Production and purification of extracellular pullulanase by Klebsilla aerogenes NCIM 2239

The present study was carried out to optimize the production of pullulanase from Klebsilla aerogenes NCIM 2239, a new strain of the bacterium. The organism was screened for its ability to synthesize and secrete extracellular pullulanase, by analysis of pullulan degrading activity. Environmental (pH and temperature) and nutritional (carbon and nitrogen) factors influencing growth and product formation were optimized through fermentation trials in shake flasks. Maximum pullulanase production (78.62 U/ml) was observed at 48 h. Optimized pH and temperature were 7.0 and 37°C, respectively. Different carbon sources (pullulan, starch, sucrose, maltose and glucose) and nitrogen sources (peptone, beef extract, yeast extract and casein) were analysed for maximum production of pullulanase. This bacterium was found to exhibit maximum pullulanase production using starch and peptone as carbon and nitrogen sources, respectively. In addition, pullulanase production by solid substrate fermentation (SSF) was investigated using K. aerogenes NCIM 2239. The extracellular pullulanase was purified by ultrafiltration method of membrane separation. The pullulanase activity after ultrafiltration was 130.21 U/ml when compared with its crude (83.08 U/ml).

Pullulanase is of great significance due to its wide area of potential application.Pullulanase is widely used in industries in the saccharification of starch (Prakash et al., 2012).It converts starch into glucose and maltose which are used in the production of glucose syrup more efficiently.The products obtained by the action of pullulanase have various pharmaceutical and neutraceutical properties.Pullulanase is inexpensive and easily available hence, it helps in the economical production of various products like resistant starch and substitutes of locust bean gum (LBG).The saccharification property of this enzyme has led to its use in various food industries for the production of glucose syrup, maltose syrup and beer (Shaw et al., 1992).Bioethanol production could also be done by the use of pullulanase thus providing a source of fuel.The basic property of breaking α linkage has been widely used by the baking industry (Prakash et al., 2012).
The aim of this research work was to optimize the parameters of fermentation such as carbon sources, nitrogen sources, pH, temperature and incubation time for the enhancement of production of pullulanase from K. aerogenes NCIM 2239 and to purify the enzyme by ultrafiltration method.In addition, the effect of submerged fermentation and solid state fermentation was studied.

Microorganism
The pullulanase-producing bacterium, K. aerogenes NCIM 2239, was procured from National Collection of Industrial Microorganisms (NCIM) and National Chemical Laboratory at Pune, India.

Inoculum preparation
The strains were maintained by subculturing consecutively in maltose limiting medium at 37°C under aerobic conditions.For inoculum preparation, a loop from the stock was sub-cultured into the nutrient broth.
Single colony from the nutrient agar was inoculated into nutrient broth and incubated to obtain initial cell concentration.This culture was used as inoculum (1% v/v) for all fermentations carried out in this process.
The pH of all media was adjusted to 7.0 and sterilized at 121°C for 20 min.After incubation of the cultures at 37°C for 2 days, the cells were removed from the culture medium by centrifugation at 10,000 rpm for 10 min.

Determination of pullulanase activity
Enzyme activity was determined by measuring the enzymatic release of reducing sugar from pullulan.In this assay, 0.5 mL of enzyme sample was added into 0.5 mL of 1% (w/v) pullulan in 0.02 M sodium phosphate buffer at pH 7.0.The reaction mixture was incubated at 40°C for 30 min and the amount of reducing sugar released at the end of the reaction was determined by dinitrosalicylic acid (DNSA) method.
Sample blank was used to correct the non-enzymatic release of reducing sugar.One unit of pullulanase activity is defined as the amount of enzyme required to produce 1 μ mol reducing sugar (equivalent to glucose) per minutes under the assay conditions (Ling et al., 2009).

Effect of incubation period
A volume of 100 ml of selected medium was taken in each 250 ml conical flask.All flasks were autoclaved at 121°C at 20 min.After cooling, the flasks were inoculated.The flasks were incubated for 1 to 3 days.The cells were precipitated by centrifugation at 10000 rpm.From the supernatant, enzyme activity was measured.

Effect of incubation temperature
The culture medium was incubated at different temperature for optimum enzyme production.For this reason, equal quantity of inoculum was added in each conical flask containing 100 ml of selected suitable medium with selected pH.The flasks were then incubated at different temperature (28, 37, 45 and 55°C), respectively, for optimum enzyme production.The effect of temperature on pullulanase production was recorded.

Effect of pH
To observe the effect of medium pH on enzyme production, 100 ml of selected medium at different pH (5, 6, 7, 8, 9 and 10, respectively) were used.The flasks were then inoculated and incubated in optimum incubation periods.The effect of medium pH on pullulanase activity was recorded.

