Simultaneous biosynthesis and purification of two extracellular Bacillus hydrolases in aqueous two-phase systems

Microbiol. Res. (2001) 156, 19–30 http://www.urbanfischer.de/journals/microbiolres

Simultaneous biosynthesis and purification of two extracellular Bacillus hydrolases in aqueous two-phase systems Viara Ivanova1, Dragomir Yankov2, Ludmila Kabaivanova1, Dimitre Pashkoulov3 1 2 3

Institute of Microbiology, Bulgarian Academy of Sciences, 26 Academician G. Bontchev str., 1113 Sofia, Bulgaria Institute of Chemical Engineering, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria Fruit Growing Institute, 4004 Plovdiv, Bulgaria

Accepted: August 6, 2000

Abstract Thermostable α-amylase with temperature optimum at 80°C, molecular mass 58 kDa and pI point 6.9 was purified from a catabolite resistant Bacillus licheniformis strain. The enzyme was sensitive to inhibition by metal ions and N-bromosuccinimide. The partition behaviour of this enzyme in aqueous two-phase systems (ATPS) of the polymer-polymer-water type was investigated and some effects of type, molecular weight and concentration of phase components were studied. Up to 100% retention in the bottom phase of polyethylene glycol 10,000–20,000/dextran 200 system was reached. Best partition conditions were obtained in PEG 10,000–20,000/polyvinyl alcohol 200 systems, where the partition coefficient K increased 750 times to 7.5. Simultaneous production and purification of α-amylase and serine proteinase in PEG-polymer-water ATPS were examined. In the system PEG 6,000/ ficoll, up to 90% of the amylase was retained in the bottom phase, whereas about 95% of the total protein (K = 22.8) and 60–75% of the proteinase were in the top phase. Similar separation of the enzymes from laboratory supernatant was obtained in system PEG/Na2SO4. Key words: hydrolases – Bacillus licheniformis – aqueous two-phase systems (ATPS)

Introduction Aqueous two-phase systems (ATPS) consist of two immiscible aqueous phases, which are polymer solutions or a polymer and a salt solution. In such systems partitioning of different components and biocatalysts Corresponding author: V. Ivanova e-mail: [email protected] 0944-5013/01/156/01-19

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occurs between the phases. They have received wide attention over the last three decades, because separation of whole cells, cell debris and nucleic acids from crude supernatant and initial protein purification could be combined in one step (Veide et al. 1983; Albertsson 1986; Baskir et al. 1989; Andersson and Hahn-Hägerdall 1991; Sikdar et al. 1991; Ariga et al. 1994). Several systems of polymer-polymer-water and polymer-salt-water types have been reported. The polyethylene glycol (PEG)-dextran-water systems and also systems prepared from modified PEG and dextran have received particular attention (Chung et al. 1994; Schmidt et al. 1994). In addition, PEG has been used with inorganic salts such as potassium phosphate, ammonium phosphate and sodium sulphate (Pathak et al. 1991; Schmidt et al. 1994) to generate two aqueous phases. There are some reports on the applicability of other polymers such as pullulan, starch derivatives and polyampholytic acrylic copolymers for aqueous twophase formation (Nguyen and Luong 1990). The studies were predominantly focused on separation and purification of proteins, cells and organelles from fermentation broth (Kroner et al. 1982; Huddleston and Lyddiatt 1990; Andersson and Hahn-Hägerdall 1991; Chen 1992; Yang et al. 1994). Another application of the aqueous two-phase systems has been the extractive bioconversion and cofactor regeneration (Smeds et al. 1983; Mattiasson 1984; Tjerneld et al. 1985; Kaul and Mattiasson 1986; Chen and Wang 1991; Krishna et al. 1991; Kim and Weigand 1992; Wang et al. 1992). Until recently, only investigations on the possibility of producing α-amylase (Andersson et al. 1985 ; Andersson and Hahn-Hägerdal 1988), cellulase (Persson et al. 1984) and protease (Lee and Chang 1990) in ATPS Microbiol. Res. 156 (2001) 1

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have been published. The oxygen supply rate to the aqueous polymer phases is the limiting factor in many aerobic bioprocesses and that could be the reason for the mild interest on simultaneous enzyme production and purification (Van Sonsbeek et al. 1993). However, to our knowledge there are no reports on the simultaneous cultivation and partial purification in aqueous two-phase systems of two hydrolases. Systems, composed of polyethylene glycol (PEG)/dextran, PEG/ficoll, PEG/polyvinyl alcohol (PVA) were examined. A carbon catabolite resistant Bacillus licheniformis 44MB82-G strain growing on glucose as carbon source was used. The strain synthesizes high levels of thermostable α-amylase with a temperature optimum of 90 °C for the native enzyme and a serine alkaline endopeptidase with a temperature optimum for the purified enzyme of 70 °C (Tonkova and Emanuilova 1989; Ivanova 1995 ; Ivanova et al. 1998). The influence of type, concentration and molecular weight (MW) of the phase-forming polymers on enzyme yield and the degree of purification of these two enzymes during cultivation of the Bacillus licheniformis cells in ATPS was studied. We report also on : (1) the partitioning of purified α-amylase in the above mentioned ATPS; (2) the separation and purification of α-amylase and proteinase from a laboratory supernatant in polymer-polymerwater and polymer-salt-water sytems.

