Production of Thermostable a-amylase by Bacillus cereus MK in solid state fermentation: Partial purification and characterization of the enzyme
S Mrudula, R Kokila
S Mrudula, R Kokila. Production of Thermostable a-amylase by Bacillus cereus MK in solid state fermentation: Partial purification and characterization of the enzyme. The Internet Journal of Microbiology. 2009 Volume 8 Number 1.
Thermostable a-amylase production under solid state fermentation was investigated using isolated thermophilic
Alpha amylases (endo-1,4-α-D-glucan glucanohydrolase, E.C. 18.104.22.168) are extracellular endo enzymes that randomly cleave the 1,4 α-linkage between adjacent glucose units in the linear amylose chain and ultimately generate glucose, maltose and maltotriose units. Among various extracellular enzymes, α-amylase ranks first in terms of commercial exploitation (Babu and Satyanarayana, 1993) and accounts 12% of the sales value of the world market (Baysal
Alpha amylases has been derived from several fungi, yeasts, bacteria and actinomycetes. However, enzymes from fungal and bacterial sources have dominated applications in industrial sectors (Pandey
Industrially important enzymes have traditionally been obtained from submerged fermentation (SmF) because of the ease of handling and greater control of environmental factors such as temperature and pH. However, solid-state fermentation (SSF) constitutes an interesting alternative since the metabolites so produced are concentrated and purification procedures are less costly (Pandey, 1992; Nigam and Singh, 1995; Chadha
Production of these enzymes using agricultural residues as substrates under SSF conditions provide several advantages in productivity, cost effectiveness in labour, time and medium components in addition to environmental advantages like less effluent production, waste minimization, etc (Pandey
In SSF, the products are formed at or near the surfaces of the solid materials with low moisture content (Selvakumar and Pandey, 1999). Therefore, it is necessary to select solvent and leaching of the product from the fermented bran. Leaching of the product from the fermented bran is a difficult process (Lonsane and Krishnaiah, 1992). Attempt has been made by researchers to isolate the desired product from the fermented bran by various techniques due to its implication on process economics (Bjurstrom, 1985 and Calton
The purpose of the present study was to investigate the production of amylase under SSF conditions by
Materials and methods
Isolation, screening and identification of thermophilic bacterium producing amylase
The soil samples for the isolation of thermophilic amylolytic organisms were collected from a potato cultivated land, Hosur, Tamil Nadu, India. The soil sample (1.0g) was suspended in saline (100 ml) and serially diluted. The diluted soil suspension (0.1ml) was spread on starch agar plates (Teodoro and Martins, 2000) and incubated at 55 ˚C for 48h. The plates were screened for amylolytic microorganisms by flooding with Grams iodine solution [2% (w/v) I2 and 0.2% (w/v) KI]. The colonies that showed largest halo-forming zones were picked up and transferred into starch broth and incubated at 55 ˚C for 24h.
The isolated microorganism was further characterized according to Bergey’s manual of determinative bacteriology (Holt
Submerged fermentation (SmF)
Solid state fermentation (SSF)
Solid state fermentation (SSF) was carried out in 250 ml Erlenmeyer flasks that contained 10 g of wheat bran and 10 ml of distilled water (moistening agent). The flasks were sterilized at 121 ˚C for 15 min and cooled to room temperature. About 1.0 ml (v/w) of exponential phase culture was added, mixed well and incubated at 55 ˚C in a humidified incubator. Flasks were periodically mixed by gentle shaking. At the end of incubation period (24 h), the flasks were taken out and the contents from the each flasks were extracted with 50 ml of sterile distilled water.
Preparation of enzyme
In solid state fermentation (SSF) the enzyme was extracted from the bacterial bran by mixing homogenously the entire bran with (1:10 w/v) distilled water and agitated on a rotary shaker at 100 rpm with a contact time of 1h at 55 ˚C. Dampened cheese cloth was used to filter the extract (Ramesh and Lonsane, 1990) and pooled extracts were centrifuged at 6000 rpm for 20 min at 4 ˚C and the clear supernatant was used as the source of extracellular enzyme.
Amylase activity was assayed by measuring the reducing sugars released from the action of amylase.
The amylase activity was routinely assayed by measuring the reducing sugars liberated in the reaction mixture. The reaction mixture (3ml) consisted of 0.5ml of (1.0% w/v) soluble starch and 0.5ml of appropriately diluted enzyme source in 2ml of 0.1M sodium acetate buffer (pH 5.6). After incubation at 60 ˚C for 30 min, 1ml of DNS was added to the tubes and the reaction was stopped by boiling the tubes in boiling water bath for 15 min. The reducing sugars released by enzymatic hydrolysis of starch were determined (Miller, 1959). A separate blank was set up to correct the non-enzymatic release of sugars.
