Partitioning of thermostable glucoamylase in polyethyleneglycol/salt aqueous two-phase system
© Ramesh and Murty. 2015
Received: 5 March 2015
Accepted: 3 June 2015
Published: 10 June 2015
A major challenge in downstream processing is the separation and purification of a target biomolecule from the fermentation broth which is a cocktail of various biomolecules as impurities. Aqueous two phase system (ATPS) can address this issue to a great extent so that the separation and partial purification of a target biomolecule can be integrated into a single step. In the food industry, starch production is carried out using thermostable glucoamylase. Humicola grisea serves as an attractive source for extracellular production of glucoamylase.
In the present investigation, the possibility of using polyethylene glycol (PEG)/salt-based ATPS for the partitioning of glucoamylase from H. grisea was investigated for the first time. Experiments were conducted based on one variable at a time approach in which independent parameters like PEG molecular weight, type of phase-forming salt, tie line length, phase volume ratio, and neutral salt concentration were optimized. It has been found that the PEG 4000/potassium phosphate system was suitable for the extraction of glucoamylase from the fermentation broth. From the results, it was observed that, at a phase composition of 22 % w/w PEG 4000 and 12 % w/w phosphate in the presence of 2 % w/w NaCl and at pH 8, glucoamylase was partitioned into the salt-rich phase with a maximum yield of 85.81 %.
A range of parameters had a significant influence on aqueous two-phase extraction of glucoamylase from H. grisea. The feasibility of using aqueous two-phase extraction (ATPE) as a preliminary step for the partial purification of glucoamylase was clearly proven.
Glucoamylase (EC 188.8.131.52) is a hydrolytic enzyme that degrades starch and related oligosaccharides, leading to the production of β-d-glucose. Other sectors that benefit from glucoamylase include brewing, textile, food, paper, and pharmaceutical industries . Glucoamylase is sourced from different microbial specimens like bacteria, yeasts, and fungi. The commercial production of glucoamylase has been mainly carried out using the genera Aspergillus and Rhizopus . For the manufacture of high-fructose corn syrups, starch needs to be first converted to glucose by high-temperature liquefaction and saccharification . A lot of focus is currently made on the high thermostability of glucoamylase used in the starch processing. Hence, a highly thermostable and environmentally compatible glucoamylase is very essential for industrial purposes . The main benefits of using thermostable enzymes in the starch processing industry include increased reaction rates, decreased contamination risk and cost-reduction in terms of cooling system [5, 6]. The thermophilic fungus, Humicola grisea possesses an efficient hydrolytic system for the production of glucoamylase. Moreover, the enzyme is stable when exposed to high temperature for a longer duration. With regard to these advantages, glucoamylase derived from the thermophilic fungus, H. grisea MTCC 352 has been used in the current study .
A variety of downstream processing techniques such ion exchange chromatography, hydrophobic interaction chromatography, and gel filtration chromatography have been exploited for the purification of glucoamylase [1, 7–11]. But the flipside of these procedures is that they are expensive, time consuming, and are often multistep low-yield protocols, not suitable for large scale production. In this regard, the use of aqueous two phase systems (ATPSs) for extraction and purification of glucoamylase has been attempted in the present investigation. Aqueous two-phase extraction (ATPE) has been widely used as a rapid and economic method for the separation and partial purification of many intracellular and extracellular enzymes [12–15].
ATPS can be formulated by mixing appropriate quantity of two hydrophilic polymers or a hydrophilic polymer and a salt. However, the use of ATPS based on hydrophilic polymer and a salt has attracted many researchers because of the following advantages: ease of separation, low cost, ease of scale-up and operation, biocompatibility, and high water content. Moreover, ATPE has high capacity and yield . The protein partitioning in any ATPS depends on many factors such as hydrophobic interactions, hydrogen bonding, ionic interactions, and van der Waals forces. Therefore, with respect to the type of polymer, polymer molecular weight and concentration, type of salt and concentration, tie line length (TLL), phase volume ratio (V R), and other processing parameters such as pH, temperature, and presence of neutral salt concentration, and the partitioning behavior varies [17, 18].