Materials and methods Bacterial strain, medium and enzyme purification. Bacillus licheniformis 44MB82-G strain was grown as described previously (Tonkova and Emanuilova 1989) in a nutrient broth supplemented with (in g/l): glucose (60.0); beef extract (Lab-Lemco powder, Oxoid, Basingstoke, Hampshire, UK, 15.0); peptone (Oxoid, 15.0); K2HPO4 (10.4); cornsteep liquor (6.6); CaCl2 (1.1), pH 6.5. This medium was used for amylase production in all experiments performed. The amylase was isolated from the cell-free supernatant by a two-phase separation and ion-exchange chromatography as described previously (Ivanova et al. 1993, 1998 ; Ivanova and Dobreva 1994 ; Ivanova 1995). The homogeneous fractions (on SDS-PAGE) were concentrated to a protein content approx. 2.0 mg/ml. Preparation of phase systems. Aqueous polyethylene glycol (PEG)/polymer two-phase systems with concentration of the phase-forming components 5 – 20% (w/v) were investigated. The PEGs had an average molecular weight from 4,000 to 20,000 (PEGs 4 – 20, respectively; Fluka Chemie AG, Buchs, Switzerland). Dextrans T200 and T500 (average molecular weight 200,000 and 500,000), ficoll 400 (average molecular weight 20

Microbiol. Res. 156 (2001) 1

400,000; Pharmacia Fine Chemicals, Uppsala, Sweden) and polyvinyl alcohol (PVA, average molecular weight 200,000, Fluka) were used as components of the bottom phase. The PEG/inorganic salts (Na2SO4, KH2PO4, Na2HPO4, (NH4)2SO4, MgSO4) systems were prepared with concentrations of the PEG and the salts 10–30%, w/v. 5.0 ml of centrifuged broth (amylase activity 1900 U/ml) or purified amylase solution (1.5 mg) were mixed for 18 h with 20.0 ml stock solutions of the polymers or salts (in 0.05 M Tris/HCl buffer, pH 7.0). Then the mixtures were left for 12 h in graduated tubes for separation of the phases and determining of their volumes. All partition experiments were conducted at room temperature (25°C) and all procedures were performed at pH 7.0 close to the amylase pI-Point 6.9. Samples of the top and bottom phases were assayed for amylase and proteinase activities and total protein after dilution with a buffer solution. When examining the effects of ATPS on enzyme production and separation, the fermentations were carried out at 40°C on a rotary shaker at 240 rpm. K2HPO4 was added to the fermentation medium after the sterilization step. Periodically and at the end of the cultivation (120 h) the phases were separated at 25°C as described and analysed. The partition coefficient K, the amount of formed product X, the recovery R, the phase volume ratio r, the purification factor PF were calculated from the equations: K = Ct/Cb; X = 100 × (Cphase × Vphase)/(Ct × Vt + Cb × Vb), %; R = 100 × (Cphase × Vphase)/(Cadded × Vadded), %; r = Vt/Vb, PF = Specific activityphase/Specific activitystarting, where Ct and Cb were the activity of amylase and proteinase or protein concentration in the top and bottom phases; Vt and Vb – volume of the top and bottom phase; Cphase and Cadded represented the activity of enzymes and protein concentration in the phases and the starting quantity; Vphases and Vadded – Volume of phases and volume of starting solution, Specific activityphase and Specific activitystarting – specific activities of the studied proteins in the phases and in the starting solution. Determination of enzyme activity and physico-chemical properties. Amylolytic activity was assayed by the method of Pantschev et al. (1981) using soluble potato starch (1.0%, w/v, Lintner starch, Serva Feinbiochemica, Heidelberg, Germany, dissolved in 0.066 M phosphate buffer, pH 6.5) as substrate. One unit of activity was defined as the amount of enzyme that produces one microequivalent of anhydrous glucose (0.162 mg) per minute at 30°C. Proteolytic activity was measured by the method of Hagihara et al. (1958) using casein (Fluka) as a substrate

(1.0%, w/v ; dissolved in 0.1 M Tris/HCl buffer, pH 7.0). The reaction was stopped after 30 min by addition of 10% (w/v) trichloroacetic acid. One unit of proteolytic activity was defined as the amount of enzyme, which liberated 1 µmole tyrosine equivalent/min at 60 °C. Protein in the phases was determined according to Bradford (1976) with BSA as the standard. SDS-PAGE was carried out according to Laemmli (1970). Bio-Rad rainbow marker (Bio-Rad Laboratories, Richmond CA, USA) was used as molecular mass standard. An ultrathin IEF was performed on 0.15 mm Servalit precotes 3 –10 at 3 W/1700 V using Serva pI-marker proteins 9. The effect of temperature was studied in the interval 30– 97 °C as previously described (Ivanova et al. 1993). Thermal stability was determined after 10 min of incubation at 80 °C. The pH-optimum and stability were measured using 12.5 µg/ml of purified enzyme per assay (Ivanova et al. 1993) and 0.05 M sodium acetate, sodium/potassium phosphate, Tris/HCl, borate and boric acid/NaOH buffers with pH values from 4.0 to 12.0. The effect of chemical reagents and metal ions (as chlorides) was determined by measuring the residual enzyme activity after 1 h of incubation at 25 °C in 0.066 M phosphate buffer, pH 6.5, containing 1.0 mM of these reagents. The kinetic constants were assayed for soluble potato starch and amylopectin, dissolved in 0.066 M phosphate buffer, pH 6.5 at 30 °C; the substrate concentration was 2.0 –10 mg/ml; the quantity of native amylase – 3.0 U/ml. Maltosaccharides were determined by HPLC (Ivanova and Dobreva 1994 ; Ivanova et al. 1998); the Lineweaver-Burk plot (Dixon and Webb 1979) was used for calculation of Km and Vmax.