One unit of amylase is defined as the amount of enzyme which releases 1μ mole of reducing sugar, per minute with glucose as standard, under the assay conditions described above.
Optimization of process parameters and leaching parameters for increased production of enzyme SSF
The protocol adopted for the optimization of process parameters and leaching parameters influencing α-amylase production was to evaluate the effect of an individual parameter. The parameters optimized were: (1) effect of solid substrates (10 substrates); (2) substrate mixture (6 combinations); (3) incubation temperature (35-75 ˚C); (4) the initial pH of the medium was varied between 4.0 and 12.0 by adding either 1N H2SO4 or 1N NaOH to distilled water before using it to moisten the medium; (5) incubation time (24 - 96 h); (6) moisture content [wheat bran: distilled water ratio (1:0.5 to 1:2 w/v)]; (7) inoculums size [5 to 25% (v/w)]; (8) the effect of carbon, nitrogen and trace mineral source on enzyme production were tested by incorporation at 1% (w/w) in the distilled water; (9) effect of type of solvent on extraction of enzyme from the fermented bran was evaluated by mixing 10 g of fermented bran with 50 ml [10% (v/v)] each of organic solvents followed by shaking at 100 rpm for a contact time of 1 h; (10) effect of solvent volume [fermented bran : sodium acetate buffer (0.1M)] between 1: 1.35 and 1: 7.5 (w/v); (11) effect of physical state; (12) effect of solvent temperature (30-80 ˚C); (13) effect of solvent pH (4-9) on extraction of enzyme from the fermented bran was carried out by mixing 10 g of fermented bran with (0.1 M) buffers of different pH.
Partial purification and characterization of enzyme
Estimation of protein
Protein content was determined by the method of Lowry (1951) with bovine serum albumin as standard.
TLC analysis of the products
Effect of temperature and pH on enzyme activity
Screening and maintenance of amylase producing bacteria
Characterization and identification of the isolate
The strain has the ability to hydrolyse both starch and gelatin. Nitrate was reduced to nitrite. Oxidase, Voges proskaeur test and citrate utilization test were found positive where as indole test, methyl red test and urease gave negative result. The strain fermented xylose, glucose, lactose, sucrose, maltose and raffinose. Carbohydrate fermentation of galactose, arabinose and mannitol was not found with the strain. The strain has showed an ability to grow on MacConkey agar. On the basis of above morphological and biochemical characteristics, it was identified as
Enzyme yields in submerged fermentation (SmF) and solid state fermentation (SSF)
The same strain when grown on wheat bran moistened with 10 ml distilled water (moistening agent) at 55 ˚C for 24h, it produced 1096 units of enzyme per kg of dry bacterial bran (DBB).
Optimization of process parameters for amylase production by Bacillus cereus MK in SSF
Screening of solid substrates
Among the 11 substrates screened for SSF, wheat bran fine gave the highest enzyme activity (3700U/kg DBB) followed by groundnut oil cake and coconut oil cake, respectively (Fig. 1). Considerable amount of enzyme production was observed when grown on gingely oil cake, maize bran, corn bran, rice bran fine and wheat bran coarse respectively and the remaining substrates tamarind seed powder, rice husk and gram bran gave low amount of yields.
Screening of substrate mixture
Among the 6 substrate mixtures screened for SSF, wheat bran + gingely oil cake (WB+GIOC) gave the highest enzyme activity (3120U/kg DBB) respectively followed by wheat bran + coconut oil cake (WB+COC). 50% of enzyme activities were observed with substrate mixtures of wheat bran + groundnut oil cake (WB+GOC) and coconut oil cake + gingely oil cake (COC+GIOC). Considerably, low amount of enzyme yields were observed with the remaining substrates (GIOC+GOC and COC+GOC) (Fig. 2).
Effect of temperature
Effect of pH
Maximum enzyme yields were observed between the pH range of 7.0 and 7.5 with an initial optimum pH of 7.0 (2070U/kg DBB). Marginal decrease in enzyme yields were observed when grown at pH below 7.0 and above 7.5, respectively (Fig. 4).
Effect of incubation time
From the figure 5, it is clear that maximum enzyme production was observed up to 24 h of incubation (2030U/kg DBB). The enzyme yield showed a gradual decrease on further extension of incubation.