Over the years, ATPSs are widely used in the purification of monoclonal antibodies, extractive fermentation, and recovery of industrial enzymes . Recent studies have employed the use of ATPS (polyethylene glycol (PEG)/potassium phosphate) for biomolecule extraction and primary purification, to a great extent. Nandini and Rastogi  dealt with the partitioning of lactoperoxidase from milk whey and studied the effect of phase-forming salt, PEG molecular weight, pH, TLL and V R, resulting in a purification-fold (PF) of 2.31. Ratanapongleka  studied the partitioning behavior of laccase from Lentinus polychrous Lev., to study the effect of PEG molecular weight and concentration, salt concentration, pH, and NaCl, leading to 99 % yield and PF of 3. Babu et al.  studied the extraction of polyphenol oxidase from pineapple and studied PEG molecular weight and concentration, salt concentration, and pH, which gave 90 % recovery and a PF of 2.7. Naganagouda and Mulimani  carried out ATPE of α-galactosidase from Aspergillus oryzae and studied the effect of PEG molecular weight, salt concentration, pH, and NaCl, resulting in a PF of 3.6 and recovery of 87.71 %. The portioning of glucoamylase from Aspergillus awamori NRRL 3112 was studied by Minami and Kilikian  using a two-step ATPE consisted of PEG/phosphate system and achieved a threefold PF. Glucoamylase from the same organism was partitioned using bioaffinity extraction with starch as a free bioligand by de Gouveia and Kilikian . To the best of our knowledge, there are no available studies based on the ATPE for glucoamylase from any known thermophilic fungi.
The present investigation was done to comprehend and augment the partition of glucoamylase. Accordingly, studies were systematically carried out by varying the stated parameters, through the one-variable-at-a-time approach. In the current study, the choice of the phase-forming salt was first done, followed by the molecular weight of PEG (fixing the concentration of PEG and salt at a constant level). Next, the influence of process parameters such as tie line length, phase volume ratio, and pH were investigated. Finally, the effect of the presence of a neutral salt (sodium chloride) on the partitioning behavior of glucoamylase was studied.
Polyethylene glycol (molecular weight (MW) 1000, 2000, 4000, and 6000), dipotassium hydrogen orthophosphate, potassium dihydrogen orthophosphate, trisodium citrate, tripotassium citrate, magnesium sulfate, magnesium sulfate heptahydrate, sodium chloride, and calcium chloride were obtained from Merck (India). Potato dextrose agar, yeast extract, and soluble starch were obtained from Hi Media Laboratories Pvt. Ltd (India). Glucose oxidase/peroxidase (GOD-POD) assay kit used was obtained from Agappe diagnostics Ltd (India). All chemicals were of analytical grade. The fungi H. grisea MTCC 352 was obtained from Microbial Type Culture Collection, Chandigarh, India.
Enzyme production and preparation of crude enzyme
The microorganism was maintained on potato dextrose agar (PDA) slant, grown at 45 °C for 10 days before being stored at 4 °C. Glucoamylase was produced through submerged cultivation in a chemically defined medium. The medium consisted of 2.84 g soluble starch, 0.96 g yeast extract, 0.05 g KH2PO4, 0.24 g K2HPO4, 0.05 g NaCl, 0.05 g CaCl2, 0.19 g MgSO4.7H2O, and 0.1 mL of Vogel’s trace elements solution. The pH of the medium was adjusted to 6 . Cultures were incubated with agitation at 150 rpm at 45 °C for 4 days. The fermented broth was further subjected to filtration using Whatman No. 1 filter paper. After the filtrate was centrifuged at 10,000 rpm for 10 min, the fungal mycelia were removed. The cell-free supernatant was referred to as crude enzyme and was used throughout the experiments.
Partitioning studies in aqueous two-phase system
Aqueous two-phase systems were prepared by mixing the requisite amounts of PEG and the various salts (trisodium citrate, tripotassium citrate, magnesium sulfate, and mono/dibasic potassium phosphate). The total weight of the systems was 10 g, and the crude enzyme amount was 10 % of the total system. The tubes were vigorously vortexed and centrifuged at 3000 rpm for 10 min to speed up the separation process. The phase equilibration was achieved by overnight incubation of the tubes, and the samples were withdrawn from the individual phase and then analyzed for total protein and glucoamylase activity. Without the incorporation of the enzyme, the samples were analyzed against blanks containing similar composition, to avoid interference of the phase components.