Results Purification and partial characterisation of the amylase After the two-phase separation step 85% of the amylase was retained in the bottom fraction. The specific activity of the enzyme increased 1.38-fold. Additional seTable 1. Summary of purification of amylase from Bacillus licheniformis 44MB82-G. Steps

Total Total Specific Yield Purification activity protein activity factor × l0–3 U mg U/mg %

Supernatant 840 Dextran 714 fraction CM700 Sepharose

280.0 172.5

3000 4140

100 85

(1) 1.38

165.0

4250

83

1.42

Fig. 1. Molecular mass determination; staining with Coomassie Brilliant Blue R 250. St – MW markers; G – amylase from Bacillus licheniformis 44MB82-G strain.

paration from other proteins was achieved by ionexchange chromatography. Thus the amylase was purified 1.42-fold with an yield 83% and its specific activity was 4250 U/mg protein (Table 1). Its molecular mass was determined to be 58 kDa (Fig. 1); the isoelectric point was 6.9. The temperature optimum at pH 7.0 was at 80°C (Fig. 2A). At 30°C, the pH optimum was at pH 7.0. The enzyme was stable in pH-interval 7.0–8.0 (Fig. 2B) and retained 80% of its activity after 10 min of incubation at 80°C. The amylase was totally inhibited by Hg+, Sn4+ and Fe2+ ions and N-bromosuccinimide (NBS). Inhibition by EDTA was not determined. The Michaelis-constant (Km) values were determined as 0.9 g/l and 1.2 g/l, amylopectin and soluble starch as substrates, respectively. Partitioning of purified α-amylase in PEG-polymerwater systems In the studied ATP systems the effect of PEG molecular weight on the partition coefficient of purified amylase was assessed by preparing them with PEG of various MW (4,000–20,000). Concentrations of the phaseforming polymers between 5 and 20% (w/v) were tested. The volume of PEG-phase was always (one exception only – PEG/ficoll, 15/20%) larger than the bottom phase volume under the experimental conditions investigated here. The partition behaviour of α-amylase in the studied PEG-polymerwater systems was dependent on the molecular weight of PEG and bottom phase polymers and on their concentration. Values of partition coMicrobiol. Res. 156 (2001) 1

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Table 2. Dependence of the phase volume ratio and partition coefficients from polymer type, concentration and molecular weight in experiments with purified amylase. Bottom phase composition and ATPS concentration (%, w/v)

Top phase composition – PEG molecular weight

Dextran 200, 10/10 Dextran 200, 15/5 PVA 200, 15/5 PVA 200, 20/5 Ficoll 400, 15/15 Ficoll 400, 15/20

3.0 6.7 2.4 1.7 2.0 1.2

4,000

6,000

10,000

20,000

2.0 8.0 1.6 2.5 2.2 1.2

1.3 6.7 1.6 3.5 2.0 0.9

< 0.01 0.16 1.22 7.50 0.17 0.25

< 0.01 0.27 1.19 6.25 0.34 0.57

Phase volume ratio 2.7 6.7 3.3 1.8 2.0 1.5

Partition coefficients Dextran 200, 10/10 Dextran 200, 15/5 PVA 200, 15/5 PVA 200, 20/5 Ficoll 400, 15/15 Ficoll 400, 15/20

Fig. 2. A – Temperature optimum (◆) and thermal stability (■); B – pH-optimum (◆) and pH-stability (■) of amylase from Bacillus licheniformis 44MB82-G strain.

efficient K in PEG/dextran and PEG/ficoll systems were less than one, ranging from <<0.01 to 0.57 (Table 2). These results indicate that a major part of amylase partitioned into the bottom phase within the limits of our experiments. In system PEG/dextran the partition coefficient K decreased from 0.33 to <<0.1 with the increase of PEG 22

Microbiol. Res. 156 (2001) 1

0.17 0.33 1.24 7.50 0.18 0.22

0.15 0.22 2.31 7.50 0.07 0.34

average molecular weight (Table 2). The values of K increased with the increase of PEG and the decrease of dextran concentrations. The values of r (phase volume ratio) were from 1.3 to 8.0 and similar effect of PEG molecular weight was observed. As shown in Fig. 3A–3D, the separation and the recovery were strongly influenced by the PEG MW and dextran concentration. Thus at high PEG (15%) and low dextran (5%) concentrations the amylase partitioning was shifted to the top phase. This effect was clearly demonstrated when using PEG 4,000 – almost 70% of it was retained in the top PEG phase (Fig. 3A). Simultaneous increase of the dextran concentration and decrease of the PEG quantity to 10/10 (%, w/v) led to retention of the amylase in the bottom dextran phase. The increase of PEG molecular weight to 10–20,000 caused a total retention of this enzyme in the dextran phase (Fig. 3C, 3D). The system PEG/PVA was proved to be suitable for enzyme separation when phosphates were not present in the buffers used in order to avoid polymerisation of PVA. In this system the amylase was separated in the top PEG phase. As shown in Fig. 3 at initial concentration of the polymers 20% (PEG) and 5% (PVA) the recovery was not influenced by the PEG molecular weight. Only the phase volume ratios increased with its increase. Almost 95% of the enzyme was retained in the top PEG phase at K = 7.50. Opposite effect of PEG MW on partitioning and recovery in ATP systems prepared with lower PEG quantity – 15% (PEG) and 5% (PVA), was determined. Moreover, the phase volume ratios increas-