Effect of moisture content
The data presented in figure 6, shows that amylase production was high in wheat bran fine to distilled water ratio of 10:10 (w/v)(3700U/kg DBB). Any further increase or decrease in the ratio resulted in decreased enzyme activity.
Effect of inoculum size
Significant enzyme yields were observed with 5.0 to 25.0% (v/w) of inoculum with maximum enzyme yield at 15.0% of inoculum (3120U/kg DBB). Higher or lower inoculum size resulted in a significant decrease in enzyme activity (Fig. 7).
Effect of carbon sources
Among the carbon sources tested, glucose (4670U/kg DBB) produced maximum amylase yield followed by maltose, fructose and lactose respectively (Fig. 8). In contrast carbon sources such as galactose, starch soluble and sucrose showed lower enzyme yields.
Effect of nitrogen sources
Among the organic and inorganic nitrogen sources tested, peptone type-I (2070U/kg DBB) showed maximum yields followed by urea and sodium sulphate, beef extract, ammonium chloride and ammonium nitrate respectively. Comparatively, less enzyme yields were observed with casein, yeast extract and ammonium sulphate (Fig. 9).
Effect of trace elements
All the trace elements tested, showed enhanced amylase yield. Calcium chloride (3980U/kg DBB) produced maximum yields followed by sodium chloride, cobalt chloride, manganous sulphate, magnesium sulphate and ferric chloride, respectively (Fig.10).
Effect of solvents
Among the various solvents selected for the extraction of amylase from the fermented bran, sodium acetate buffer (0.1M, pH 5.6) (9000U/kg DBB), leached maximum amylase enzyme from the fermented bran. Considerable amount of enzyme yields were obtained with the other solvents (Fig. 11).
Effect of solvent volume
The optimum solvent volume was found to be 1:2.5 (w/v) (8500U/kg DBB) and was capable of extracting maximum enzyme from the fermented bran (Fig. 12).
Effect of physical state
From the figure 13, it is observed that the effect of agitation on extraction of amylase from the fermented bran was found to be best condition for the maximum recovery when compared to static position.
Effect of solvent temperature
To study the effect of temperature on the leaching process, the temperature was varied from 30 to 80 ˚C each at 10 ˚C intervals. From the figure 14, it is observed that 50 ˚C (10000U/kg DBB) was found to be the most effective condition for the leaching of the enzyme. Further increase or decrease in temperature resulted decrease in enzyme yield.
Effect of solvent pH
The effect of solvent pH on the extraction of enzyme was studied by incubating the fermented bran with buffers of different pH ranging between 4.0 and 10.0 (10000U/kg DBB). From the figure 15, it is clear that as the pH of the buffer increases from 4.0 to 7.0, the leaching of the enzyme from the fermented bran increased and found maximum at pH 7.0. Further increase in pH showed decreased enzyme leaching from the fermented bran.
Evaluation of optimized process parameters on amylase production by Bacillus cereus MK in SSF
Under the optimum conditions described above, the strain produced 15250 units of amylase per kg of dry bacterial bran (Fig. 16).
Partial purification of the enzyme
The enzyme produced by
TLC analysis of the products
TLC analysis of reaction products of
Effect of temperature on enzyme activity and stability
Effect of pH on enzyme activity and stability
The pH optimum of the amylase determined at 90 ˚C, where in the range of 6.0 to 7.0. At pH 5.0 and 6.0 amylase retained 67% and 80% activity, respectively. The study on pH stability of the amylase enzyme at high temperature (90 ˚C) for 1h indicated that the enzyme exhibited good stability over a pH range (2.0 to 7.0), retaining 100% activity at pH 7.0 (Fig. 20).
The production of enzyme was determined at different temperatures ranging from 35 to 75 °C and optimum enzyme production was obtained at 55 °C. Similar patterns were reported for
pH of the growth medium plays an important role by inducing morphological changes in microbes and in enzyme secretion. The pH change observed during growth of microorganism also effects the product stability in the medium (Rani Gupta
The incubation time for achieving the maximum enzyme level is governed by the characteristics of the culture and is based on the growth rate and enzyme production. Maximum amylase production was observed at 24h of incubation time. Further increase in incubation time showed decreased enzyme yields. The decrease in enzyme yield may be because of the decomposition or degeneration of amylase due to interaction with other components in the media (Ramesh and Lonsane, 1990). These results are in accordance with the observations made by Dharani Aiyer (2004).
