Partitioning and purification of extracellular β-1,3-1,4-glucanase in aqueous two-phase systems

Partitioning and purification of extracellular β-1,3-1,4-glucanase in aqueous two-phase systems first-author †E-mail:gqhe@zju.edu.cn; tangchunxi@zju.edu.cn †E-mail:gqhe@zju.edu.cn; tangchunxi@zju.edu.cn gqhe@zju.edu.cn †E-mail:gqhe@zju.edu.cn; tangchunxi@zju.edu.cn †E-mail:gqhe@zju.edu.cn; tangchunxi@zju.edu.cn gqhe@zju.edu.cn School of Biosystem Engineering and Food Science, Zhejiang University, Hangzhou 310029, China School of Biosystem Engineering and Food Science, Zhejiang University, Hangzhou 310029, China The partition behaviors of β-1,3-1,4-glucanase, α-amylase and neutral proteases from clarified and whole fermentation broths of Bacillus subtilis ZJF-1A5 were investigated. An aqueous two-phase system (polyethylene glycol (PEG)/MgSO 4 ) was examined with regard to the effects of PEG molecular weight (MW) and concentration, MgSO 4 concentration, pH and NaCl concentration on enzyme partition and extraction. The MW and concentration of PEG were found to have significant effects on enzyme partition and extraction with low MW PEG showing the greatest benefit in the partition and extraction of β-glucanase with the PEG/MgSO 4 system. MgSO 4 concentration influenced the partition and extraction of β-glucanase significantly. pH had little effect on β-glucanase or proteases partition but affected α-amylase partition when pH was over 7.0. The addition of NaCl had little effect on the partition behavior of β-glucanase but had very significant effects on the partitioning of α-amylase and on the neutral proteases. The partition behaviors of β-glucanase, α-amylase and proteases in whole broth were also investigated and results were similar to those obtained with clarified fermentation broth. A two-step process for purifying β-glucanase was developed, which achieved β-glucanase recovery of 65.3% and specific activity of 14027 U/mg, 6.6 times improvement over the whole broth. Aqueous two-phase system Partition β-1,3-1,4-glucanase INTRODUCTION 1 β-1,3-1,4-glucan is the major cell wall component of barley and other cereal endosperms. It consists of a polymer of glucose monomers joined by β-1,3- and β-1,4-glycosidic bonds in an irregular fashion (Woodward et al., 1983 1983 ; Edney et al., 1991 1991 ). Due to its unique structure, barley β-glucan can be dissolved in water in high molecular mass and resulting in a high viscosity solution. The high-molecular-mass β-glucan released during mashing causes severe filtration problems and may lead to gelatinous precipitates in the resulting beer. The use of barley varieties of high β-glucan content, the addition of adjuncts or the use of under modified malt can result in beer with troublesome levels of high molecular weight β-glucan. Barley β-glucan can also cause high intestinal viscosity that affects the digestibility of feed and absorption of nutrients by chicks and piglets fed barley (Planas, 2000 2000 ). The addition of exogenous enzymes during mashing or feed preparation can reduce the negative effects of barley β-glucan (Stone and Clarke, 1992 1992 ; Godfrey and Reichely, 1983 1983 ; White et al., 1983 1983 ). Some bacteria and fungi can produce β-glucanase that has the same substrate specificity as malt β-glucanase and, thus, the ability to hydrolyze barley β-glucan specifically. And β-1,3-1,4-glucanase produced by Bacillus has great industrial potential because of its higher thermal stability. Generally, industry uses crude raw materials to produce β-glucanase from Bacillus . The high viscosity and solid mass content of the resulting broth, and the simultaneous production of α-amylase and neutral proteases, cause difficulties in selectively purifying enzymes using conventional techniques, such as filtration and centrifugation. It is also difficult to scale-up these techniques because of particulate materials in particular bacteria, compressible filter cakes and viscous slurries (Schmidt et al., 1994 