ed with the increase of PEG’s molecular weight up to 6,000 and then decreased twice. Efficient separation was reached (K = 2.31, Rtop-89%) when PEG 6,000 was used (Fig. 3B). The optimal molecular weight of PEG in the system PEG/ficoll 400 at composition 15/15% (w/v) was 6,000. Almost 90% of the enzyme was retained in the bottom ficoll phase at K= 0.07 (Fig. 3B ). About 74% of the enzyme was separated in the ficoll phase when PEGs 4,000 and 10,000 were used (Fig. 3A, 3C). The partition coefficient was in the range of 0.17–0.18. Similar results were obtained also with these PEGs at phase composition 15/20% (w/v). In the experiments with PEG 6,000, the separation was better at polymer concentration 15/15% than at 15/20% (K–0.34; Fig. 3B). The PEG 20,000 was proved to be not suitable for separation in combination with ficoll and the enzyme distributed equally in both phases (Fig. 3D). No significant differences in the phase volume ratios, depending on the MW of the PEG used, were obtained. Partitioning of laboratory supernatant in PEG-salt-water systems

Fig. 3. Effect of the system composition and PEG molecular weight on the recovery R of purified amylase. ATP system 1: – 15% PEG/5% dextran 200; 2: – 10% PEG/10% dextran 200; 3: – 15% PEG/5% PVA; 4: – 20% PEG/5% PVA; 5: –15% PEG/15% ficoll 400; 6: – 15% PEG/20% ficoll 400.  – top and  – bottom phases

In order to assess the separation performance of PEG-salt- water systems, the effects of some parameters (salt type and concentration, PEG average molecular weight and concentration) on the partition coefficients of amylase, proteinase and total protein from laboratory supernatant were investigated. In such systems phase separation can be achieved more rapidly than in polymer-polymer-water systems since the viscosities of the phases are lower. The partition coefficients for α-amylase in PEG/salt systems were dependent from the PEG molecular

Table 3. Influence of PEG molecular weight and salt type on partition behaviour of proteins from laboratory supernatant. System 20% PEG/ 20% Na2SO4

Influence of PEG’s molecular weight

Purification factors

Volume

Partition coefficients

Amylase

ratio

Amylase

Proteinase

Protein

Top

Bottom

Top

Bottom

PEG 4,000 PEG 6,000 PEG 10,000 PEG 20,000

0.7 0.6 0.5 0.7

4.67 2.45 2.30 4.29

12.05 29.75 14.75 20.40

8.13 8.40 6.00 7.50

0.75 0.65 0.73 0.88

1.32 2.00 1.90 1.54

1.23 0.84 1.05 1.13

0.83 0.21 0.43 0.42

System 20% PEG 4,000/ 20% salt

Influence of salt type

Purification factors

Volume

Partition coefficients

Amylase

ratio

Amylase

Proteinase

Protein

Top

Bottom

Top

Bottom

0.7 1.8 1.7 1.2 0.6

4.67 2.90 1.76 1.00 0.87

12.05 4.67 6.00 1.76 4.56

8.13 5.00 1.64 0.44 0.15

0.75 1.02 1.02 3.17 1.50

1.32 1.75 1.81 1.43 2.00

1.23 0.75 1.04 1.89 1.41

0.83 0.80 0.28 0.48 0.36

Na2SO4 KH2PO4 Na2HPO4 (NH4)2SO4 MgSO4

Proteinase

Proteinase

Microbiol. Res. 156 (2001) 1

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Fig. 4. Effect of ATP system composition and concentration on the recovery R of amylase, proteinase and total protein from laboratory supernatant. (A) PEG-salt-water system 1: – 10% PEG 4,000/15% Na2SO4 ; 2: – 20% PEG 6,000/20% Na2SO4 ; 3: – 20% PEG 4,000/20% (NH4)2SO4 ; 4: – 20% PEG 4,000/20% Na2HPO4 ; 5: – 20% PEG 4,000/20%MgSO4. (B) PEG-polymer-water system 1: – 10% PEG 4,000/10% dextran 200; 2: – 10% PEG 10,000/10% dextran 200; 3: – 15% PEG 4,000/20% ficoll 400; 4: – 15% PEG 6,000/20% ficoll 400. – Amylase in top phase (T); = amylase in bottom phase (B); – proteinase in (T); – proteinase in (B); – total protein in (T); – total protein in (B).

weight (Table 3). K decreased with the increase of PEG molecular weight from 4,000 to 10,000. The decrease in K in the MW range 4,000 – 6,000 was more marked. The amylase recovery R in the top phase reached from 50% 24

