of cyclodextrins and low-calorie beer. It can also be used as a dental plaque control agent [16]. The use of this enzyme for a variety of applications has been inhibited due to the high cost of production and low yield [5]. This limitation required the use of cheap substrates that are high in starch for the potential large-scale production of this enzyme. The substrates considered are two commonly grown plants in India i.e. Ipomoea batatas(sweet potato) and Phaseolus vulgaris (Local red kidney beans). The other two novel substrates are pest plants i.e. Pistia stratiotes(water cabbage) and Eichhornia crassipes(water hyacinth). Ipomoea batatas(sweet potato) is a dicotyledonous plant that belongs to the family Convolvuceae[17]. It has a tuberous growth that is a rich source of starch [17]. Another common source of starch and one of the most commonly consumed legumes is Phaseolus vulgaris (Local red kidney beans) [18], [19]. It mainly consists of carbohydrates (50%-70%) in its dry state [18]. Eichhornia crassipes(water hyacinth) and Pistia stratiotes(water cabbage) are classified as pest plants worldwide [20], [21]. They form dense matt growth over the surface of the water bodies often covering them. These plants are free floating plants with leaves growing above the water. Water hyacinth grows tall leaves with a single lavender colored flower whereas; water cabbage leaves grow in the form of a rosette. Both have a high reproduction rate as the seeds of water hyacinth may remain viable for over 28 years[22]. This profound growth causes numerous problems such as eutrophication, blockage of rivers, hampers fishing and endangers the existing flora and fauna by blocking the penetration of sunlight. One of the major rivers in Pune, India i.e. the Mulariver is also facing a problem due to the weed infestation and a huge amount is being spent for the control of these weeds that are causing problem environmentally, economically and socially [23]. Despite the attempts, they still continue to be a hazard. The use of these plants as a suitable substrate is being considered as they do not compete for land, have a negligible cost and grow at a fast rate[20]. This study attempts to test the use of cheap substrates that are well known to contain a high level of starch i.e. sweet potato and local red kidney beans. It also tests the use of novel substrates i.e. water hyacinth and water cabbage as a carbon source for pullulanase production.

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