An appropriate amount of the crude enzyme was allowed to react with 1 % (w/v) soluble starch solution in 50 mM citrate buffer (pH 5.5), at 60 °C for 10 min. The concentration of the glucose produced was estimated by GOD-POD method using a standard glucose curve prepared under similar conditions. One unit of glucoamylase activity was defined as the amount of enzyme that releases 1 μmol of glucose from soluble starch per minute under assay conditions.
The total protein was estimated, as described by Bradford , using bovine serum albumin as a standard.
Estimation of partition parameters
The partitioning parameters in ATPS were calculated as follows.
where C PT and C PB are the PEG concentrations (% w/w) in the top and bottom phases, respectively, and C ST and C SB are the salt concentrations (% w/w) in the top and bottom phases, respectively.
Results and discussion
The essence of ATPE lies in the differential partitioning of the target biomolecule to one phase and the contaminants to the other phase. It is this mechanism that leads to the purification of a target biomolecule. Extraction of biomolecules using ATPS could be tougher using theoretical predictions, primarily due to the fact that a complex set of parameters decide the extent of partitioning in an ATPS. They include the properties of the biomolecule (size, charge, and hydrophobicity) and the properties of the system like (i) type and concentration of phase-forming salt, (ii) concentration and molecular weight of phase-forming polymer, (iii) tie line length, (iv) phase volume ratio, (v) pH of the system, and (vi) concentration of neutral salts. Details of the selection of each of these parameters and their effect on partitioning of glucoamylase have been presented in the following sections.
Effect of phase-forming salts
Effect of phase-forming salts on glucoamylase partitioning
Phase volume ratio
Specific activity (U/mg)
16.8 ± 0.73
21.06 ± 1.66
82.23 ± 1.10
1.12 ± 0.09
12.92 ± 1.23
19.22 ± 1.02
75.75 ± 2.19
1.02 ± 0.01
16.98 ± 1.72
15.88 ± 0.16
69.73 ± 1.77
0.85 ± 0.04
10.82 ± 1.58
27.34 ± 0.51
82.48 ± 0.97
1.46 ± 0.09
Effect of PEG molecular weight
Effect of PEG molecular weight on glucoamylase partitioning
PEG molecular weight
Phase volume ratio
Specific activity (U/mg)
15.06 ± 1.33
19.16 ± 0.92
61.68 ± 0.94
1.14 ± 0.02
13.21 ± 0.43
21.38 ± 1.23
73.31 ± 1.69
1.28 ± 0.11
10.82 ± 1.58
27.34 ± 0.51
82.48 ± 0.97
1.46 ± 0.09
9.57 ± 1.02
21.87 ± 0.83
84.01 ± 1.24
1.31 ± 0.05
Effect of TLL
Effect of phase volume ratio
Effect of pH
Effect of NaCl
Based on the above observations, it is clear that PEG 4000 and KH2PO4/K2HPO4 phase system can be used as a potential technique for the separation and partial purification of glucoamylase.
The recovery of glucoamylase from thermophilic fungal sources using aqueous two-phase extraction was reported for the first time. The influence of various parameters on separation and partial purification of glucoamylase from H. grisea in aqueous two-phase systems was revealed. The PEG 4000/potassium phosphate phase system was found to be the most efficient for the extraction of glucoamylase, when compared to other salt systems. It was noted that glucoamylase preferentially partitioned to the salt-rich bottom phase. The optimized conditions of tie line length were at 30.62 %, phase volume ratio 0.53, pH 8, and 2 % w/w NaCl. The said conditions provided a maximum yield of 85.81 % and purity of 2.68-fold compared to crude extract. Overall, the results demonstrated the feasibility of using ATPE as a preliminary step for the partial purification of glucoamylase.
The authors gratefully acknowledge the Department of Biotechnology, MIT, Manipal University for providing the facilities to carry out the research work.
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