1994 ). Partitioning in aqueous two-phase systems (ATPS) has been shown to provide a powerful method for separating and purifying mixtures of proteins. These systems are composed of aqueous solutions either of two water-soluble polymers, usually polyethylene glycol (PEG) and dextrin, or of a polymer and a salt, usually PEG and phosphate or sulfate. Some new systems have recently been developed which, for example, rely on a system of surfactant and surfactant or polymer and surfactant (Xiao et al., 2001 2001 ). Compared with other commonly used separation and purification techniques, ATPS has a number of advantages, such as ease of scale-up, the ability to handle particulate materials and the ability to process streams continuously. ATPS can remove undesirable by-products in crude supernatants, for example, undefined polysaccharides and pigments and interfering proteins that lower the activity of the enzyme. This makes ATPS a potentially attractive technique for separation and purification of industrial enzymes, extracellular as well as intracellular. There are many factors influencing the partitioning in ATPS. Differences in partitioning result from the interaction of factors inherent in the system itself, such as choice of system components, polymer molecular weight, concentration of polymers and salts, ionic strength, and pH, and those of the target protein, such as hydrophobicity, charge and molecular weight. The causative mechanisms of uneven biomolecules distribution are poorly understood. Fundamental theories of protein partition derived from classical polymer solution thermodynamics are being developed. Although the present models provide some information, no comprehensive theory currently exists to guide the design of systems for separation of specific mixtures of proteins and particles. Therefore application of the technique requires experimentation to design an adequate phase system for optimal partitioning of a particular protein. The success of a technique can be monitored with the use of partition coefficient K , the ratio of biomolecule concentration in the top phase to that in the bottom phase. ATPS has been successfully used to purify extracellular enzymes, for example, xylanase (Jain and Johri, 1999 1999 ), β-xylosidase (Pan and Li, 2001 2001 ), β-glucosidase (Johansson and Reczey, 1998 1998 ), α-amylase (Planas, 2000 2000 ; Jiao et al., 1998 1998 ). There is no report for purification of β-glucanase of Bacillus with ATPS. Schmidt et al.( 1994 1994 ) studied the partition behavior of pure α-amylase and α-amylase from supernatant and whole broth of Bacillus subtilis fermentation in PEG/dextrin, PEG/phosphate, and PEG/sulfate systems, but did not investigate the partition behavior of the β-glucanase and proteases. Jiao et al.( 1998 1998 ) studied the purification of α-amylase and proteases from supernatant of Bacillus fermentation in PEG/sulfate system but did not mention β-glucanase activity. The main purpose of this paper was to study the partition behaviors of β-glucanase, α-amylase and neutral proteases in a PEG and MgSO 4 aqueous two-phase system and to investigate the effects of PEG molecular weight and concentration, pH, MgSO 4 concentration and NaCl concentration on extraction of enzymes from clarified or whole fermentation broth. MATERIALS AND METHODS 1 Bacillus subtilis ZJF-1A5 was a wild strain isolated from soil that was mutated in our Lab with UV and diethyl sulfate. Chemicals 2 PEG with average MW of 2000, 4000 or 6000 was obtained from Pudong Gaonan Chemicals Ltd. Shanghai, China. Lichenin was purchased from Sigma Corporation of America and used as substrate for the β-glucanase assay. All other salts and chemicals used were of analytical grade. Crude enzyme preparation 2 The medium used for seed culture contained: dextrin, 20 g/L; yeast extract, 20 g/L; KH 2 PO 4 , 1.0 g/L; MgSO 4 ·7H 2 O, 0.1 g/L; CaCl 2 , 0.1 g/L. The medium used for enzyme production contained: barley flour, 63.5 g/L; corn flour, 44.8 