Microbiol. Res. 156 (2001) 1

(PEG 4,000; Fig. 4A) to 80% (PEG 10,000) of the added activity (data not shown). However, the proteinase and the other proteins showed higher values of K (up to 30), which resulted in poor separation and purification (recovery in the top phase from 70 to 90% 5 Fig. 4A). On the base of these results the next experiments were carried out using PEG 4,000. Higher partition coefficients for amylase and total protein were determined with Na2SO4 as phase-forming component (Table 3). Its replacing with other salts markedly decreased the partition coefficients to values lower than 1 (K = 0.87 and 0.15 in system with MgSO4, K = 1.0 and 0.44 in system with (NH4)2SO4). Simultaneously, in these systems a satisfactory separation of the studied hydrolases was achieved (Fig. 4A). However, the results for recovery of total protein and proteinase showed that in these sulphate systems the separation was influenced by the protein concentration and precipitation of the protein out of the solution occurred. Nevertheless, the bigger part of amylase (MgSO4) and total protein ((NH4)2SO4) was retained in the bottom phase (Fig. 4A). Increasing the PEG concentration in the PEG 4,000/Na2SO4 system caused an increase (3.1-fold) in the partition coefficient for proteinase (Table 4). Significant changes of K for amylase and total protein were not observed. The protein partitioning was strongly dependent on the salt concentration (respectively ionic strength). All proteins partitioned in the top phase, the separation of the amylase from the proteinase and the contaminants was not satisfactory, even at a good value of the ratio between the partition coefficients (K amylase/K proteinase > 100). At low Na2SO4 concentration (15%), the enzymes partitioned in different phases. The amylase was retained in the bottom phase (55%), the proteinase and the total protein – in the top phase (64% and 68%, respectively, Fig. 4A). For PEG 4,000 (10–15%) and Na2SO4 10% phase formation was not observed. The salt and PEG type and concentration influenced the phase volumes. An effect of the Vt and Vb on the purification of the studied proteins was observed. At Vt < Vb, the purification factors for amylase in the bottom phase were 1.3–2.0 and that of proteinase in the top phase 1.0–1.4. At Vt > Vb, the purification factors were similar and the amylase distributed in both phases. Partitioning of laboratory supernatant in PEG-polymerwater systems The partition coefficients of amylase decreased to values lower than 1 when the polymer-salt-water systems were replaced with polymer-polymer (dextran or ficoll)-water systems. In systems PEG/ficoll 400 and PEG/dextran

Table 4. Influence of PEG 4000 and Na2SO4 concentration on partition behaviour of proteins from laboratory supernatant. System 10% PEG 4,000/ Na2SO4

Influence of PEG concentration

Purification factors

Volume ratio

Amylase

20% PEG 25% PEG 30% PEG

2.4 2.8 3.1

System 10% PEG 4,000/ Na2SO4

Influence of salt concentration Volume ratio

15% Na2SO4 20% Na2SO4 25% Na2SO4 30% Na2SO4

0.4 0.3 0.3 0.2

Partition coefficients Amylase 0.76 0.61 0.59

Proteinase

Proteinase

Protein

Top

Bottom

Top

Bottom

1.25 1.25 1.46

1.07 0.85 0.70

1.75 1.75 1.70

0.51 1.03 1.10

0.52 0.33 0.43

1.23 3.78 3.70

Purification factors

Partition coefficients

Amylase

Proteinase

Amylase

Proteinase

Protein

Top

Bottom

Top

Bottom

1.15 9.56 20.03 270.10

7.32 14.08 17.89 19.78

4.79 10.0 23.50 33.33

0.40 0.35 0.25 0.29

1.68 0.37 0.35 0.15

0.93 0.76 0.46 0.59

0.61 0.54 0.71 1.00

In system PEG 4,000/Na2SO4 and at concentration 10/10% and 15/10% (w/v), two-phases were not formed. Table 5. Partition coefficients as function of phase-forming polymer concentration, type and molecular weight in experiments with laboratory supernatant. ATP System

Partition coefficients K in PEG/polymer ATPS

%, w/v

A

P

Pr

A

P

Pr

A

P

Pr

A

P

Pr

1; 10/10 1; 15/5 2; 15/15 2; 15/20

0.19 0.35 0.28 0.03

1.17 1.40 0.60 1.00

0.42 0.71 0.18 0.39

0.16 0.98 0.38 0.27

1.14 1.17 0.71 1.27

0.50 0.15 0.36 0.28

0.07 0.16 0.54 0.74

0.93 0.97 1.63 1.05

0.62 0.33 0.15 0.40

0.18 0.36 0.17 0.66

0.85 0.81 0.50 1.00

0.50 0.28 0.23 0.57

PEG 4,000

PEG 6,000

PEG 10,000

PEG 20,000

1, system PEG/dextran 200; 2, sytem PEG/ficoll 400; A, amylase; P, proteinase; Pr, total protein