Effect of carbon sources
The effect of carbon sources on pullulanase production was tested by using different carbon sources namely, sucrose, starch, pullulan, glucose and maltose.They were tested individually at the concentration of 1% in the carbon source-optimized medium.

Effect of nitrogen sources
Nitrogen source is one of the most common factors which affect the pullulanase enzyme production.The organic nitrogen source used greatly influenced both growth and pullulanase production.The growth medium was initially supplemented with different organic nitrogen sources (0.5%), that is, beef extract, peptone, yeast extract and casein.Then, the nitrogen source that gives maximum enzyme production was optimized.
The enzyme was extracted by using 25 ml of distilled water to the solid substrate culture and well mixed by agitation for 5 min, then filtered through a Whatmann paper.The filtrate was centrifuged at 8000 rpm for 20 min.The supernatant was used as crude enzyme and the enzyme activity was assayed.

Purification of pullulanase by ultrafiltration
The enzyme was purified by ultrafiltration method.Since the molecular weight of pullulanase was 98 kDa approximately (Malakar et al., 2012;Shehata et al., 2016), the polyether sulphone membrane of molecular weight cut-off 100 kDa was used.
In this method, the polyether sulphone membrane was fitted in membrane support.The experimental setup was placed above the magnetic stirring plates.In the feed reservoir, 150 ml of crude enzyme was loaded through the peristaltic pump and the pressure was passed for stirring.Simultaneously, permeate was collected through an outlet (Liu et al., 2009).The collected volume of permeate was analyzed using DNSA method.

Effect of incubation period
The growth of microorganism and their enzymatic activities were estimated during incubation period which extended up to 48 h.The results are shown in Figure 2. The biosynthesis of extra cellular enzyme increased almost linearly until the stationary phase of growth was attained.Growth as well as pullulanase activity reached maximum value after 48 h of incubation.
The effect of incubation period was checked at an interval of 12 h till 72 h.The maximum enzyme activity was observed at 48 h after which the enzyme activity started to decrease.Similar result was reported from pullulanase enzyme from Bacillus cereus (Waleed et al., 2015).
The reason for this is the denaturation of the enzyme caused by the interaction with other components in the medium (Lonsane et al., 1990).It could have also been due to the fact that, the microorganism was on its decline  phase during the third day of fermentation which resulted in the decreased enzyme production (Figure 2).During long time exposure, proteins component would denature in the submerged fermentation medium.

Effect of temperature
Temperature is one of the most important parameter governing the growth of organism and enzyme biosynthesis.The enzyme production pattern shows a gradual increase in the enzymatic activity giving maximum production at 37°C.Similar result was reported from pullulanase enzyme from B. cereus (Waleed et al., 2015).
At higher temperature due to the production of the large amount of the metabolic heat, the fermenting substrate temperature shoots up, thereby inhibiting microbial growth and enzyme production.The effect of temperature was observed in the range of 28, 37, 45 and 55°C.The maximum enzyme activity was observed at 37°C after which, the enzyme loses its stability and activity decreases (Figure 3).
Similar results were reported for Aspergillus niger at 35°C (Lonsane et al., 1985), and for amylase production by Bacillus sp. at 37°C, others showed a high level of enzyme production from Raoultella which was 30°C (Hii et al., 2009).The optimum condition for amylopullulanase production by Clostridium thermosulfogenes was observed at 60°C (Swamy and Seenayya, 1996).

Effect of pH
Microorganisms were grown at different pH in the range of 5 to 10, to determine the effect on pullulanase production.The degree of enzyme production was highest at pH 7and reduces with the increase in pH as described in Figure 4. Similar result was reported from pullulanase enzyme from B. cereus (Waleed et al., 2015).
pH can influence the growth and product formation due to its effect on the solubility of nutrient, ionization of the substrate and its availability to the microorganism (Kotwal et al., 1998).Also, the acidic or basic media may lead to denaturation of the enzyme (Bertoldo et al., 2002).
An attempt to overcome the problem of pH variability during the solid substrate fermentation (SSF) process was obtained by the substrate formulation, considering the buffering capacity of the different components employed by the use of buffer formulation with components that have no deleterious influence on the biological activity (Kotwal et al., 1998).