g/L; KH 2 PO 4 , 1.0 g/L; MgSO 4 ·7H 2 O, 0.1 g/L; CaCl 2 , 0.1 g/L. Bacillus subtilis ZJF-1A5 was activated at the first preculture with 10 ml medium in a 100 ml culture flask in a 200 r/min rotary shaker at 37 °C. After 18 h cultivation, the first culture broth was divided and inoculated (4%) into 30 ml seed culture medium in 250 ml culture flask. The flasks were shaken at 200 r/min for 18 h at 37 °C. The seed culture was inoculated (3.82%) into 5 L fermentor (B. Braun) containing 3 L medium at 500 r/min, 37 °C, and 1 L/(L·min) for 40 h. After fermentation, the culture broth was centrifuged at 1500 g for 15 min. Cells and insoluble components were discarded. The supernatant was used as a crude enzyme solution. Enzyme assays 2 β-glucanase: Enzyme preparation (100 μl), diluted with phosphate buffer (0.2 mol/L, pH 6.0), was incubated at 50 °C for 10 min with 0.9 ml 0.2% ( w / V ) lichenin in the same buffer. Reducing sugars were estimated as described by Miller ( 1959 1959 ). One unit of β-glucanase activity (U) was defined as the amount of enzyme required to form 1 μmol reducing sugar (glucose as standard) per minute. α-amylase (Syu and Chen, 1997 1997 , but modified): 1.2% buffered starch solution was used as the substrate for the assay of α-amylase activity. The reagent was I 2 -KI solution (0.5%:5%). One millilitre phosphate buffer (0.2 mol/L, pH 6.0) was added into 2 ml of 1.2% ( w / V ) starch solution and the mixture was incubated in 40 °C water bath for 5 min. One millilitre sampled enzyme solution was added into the starch solution, and then incubated at 40 °C for 30 min. After that, 10 ml of 0.5 mol/L acetic acid was added to stop the reaction. One millilitre of such solution was mixed with 10 ml I 2 -KI reagent and the absorbance of the solution was measured at wavelength of 660 nm. The α-amylase activity was measured by its dextrinizing power (D.P.). α-amylase unit (1 D.P.) is defined as the amount of amylase which will produce 10 percent decrease in the intensity of amylase’s blue color under the conditions mentioned above. Neutral proteases (Han and Lee, 1997 1997 , but modified): the substrate was 0.6% ( w / V ) casein in Tris-HCl (0.05 mol/L, pH 7.5). Enzymatic hydrolysis was initiated by addition of 1 ml properly diluted enzyme solution in 5 ml substrate. After 5 min incubation at 40 °C, 5 ml of 0.4 mol/L trichloroacetic acid (TCA) was pipetted into the solution to terminate the reaction. The solution was incubated at 40 °C for 20 min to precipitate the residue substrate, and filtrated. The tyrosine concentration of the filtrate was determined spectrophotometrically at 275 nm. One unit of proteases activity is based on the tyrosine (per μg tyrosine enzyme per min). Protein determination 2 Protein content was determined by the Bradford method using bovine serum albumin (BSA) as a standard (Zhang et al., 1997 1997 ). Blank systems, without protein, were used as reference, and no interference from phase components was observed. Aqueous two-phase extraction 2 Phase systems were prepared from stock solutions of PEG (50%, w / w ) and MgSO 4 (20%, w / w ). The pH was set using NaOH or HCl. Stock solutions were stored at 4 °C. Before use, the temperature of all stock solutions was equilibrated to room temperature. Low-speed centrifugation (1000 g) was used to speed up phase separation after thorough but gentle mixing of the system components. The phase volume ratios were determined in graduated centrifuge tubes. Samples of the top and bottom phases were then assayed for activities of enzymes and total protein. All partition experiments were done at room temperature. RESULTS AND DISCUSSION 1 Effect of PEG molecular weight on enzymes partitioning 2 The phase volume ratio ( R =volume of top phase/volume of bottom phase) decreased with increasing PEG MW. The partitioning of total protein and enzymes depended on the PEG MW. Partition coefficient ( K =concentration in top phase/concentration in bottom phase) and extraction rate ( E =the