200, the PEG molecular weight did not affect markedly the partition coefficients K for α-amylase, proteinase and contaminating proteins (Table 5). The phase volume ratio did not affect the partition coefficients and the purification factors for α-amylase (Tables 5, 6). The purification factors were similar and both enzymes partitioned in the top phase when the systems contained 15% PEG 4,000 –10,000/15% ficoll 400 and 15% PEG 10,000 –20,000/20% ficoll 400. As shown in Table 6 and Fig. 4B, the enzymes were separated in different phases when systems were formed from 15% PEG 4,000 – 6,000/20% ficoll 400 and 15% PEG 20,000/15% ficoll 400. Amylase recovery in the bottom phase reached 70 – 95%; the proteinase recovery in the top phase – 35 – 65%. The ratio of the partition coefficients for α-amylase to that for total protein was 8.8 (PEG 10,000/dextran) and 13 (PEG 4,000/ficoll; Table 5). Since the total protein partitioned to the bottom phase, the separation of α-amylase from the contaminants was not adequate, even though a good value of the

ration between the partition coefficients was obtained (Table 6). The values of K for amylase increased (1.8–6.1-fold) and in most cases the values of K for contaminating proteins decreased (1.7–3.3-fold) with the increase of PEG concentration in PEG/dextran 200 systems. Better combination of purification factors and partition coefficients for amylase (separated in bottom phase) and proteinase (retained in top phase) was achieved in systems composed from 10% PEG 4,000 or 10,000 and 10% dextran 200; and from 15% PEG 4,000–6,000 and 20% ficoll 400 (Table 6). However, as seen from Fig. 4B, the recovery of total protein in bottom phase was approx. 30–40%, corresponding to 70–90% recovery of the amylase in the same phase. From the view point of α-amylase, about 50–60% of the contaminants were eliminated using systems PEG 4,000/ficoll 400 (15/20, % w/v) and PEG 10,000/dextran 200 (10/10%). Their elimination reached 70–75% in the case of proteinase in system PEG Microbiol. Res. 156 (2001) 1

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Table 6. Purification factors and phase volume ratio as function of phase-forming polymer concentration, type and molecular weight in experiments with laboratory supernatant. ATPS composition

Purification factors in PEG/polymer ATPS PEG 4,000

PEG 6,000

PEG 10,000

PEG 20,000

%, w/v

A

P

A

P

A

P

A

P

1; 10/10

2.29 (B) 1.50 (B)

1.64 (T) 1.58 (T)

1.70 (B) 3.92 (T)

1.50 (T) 3.96 (T)

2.41 (B) 1.79 (B)

1.46 (T) 1.54 (T)

1.67 (B) 1.24 (T)

1.45 (T) 1.49 (T)

2.70 (T) 2.52 (B)

3.08 (T) 3.07 (T)

2.43 (T) 2.16 (B)

3.21 (T) 3.93 (T)

2.64 (T) 3.12 (T)

3.84 (T) 3.52 (T)

1.69 (B) 2.30 (T)

2.48 (T) 2.17 (T)

1; 1515 2; 15/15 2; 15/20

Phase volume ratio PEG 4,000

PEG 6,000

PEG 10.000

PEG 20,000

1; 10/10 1; 1515

1.8 7.1

1.9 6.5

1.9 5.7

1.9 6.1

2; 15/15 2; 15/20

8.8 1.0

2.0 1.1

2.0 1.5

1.9 1.5

1, system PEG/dextran 200; 2, sytem PEG/ficoll 400; A, amylase; P, proteinase; (T) and (B), top and bottom phases. Table 7. Biosynthesis, partition coefficients, phase volume ratio and purification factors in ATPsystems (PEG 6,000 as top phase). ATPS composition top/bottom, %

Biosynthesis Amylase

Partition coefficients Proteinase

Protein

AmyIase

Proteinase

Protein

0.08 0.04 0.10 0.08 0.07 0.09

0.48 0.51 0.35 0.57 1.74 1.52

0.09 0.08 0.10 0.06 21.50 22.80

Phase volume ratio

as % from the control experiments 10/5 Dextran 200 15/5 Dextran 200 10/10 Dextran 200 15/5 Dextran 500 15/15 Ficoll 400 15/20 Ficoll 400

78.0 58.0 63.0 66.0 48.0 41.0

68.0 59.0 117.0 76.0 88.0 73.0

78.0 73.0 55.0 62.0 110.0 73.0

2.3 1.6 3.6 4.0 1.6 1.0

Purification factors* Amylase

10/5 Dextran 200 15/5 Dextran 200 10/10 Dextran 200 15/5 Dextran 500 15/15 Ficoll 400 15/20 Ficoll 400

Proteinase

Top phase

Bottom phase

Top phase

Bottom phase

0.91 0.96 0.50 1.20 0.11 0.08

1.02 1.02 1.06 0.96 32.5 22.7

3.0 2.07 4.05 3.48 0.76 0.64

0.57 0.61 0.61 0.38 9.43 9.57

* Calculated on the base of total enzyme quantity.

4,000 – 6,000/ficoll 400. In the same time, both enzymes were purified from 70 – 75% of the contaminating proteins in the top phase when the systems contained PEG 6,000 and dextran 200 (15/5, % w/v); PEG 6,000 and ficoll 400 (15/15%); PEG 10,000 and ficoll 400 (15/20%). 26

Microbiol. Res. 156 (2001) 1

Production and separation of enzymes in aqueous twophase systems Production and simultaneous partition of hydrolases in two-phase batch fermentations was tested in systems PEG/dextran and PEG/ficoll. High viscous two-phase systems with PEGs 10–20 were not investigated be-