Among the several factors that are important for microbial growth and enzyme production under solid state fermentation using a particular substrate, moisture level / water activity is one of the most critical factors (Pandey
Inoculum level selected for this study ranged from 5 to 25% of 24h. Enzyme production is varied with inoculum level and showed parabolic nature in the present study. Maximum amylase synthesis was noticed with 15% inoculum size.
Supplementation of carbon sources in the form of mono saccharides, di saccharides and poly saccharides to solid medium at 1.0% level showed different impact on enzyme production with different compounds. Glucose supported maximum production followed by maltose. These data suggested that glucose was not a repressor of a amylase enzyme in this bacterial strain unlike the observed catabolic repression by glucose in
Addition of nitrogen sources have been reported to have an inducing effect on the production of various enzymes including α-amylase in a SSF medium (Pedersen and Nielsen, 2000). Results revealed that complex nitrogen sources supported better for α-amylase production over inorganic nitrogen sources. Peptone showed maximum influence in enhancement of enzyme production. Similar observations were noticed in case of amylase production by different microbial species (Gangadharan
All trace elements tested enhanced amylase yield significantly and calcium chloride was found to be best metal ion for the production of enzyme followed by sodium chloride. Allan
In SSF the products are formed at or near the surfaces of solid materials with low moisture content (Selvakumar and Pandey, 1999). So it is necessary to select a solvent for leaching out the product from the fermented bran. Leaching of enzymes from the fermented bran is a different task, and is an important aspect for the development of cost effective process for enzyme production in SSF. Among the solvents tested, sodium acetate buffer (0.1M, pH 5.6) gave the best result. In contrast, glycerol (10%) in sodium acetate buffer was found to be the best solvent for extraction of α-amylase from the fermented bran of
In SSF system as the free flowing solvent is limited, therefore sufficient amount of solvent is required to leach out the product from the fermented bran. In the present study, 1:2.5 (w/v) fermented bran to solvent ratio was found optimum. Palit and Banerjee (2001) reported an optimum solid to solvent ratio of 1:3 for extraction of α-amylase from the fermented bran of
The leaching of α-amylase increased upon agitation. This may be because on agitation, fermented bran gets homogenously distributed in a continuous phase of solvent (Tunga
The effect of temperature on leaching process was carried out by varying solvent temperature from 30 to 80 oC each at 10 oC intervals, the solvent temperature of 50 oC was found most effective for leaching of enzyme. Whereas temperature did not showed a significant effect on leaching of thermostable pullulanase from the fermented bran (Rama Mohan Reddy
The solvent pH of 6.0 was found to be optimum for the leaching of α-amylase and any increase or decrease in the pH of the solvent resulted in sharp decrease in the leaching of the amylase. Similar results were reported by Rama Mohan Reddy
Fractionation of culture supernatants showed the existence of α-amylase in the type strain of
The amylase activities at different temperatures were active in a broad temperature range (50 to 100 oC) and displayed optimum activity at 90 oC. This attribute can be exploited in starch processing industries which requires a broad temperature range. Similar findings have been reported by Melasniemi (1987) for
The effect of temperature on heat stability on amylase in the absence of substrate showed that the enzyme activities were entirely stable up to 90 oC for 30 min. Similarly, thermostability of pullulanase from
The presence of 4% starch increased in thermostability of amylase at 100 oC to 2h. The heat stability of enzymes reported for thermostable β-amylases (Hyun and Zeikus, 1985 and Rama Mohan Reddy
The pH optima were determined in three buffer systems the enzyme showed good activity at pH 7.0. The optimum activity was around 79% between 7.0 and 8.0 pH ranges. The effect of pH on α-amylase activity indicates that the amylase is active in the wide pH range of 4.0 to 8.0. This suggests that the enzyme would be useful in processes that require a wide pH range from acidic to slightly alkaline and retained activity about 58% and 80% activities at pH 4.0 and 8.0 respectively. β-amylase from
The enzyme was found to be stable at pH 7.0 for 2h. Oboh (2005) reported that the amylase from fermented cassava showed pH stability for 4h at pH 6.0 and 7.0.
In the present study, initially the strain produced 1096 units of amylase per ml of culture broth. After optimization, the same strain produced 15250 units of amylase per gram of dry bacterial bran in solid state fermentation. The enzyme yields obtained were 14 times more in SSF than before optimization. Therefore these results clearly indicated the scope for utilization of
The authors wish to acknowledge the Management, M.G.R. College, Hosur for providing laboratory facilities.