Effect of carbon sources
The effect of different carbon sources on the production of pullulanase was studied using the basal culture medium, supplemented with 1% of carbon sources.The culture containing 1% of each carbon source at a time was incubated and tested for enzymatic activity.
Among these glucose, maltose, pullulan, sucrose and starch were used as 1% at a time.Starch enhanced the pullulanase activity (92.76 U/ml) when compared with other carbon sources.Carbon source is the most effective factor for the production of pullulanase from microorganism.
In the presence of soluble starch, the production of pullulanase was increased as compared to other carbon sources.Maximum production of pullulanase was observed with soluble starch and hence, it is a good source of carbon used in production of pullulanase (Malviya et al., 2010).
The highest pullulanase activity may be due to inducible nature of maltose, being hydrolytic product of starch.Various carbon sources were checked as media component and pullulan was observed with best enzyme  activity, while maltose, sucrose and glucose showed gradual decrease but significant in enzyme activity (Figure 5).

Effect of nitrogen sources
The effect of different nitrogen sources on the production of pullulanase was studied using the basal culture medium supplemented with 0.5% of nitrogen sources.Among the four organic nitrogen sources, peptone had more influence on pullulanase production (79.61U/ml) than others (Figure 6).This may be due to the low molecular weight of peptone, which can be easily degraded and can be absorbed.
Different nitrogen sources were checked for enzyme activity among which peptone gave the maximum activity.
The highest value of pullulanase activity was achieved in medium containing peptone which indicates that, peptone could be one of favorable organic nitrogen source that enhances the cell growth of the culture as well as the pullulanase production (Hii et al., 2009).

Determination of optimal conditions for pullulanase production in submerged fermentation (SmF)
The result depicted in Table 1 showed that, media containing starch as carbon source with peptone as nitrogen source, was inoculated with 1 ml of bacterial suspension when incubated at 37°C for 48 h at pH 7, which was the best media for pullulanase production.

Solid substrate fermentation (SSF)
Different solid substrates were used for pullulanase production from K. aerogenes NCIM 2239, as shown in Figure 7, which gave the highest enzyme activity in media containing mixture of rice bran and corn bran as substrate in SSF.
The enzyme activity reached 151.54 U/ml, while the media containing rice and corn bran gave the lowest enzyme activity.Similar results are also reported for  Bacillus lichenoformis (BS18) (Khalaf and Aldeen, 2013).

Comparison between SSF and SmF
SSF had given higher production of pullulanase than submerged fermentation (SmF).The results are shown in Figure 8.
SSF have been defined as the microbial transformation of biological materials in their natural state, in contrast with liquid or submerged fermentation that is carried out in dilute solution or slurries (Kotwal et al., 1998).The solid substrate in SSF provides a rich and complex source of nutrient, which may be sufficient or sometimes inadequate and incompete, with respect to the overall nutritional requirement of that particular microorganism that is cultivated on that substrate (Kotwal et al., 1998).SSF is preferred to SmF because of the simple technique, low capital investment, lower levels of catabolic repression and end product inhibition, low waste output, better product recovery, and high quality production (Mahanta et al., 2008).

Purification of crude enzyme by ultrafiltration method
The enzyme was purified by ultrafiltration method of membrane separation and enzyme activity, which was measured by DNSA method.The nature of solid substrate is the most important factor in SSF, which not only supplies the nutrient to the culture but also serves as an anchorage for microbial cells.
Therefore, the particle size and the chemical compositions of substrate are of critical importance (Kim et al., 1985) (Figure 9)

Conclusion
Pullulanase was successfully produced and purified from K. aerogenes NCIM2239 strain.As it is very stable at higher range of temperature and pH, this can be used in many industrial processes.The media preparation, which is one of the most important objective of this study was achieved and the most appropriate combinations of media components where stabilised.In order to study enzyme activity, the enzyme substrate reaction was performed based on various parameters such as temperature (37°C), incubation time (48 h) and pH 7. Various carbon sources were checked as media component among which starch gave best enzyme activity, while maltose, sucrose and glucose showed significant enzyme activity.Peptone gave the maximum activity among different nitrogen sources.The study of the SSF medium, SmF and comparison between them indicated that, the use of SSF gave higher production of enzyme than SmF.SSF technique is a suitable and economic method for pullulanase production.The pullulanase activity of the crude form was 83.08 U/ml.The crude enzyme under purification using ultrafiltration gave rise to 130.21 U/ml enzyme activity, and thus can be used in various industrial processes (Table 2).

Figure 2 .
Figure 2. Effect of incubation time on pullulanase production.

Figure 4 .
Figure 4. Effect of pH on pullulanase production.

Figure 5 .
Figure 5.Effect of carbon sources on pullulanase production.

Figure 6 .
Figure 6.Effect of nitrogen sources on pullulanase production.

Figure 7 .
Figure 7. Enzyme activity of different sources in SSF.

Figure 9 .
Figure 9. Enzyme activity between the crude and purified enzymes.

Table 1 .
Optimal conditions for pullulanase production by using submerged fermentation medium.

Table 2 .
Enzyme activity between the crude and purified enzymes.