Fig. 5. Separation of the formed products in top and bottom phases. ATP system PEG 6,000/dextran 200; 1: – 10% PEG/5% dextran 200; 2: – 15% PEG/5% dextran 200; 3: – 10% PEG/10% dextran 200; 4: – 15% PEG/5% dextran 500; 5: – 15% PEG/15% ficoll 400; 6: – 15% PEG/20% ficoll 400. – Amylase in top phase (T); – amylase in bottom phase (B); – proteinase in (T); – proteinase in (B); – total protein in (T); – total protein in (B).

cause oxygen mass-transfer limitations and respectively lower biosynthesis could be expected (Persson et al. 1984). An intermediate value of PEG molecular weight was chosen (6,000). PVA was not used as component of ATPS because of the high potassium phosphate concentration in the fermentation medium and the growth temperature (40°C). In such system, immobilization of the Bacillus licheniformis cells could be obtained. The partition of proteins in ATPS depends on various factor such as the ionic composition, pH, size of protein molecules, concentration and molecular weight of the phase-forming polymers. The accumulation of Bacillus

licheniformis cells and their lysis, medium components (phosphates, calcium ions), change of pH from 7.0 to 8.5–9.5 at the fermentation end and the production of various metabolites by the cells during the process are part of the factors, affecting the partition behaviour of the studied enzymes. The use of two-phase systems for cultivation of Bacillus licheniformis 44MB82-G cells affected the biosynthetic abilities of the cells. Effects of the molecular weight, the type of the bottom phase and the concentrations of phase-forming polymers were proved. Two-phase systems produced less hydrolases and protein than the control cultivations. Better amylase production was obtained in systems with dextran as component of the bottom phase. When the bottom phase was composed from ficoll 400, the yield represented 50% of that, obtained from the control cells. lt has been proved (Kim and Weigand 1992) that the presence of PEG inhibits growth and thus the amylase and proteinase production. From the view point of amylase synthesis, the optimal concentrations and the bottom phase composition were established to be 10/5 (%, w/v) and dextran 200 (Table 7). The production of total protein was from 55 to 110% of the control. An increase of the proteinase yield related to the increase of the bottom phase molecular weight (dextran 500 and ficoll 400) was observed. The proteinase production was influenced also by the concentration of phase-forming polymers. Thus at equal polymer concentration (10%PEG/10% dextran 200, 15% PEG/15% ficoll) the proteinase yield reached 117% of the control (Table 7). Relatively poor separation of the amylase from the proteinase and other contaminants in the PEG/dextran 200 systems was observed. The proteinase was distributed equally into both phases (Fig. 5). The cells, spores, debris and proteins were retained principally in the bot-

Table 8. Biosynthesis, partition coefficient and partition of formed products as function of fermentation time and phase-forming polymer concentration in ATPS composed from PEG 6,000 and dextran 200. ATP System

Time

Biosynthesis

A

P

Partition coefficients

Partition of products formed (top/bottom)

Pr

A

P

Pr

A

P

Pr

% (w/v)

h

as % from the control experiments

10/5

72 96 120

62.0 58.0 78.0

50.0 73.0 68.0

65.0 79.0 78.0

0.33 0.09 0.08

0.76 0.52 0.48

0.35 0.14 0.09

44/56 18/82 16/84

64/36 55/45 53/47

45/55 25/75 18/82

15/5

72 96 120

27.0 44.0 58.0

26.0 46.0 59.0

49.0 72.0 73.0

0.03 0.10 0.10

0.19 0.38 0.35

0.17 0.13 0.10

9/91 27/73 26/74

40/60 58/42 55/45

37/63 32/68 27/73

A, amylase; P, proteinase; Pr, total protein. Microbiol. Res. 156 (2001) 1

27

tom phase. Better were the results in PEG/dextran 500 system. About 75% of the amylase was separated in the bottom phase and 70% of the proteinase – in the top phase. Nevertheless only the proteinase was purified from 2 to 4 times (Table 7). Values of K were less than one – from 0.04 to 0.1 for amylase and total protein and from 0.35 to 0.57 for the proteinase. Higher PEG/ dextran ratio resulted in lower phase volume ratio and was found to influence the biosynthesis, the partition of the amylase and the protein in the phases, but not their partition coefficients (Table 7). Best partition conditions were found in PEG/ficoll system as a combination of very low partition coefficients of amylase and the tendency of the proteinase, cell debris and contaminating proteins to partition to the top PEG phase at both polymer concentrations examined. Up to 90% of the amylase (Fig. 5) were retained in the bottom phase at an extremely higher degree of purification – the enzyme was concentrated up to 32 times. About 70% of the proteinase were separated in the top phase. Nevertheless its purification was unsatisfactory because up to 90% of the total protein was also retained in this phase (Table 7). The production of these hydrolases in ATP system PEG 6,000/dextran 200, the partition coefficients and the partition of products formed (top/bottom phase) are compared on Table 8 as a function of the cultivation time and of the concentration of phase-forming polymers. After 5 days (120 h) of cultivation of Bacillus licheniformis cells in fermentation medium containing 10% PEG and 5% dextran, about 84% of the amylase were determined in the bottom phase. The amylase quantity increased in the dextran phase from the 72 to the 120 hour with the decrease of the partition coefficient from 0.33 to 0.08. Similar were the results for the total protein in the two studied systems. Different situation was observed in the second system – 15% PEG/5% dextran 200, where the amylase quantity decreased from 90 (72 h) to 74% (120 h) in the bottom phase with the increase of partition coefficient. In both systems studied, an equalisation of the proteinase concentration in the phases was observed. This tendency was from top to bottom in the 10%/5% system and from bottom to top phase in the 15%/5% system. As it was demonstrated earlier (Kim and Weigand 1992), the presence of PEG inhibits cell growth and this fact might explain the lower production of amylase and proteinase during the two-phase cultivation (Table 8).