total enzyme activity in top phase/the total enzyme activity in system) of total protein increased as PEG molecular weight increased from 2000 to 6000 (Table 1 1 ). The partitioning and extraction of β-glucanase in PEG and MgSO 4 systems was strongly depended on the PEG MW. Lower PEG MW led to higher partition coefficients and extraction rates, the difference being especially significant when the PEG concentration increased (results not shown). While PEG MW had less effect on the partitioning and extraction of the α-amylase and neutral proteases, most of the α-amylase and proteases was partitioned to the bottom phase. High MW PEG would not be a reasonable choice for further investigation, as all enzymes had low K values and extraction rates at high PEG MW, which would lead to very poor separation and purification of enzymes. Therefore, low MW PEG (PEG 2000) was chosen as phase component for further studies. E e , E g , E a and E p represent the extraction rate of total protein,β-glucanase, α-amylase and neutral protease respectively; R represents phase ratio; K e , K g , K a and K p represent the partition coefficients of total protein, β-glucanase, α-amylase and neutral protease respectively PEG MW R K e E e (%) K g E g (%) K a E a (%) K p E p (%) 2000 1.160 0.550 38.79 1.44 73.94 0.157 15.36 0.27 24.02 4000 0.975 0.660 40.49 0.44 27.70 0.525 31.48 0.22 14.36 6000 0.915 0.802 42.32 0.26 19.40 0.219 16.66 0.27 20.00 Effects of PEG 2000 concentration on enzymes partitioning 2 Results (Fig. 1 1 ) showed that increasing PEG 2000 concentration caused an increase (2.30-fold) of the partition coefficient for β-glucanase (from K =0.86 to K =2.84), and the extraction rate from 26.84% to 75.66%. Increasing PEG 2000 concentration had no significant effects on partition coefficients or extraction rates for α-amylase or neutral proteases, and so PEG 2000 offers a convenient method for separating β-glucanase from α-amylase and neutral proteases. Increasing the PEG concentration to 20% ( w / w ) caused a 2.30-fold increase in the partition coefficient for β-glucanase, although extraction rate and partition coefficient reached a relatively constant level at a PEG concentration of 16% ( w / w ). Therefore, the PEG 2000 concentration should be kept at 16%. E e , E g , E a and E p represent the extraction rate of total protein, β-glucanase, α-amylase and neutral proteases respectively; R represents phase ratio; K e , K g , K a and K p represent the partition coefficient of total protein, β-glucanase, α-amylase and neutral proteases respectively Effect of MgSO 4 concentration on enzymes partitioning 2 The effects of MgSO 4 concentration on enzyme partitioning and extraction are shown in Table 2 2 . In the PEG 2000/MgSO 4 system, although volume ratios decreased as MgSO 4 concentration increased, K value and extraction rate of β-glucanase, α-amylase and neutral proteases all increased. But the α-amylase transferred to the bottom phase and extraction rate was only 15.47% when the MgSO 4 concentration was 16%. Additionally, when the MgSO 4 concentration was lower than 14%, the E value and K value of neutral proteases had no significant effect ( E p varies from 22.56 % to 29.55 %, K p varies from 0.29 to 0.64), but an increase of MgSO 4 concentration from 14% to 16% significantly increased the partition coefficient of neutral proteases from 0.64 to 3.35, and the extraction rate from 29.55% to 72.40%. These results indicated that increasing MgSO 4 concentration could increase the extraction rate of β-glucanase, and that the α-amylase extraction rate increased a little. But when the MgSO 4 concentration was more than 14%, a lot of neutral proteases were extracted into the top phase, which was detrimental to the purification of β-glucanase. Therefore, an MgSO 4 concentration of 14% was chosen for all further investigations. E e , E g , E a and E p represent the extraction rate of total protein, β-glucanase, α-amylase and neutral proteases respectively; R represents phase ratio; K e , K g , K a and K p represent the partition coefficient of total protein, β-glucanase, α-amylase and neutral proteases respectively MgSO 4 (%, w / w ) R K e E e (%) K g E g (%) K a E a (%) K p E p (%) 8 1.32 0.57 43.05 1.23 61.76 0.0079 1.04 0.29 27.93 10 1.16 0.77 38.26 1.68 57.32 0.0446 3.45 0.36 22.56 12 1.00 0.89 46.96 2.47 71.14 0.1050 9.54 0.41 29.26 14 0.65 1.16 43.06 4.00 72.34 0.1190 6.44 0.64 29.55 16 0.78 1.94 60.24 5.45 81.01 0.2340 15.47 3.35 72.40 Effect of pH on the enzymes partitioning 2 Results shown in Table 3 3 show that R was independent of pH in the PEG 2000/MgSO 4 system. As pH increased from 4.3 to 9.0, the partition coefficients and the extraction rates of β-glucanase and neutral proteases changed little. However, K values and extraction rates for α-amylase increased as pH increased. The differences could be explained by the fact that the isoelectric points (pI) of β-glucanase and neutral proteases of Bacillus are basic, whereas, pI of α-amylase is acidic (Han and Lee, 1997 1997 ; Huddleston and Veide, 1991 1991 ; Schmidt et al., 1994 1994 ). As a general rule, negatively charged proteins partition to the top phase and positively charged proteins to the bottom phase. At higher pH, the protein was more negatively charged than at low pH, and therefore, interactions between α-amylase and PEG units became stronger. The result indicated that the pH 7.0 PEG 2000/MgSO 4 systems should be chosen to separate the β-glucanase from the other two enzymes. E e , E g , E a and E p represent the extraction rate of total protein, β-glucanase, α-amylase and neutral proteases respectively; R represents phase ratio; K e , K g , K a and K p represent the partition coefficient of total protein,β-glucanase, α-amylase and neutral proteases respectively pH R K e E e (%) K g E g (%) K a E a (%) K p E p (%) 4.3 0.73 1.58 53.48 3.60 72.31 0.14 14.16 0.50 34.58 5.0 0.73 1.63 56.03 4.45 71.70 0.16 15.82 0.48 26.73 6.0 0.77 0.96 42.43 5.05 79.56 0.13 15.07 0.54 27.18 7.0 0.72 1.17 45.82 5.07 77.46 0.13 13.70 0.54 27.89 8.0 0.75 0.58 30.43 5.00 78.95 0.23 14.65 0.82 28.71 9.0 0.72 1.07 55.75 5.28 79.17 0.27 24.82 0.73 36.40 Effect of NaCl concentration on the enzymes partitioning 2 Generally the addition of neutral salts, such as NaCl, affects partitioning in ATPS by speeding up phase separation, by influencing the phase potential or by decreasing protein hydrophobicity. Results shown in Fig. 2 2 show that NaCl concentration in the PEG 2000/MgSO 4 system had little effect on β-glucanase partition and extraction, although, it did change slightly the partition coefficient and extraction rate when the concentration of NaCl increased from 0 to 10%. However, the NaCl concentration did have significant effects on the partitioning and extraction of α-amylase and neutral proteases. Partition coefficient and extraction rate for the neutral proteases increased very rapidly when 1% ( w / w ) NaCl was added. The extraction rate reached 97.1% when NaCl concentration was increased to 2% ( w / w ). At these concentrations, NaCl had only slight effects on α-amylase partition coefficient and extraction rate. When the NaCl concentration reached 5% ( w / w ), however, partition coefficients and extraction rates of α-amylase increased significantly and when the NaCl concentration was over 8%, the extraction rate of α-amylase exceeded 80%. This could have been caused by the different partitioning of Na + and Cl − ions between top and bottom phase resulting in a increase of potential difference between the two phases. As a result, adjustments of the NaCl concentration in PEG 2000/MgSO 4 system can be used to efficiently separate the three enzymes studied. E e , E g , E a and E p represent the extraction rate of total protein, β-glucanase, α-amylase and neutral proteases respectively; R represents phase ratio; K e , K g , K a and K p represent the partition coefficient of total protein, β-glucanase, α-amylase and neutral proteases respectively