Discussion The protein partitioning in two-phase aqueous polymer systems depends on protein size, conformation, surface structure, the interaction of salts with the proteins and 28

Microbiol. Res. 156 (2001) 1

the phase polymers, the interaction of the polymer chains with the proteins. However, no model of the two-phase partitioning has yet been developed which allows the a priori calculation of protein partitioning for a wide range of phase polymer molecular weights and polymer and salt concentrations. It was demonstrated that the molecular weight of phaseforming polymers influences the composition of the phases and the polymer-protein interactions (Baskir et al. 1989) and therefore influences the partitioning. In systems PEG/dextran the values of partition coefficient K decreased with the increase of their average molecular weights and concentrations (Albertsson 1986; Hayashida et al. 1990). It has been shown that increasing the molecular weight of one of the phase polymers will cause partition of the blomaterial more strongly into the other phase (Albertsson 1986; Baskir et al. 1989). This study has demonstrated that the partitioning of thermostable α-amylase from Bacillus licheniformis 44MB82-G strain could be carried out successfully in the studied ATPS of polymer-polymer-water type. The value of K for α-amylase was dependent on the molecular weight of PEG in PEG/dextran systems. The lower PEG molecular weight resulted in smaller enzyme quantity in the bottom phase. In systems 10% PEG 10–20,000/10% dextran 200 the amylase was retained totally in the dextran fraction. The system PEG/PVA was proved to be suitable for separation of this enzyme in the top PEG phase at K = 7.50. The molecular weight of the PEGs used was not an affecting factor on the recovery. The best results for PEG/ficoll system were obtained when PEG 6,000 and phase concentration ratio 15/15 (%, w/v) were applied. More than 90% of the enzyme was retained in the top PEG phase. The optimal molecular weight of the PEG in the system PEG/ficoll was proved to be 6,000 at equal polymer concentration – 15%. At higher ionic strengths in polymer-salt-water systems the partitioning shows a strong dependence on the salt concentration. Two-phase systems may be produced by combining PEG with Na2SO4 and MgSO4. The present study demonstrates the feasibility of using polymer-polymer-water two-phase systems for separation and partial purification of hydrolases from laboratory supernatant. Simultaneous cultivation of Bacillus licheniformis 44MB82-G cells, synthesis of thermostable amylase and proteinase and purification in a PEG-polymer aqueous two-phase system were proved to be possible. The enzymes were produced and extracted at a high purity in one step. In the system 10% PEG/10% dextran 200 more than 90% of the amylase was retained in the bottom phase. The proteinase was separated between the phases. A better separation of these enzymes was obtained in system using dextran 500 (with higher molecular

weight) – 76% of the amylase were extracted in the bottom phase and 70% of the proteinase-in the top phase. The cells, spores and cell debris were retained in the bottom phase. The replacement of the dextran with ficoll was successful. Similarly to the separation of the purified enzyme, 90% of the synthesised amylase was retained in the bottom (ficoll) phase and 60 – 75% of the proteinase was in the top (PEG) phase depending from the polymers concentration. In these system the cells, spores and cell debris were recovered in the top phase. The bottom phase consisted from ficoll, amylase and a little quantity of proteinase, but the concentration of other proteins was very low (< 4% of the total amount). The use of two-phase systems for cultivation of Bacillus licheniformis 44MB82-G cells affected the biosynthetic abilities of the cells. From the view point of amylase synthesis, the optimal ratio of polymer concentrations was 10% PEG 6,000/5% dextran 200. The obtained amylase yield was lower than the respective yield from the control batch fermentations. Better amylase production was obtained in the systems with dextran as component of the bottom phase. When the bottom phase was composed from ficoll 400, the yield was about 50% lower than the obtained in the control experiments. The proteinase production was influenced by the ratio between PEG and bottom phase-forming polymer concentration. In these systems, the increase of the top phase led to decrease of hydrolase production and together with the lower aeration rate (as a result from the higher viscosity of the two-phase systems) is the mean reason for the lower biosynthetic activity of Bacillus licheniformis cells. The study has also shown that only some of the characteristics of this α-amylase from Bacillus licheniformis 44MB82-G strain seem to be similar to those of the thermostable amylase from Bacillus licheniformis 44MB82-A strain (Ivanova et al. 1993 ; 1998) – equal molecular masses and pI-points, inhibition by NBS. Nevertheless, some clear distinctions were determined (1) – the pH- and temperature optima were different; (2) – amylase G was resistant to inactivation by EDTA and more sensitive to inhibition by metal ions, (3) – amylase G was more stable in absence of calcium and (4) – this enzyme had a greater affinity to amylopectin – Km 0.9 g/l compared to 2.0 g/l for amylase A (Ivanova et al. 1998).

Acknowledgements This work was supported in part by a Project grant MU10B from the National Scientific Foundation, Bulgarian Ministry of Science and Education. We also thank Dr. Tonkova from the Institute of Microbiology of the Bulgarian Academy of Sciences for kindly supplying the bacterial strain, consultations and discussions.

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