Partitioning of enzymes from whole broth 2 The PEG 2000/MgSO 4 system was used with whole broth to purify β-glucanase, α-amylase and neutral proteases and to separate typical B. subtilis contaminants. Results in Table 4 4 show that the partition behavior of enzymes in whole broth was superior to that obtained from clarified broth. Most of the β-glucanase was partitioned to the top phase (Extraction rate reached 79.6%) and almost all of the α-amylase ( > 99%) and most of the neutral proteases ( > 80%) was partitioned to the bottom phase of whole broth. The specific activity of β-glucanase was 4903 U/mg, 2.66 times that in unpartitioned whole broth. Cells and solid mass were absorbed to the interface but were easily removed by centrifugation. E e , E g , E a and E p represent the extraction rate of total protein, β-glucanase, α-amylase and neutral proteases respectively; R represents phase ratio; K e , K g , K a and K p represent the partition coefficients of total protein, β-glucanase, α-amylase and neutral proteases respectively R K e E e (%) K g E g (%) K a E a (%) K p E p (%) Clarified broth 0.83 0.84 41.03 3.22 72.76 0.196 14.01 0.72 37.28 Whole broth 0.72 0.39 22.08 2.80 79.55 0.047 3.25 0.33 18.95 Purify β-glucanase in two steps 2 The PEG 2000/MgSO 4 system is extremely attractive for purifying β-glucanase in two steps. In the first step, a system with 16% PEG 2000 and 14% MgSO 4 was used, where most of β-glucanase (72.0%) partitioned to the top phase along with some neutral proteases and other contaminants. Almost all of the α-amylase ( > 99%) and some of the neutral proteases ( > 70.00%) partitioned to the bottom phase. Cell debris and other contaminants partitioned to the bottom and absorbed to the interface. The top phase was then repartitioned (back extraction) by adding magnesium sulfate and water. When the MgSO 4 concentration was increased to 14.67%, most of the β-glucanase (82.20%) in the top phase was salted-out and precipitated in the bottom phase. β-glucanase recovery rate and specific activity was 65.34% and 14027 U/mg respectively, 6.6 times higher than that in whole broth. CONCLUSION 1 Based on the experimental results above, it can be concluded that: in PEG/MgSO 4 system, low MW PEG (PEG 2000) is beneficial for purification of β-glucanase; 16% PEG can be used to achieve a high extraction rate of β-glucanase and significantly decrease the proportion of α-amylase and neutral proteases in the upper phase. MgSO 4 concentration should be maintained at 14% to increase the extraction rate of β-glucanase and decrease the contamination by neutral proteases. In order to separate the β-glucanase from the other two enzymes, the pH 7.0, PEG 2000/MgSO 4 system should be used. It was considered that the hydrophobicity difference of the three enzymes caused the differences of NaCl concentration required for the extraction in the upper phase. So adjustment of NaCl concentration in PEG 2000/MgSO 4 system is an efficient way to separate the three enzymes. Partition behaviors of β-glucanase, α-amylase and protease in whole broth are similar to those in supernatant based on the experimental results. Most of the β-glucanase is partitioned to the top phase (Extraction rate reached 79.6%) and almost all of the α-amylase ( > 99%) and most of the neutral proteases ( > 80%) was partitioned to the bottom phase. The two-step process for purifying β-glucanase was established, and β-glucanase recovery was 65.34% and specific activity of β-glucanase was 14027 U/mg. These results indicated that this ATPS provided mild aqueous environments for biological molecules separation. 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Method and Technology of Biochemistry Experiments 10.1631/jzus.2005.B0825 Project (No. 20276064) supported by the National Nature Science Foundation of China 2005-03-17 2005-04-28 2005-07-29 2022-01-05 Copyright © 2005, Journal of Zhejiang University Science 2005 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 08 6B