Chapter 3 Materials and methods
Low heat skim milk powder (SMP) was supplied by Synlait Milk Ltd., Rakaia, New Zealand. Natural calf rennet [Strength, 280 International Milk-clotting Units (IMCU) mL-1] was obtained from RENCO New Zealand Laboratory (RENCO New Zealand, Eltham, New Zealand). Tamarillo fruit, including the red cultivar Laird`s Large tamarillo and the yellow cultivar Amber tamarillo, were purchased from a farm in Maungatapere, New Zealand.
All chemicals used in this thesis were of analytical grade, while the acetonitrile and trifluoroacetic acid were of HPLC grade.
Milk sample preparation
Preparation of reconstituted milk samples
Reconstituted skim milk was obtained by mixing skim milk powder with Milli-Q water to obtain a desired total solid concentration (e.g. 10%, 11.25% and 20% (w/w)). The mixtures were stirred using a magnetic stirring for at least 2 h at room temperature to ensure dispersion. The mixtures were kept in a 4°C fridge overnight to ensure ful hydration. Prior to use, the reconstituted milks were left at room temperature for at least 4 h to ensure temperature equilibration to room temperature.
pH adjustment of the reconstituted milk samples was achieved by the addition of glucono-δ-lactone (GDL) known to achieve accurate acification. GDL was mixed with the milk samples by magnetic stirring for 2 min to ensure full dissolution. Table 3.1 reports the amounts of GDL added to 10% (w/w) reconstituted skim milk to achieve the desired pH values.
Preparation of milk sample for ultra-small-angle neutron scattering and small-angle X-ray scattering.
Milk samples for ultra-small-angle neutron scattering (USANS) and small-angle X-ray scattering (SAXS) were prepared by mixing skim milk powder with deuterium oxide (D2O). D2O instead of H2O was used because it allowed a better contrast for USANS measurements. The mixtures were stirred using magnetic stirring for at least 2 h at room temperature. The mixing was performed in a container that was sealed by para-film to minimise D2O degradation. The mixtures were kept at 4°C overnight to ensure completely hydration. The mixtures were equilibrated to room temperature for at least 4 h before use.
Sodium caseinate preparation
The method for the preparation of sodium caseinate was adapted from Lucey et al. (2000). Sodium caseinate was obtained from 10% (w/w) reconstituted skim milk as prepared in Section 3.2.1. The milk was acidified to pH 4.6 by adding 2 M HCl at room temperature. The formed curd was separated from the whey using two layers of cheesecloth. The filtered curd was washed five times with Milli-Q water and dewatered using two layers of cheesecloth. The washed curd was re-dissolved with Milli-Q water in a 1:1 mixture, and the pH of the mixture was adjusted to approximately 6.8 by 2 M NaOH with gentle magnetic stirring. Sodium caseinate solution was lyophilized using a freeze-drier (Labconco Corporation, Missouri, USA).
Preparation of crude protease extract from tamarillo fruit
Laird’s Large or Amber tamarillo fruit (300 g) were homogenized to break the plant cells and release the protein into solution using a Polytron PCU2 laboratory homogeniser (Brinkmann Instruments, Luzern, Switzerland). The filtrate (~85 mL) was filtered through two layers of cheesecloth to remove the insoluble material, and then mixed with 80 mL 0.05 M sodium citrate buffer (pH 5.5) to electricaly charge the proteins. The mixture was centrifuged at 15,000 ×g at 4°C for 20 min in a Sorvall LYNX superspeed centrifuge (Thermo Fisher Scientific, USA) with a Fiberlite rotor (F12-6×500 LEX, Thermo Fisher Scientific, USA). The supernatant was collected and the precipitate was discarded.
Ammonium sulphate precipitation and dialysis
Ammonium sulphate was used to salt out the proteins because of its relatively high solubility in water. According to the method of Burgess et al. (2009), 61.69 g of ammonium sulphate was slowly added to 155 mL of the supernatant to make a 65% saturated ammonium sulphate solution using a magnetic stirrer to mix gently for 20 min. The solution was held in an ice bath overnight to allow the proteins to precipitate. The precipitated proteins were collected by centrifuging at 15,000 × g at 4°C for 20 min and then re-dissolved in 30 mL 0.05 M citrate buffer pH 5.5. One volume of protein solution was dialysed against at least five volumes of citrate buffer using a cellulose dialysis tube (molecular weight cut-off of 12,000 Da) to remove ammonium sulphate salts at 4°C for 24 h with at least three changes of the buffer solution. Tamarillo crude extract solution was stored in a -80°C freezer.
Purification of tamarillo protease
The method of purification of the tamarillo protease was based on the purification of actinidin from kiwifruit (McDowall, 1970; Aminlari et al., 2009). Ion-exchange chromatography, both cation and anion, is frequently used in the separation and purification of proteins and polypeptides. For anion ion-exchange chromatography, the gel matrix beads are linked to diethylaminoethanol (DEAE) groups, imparting positive charges to the resin. This allows proteins or peptides with negative charges to adsorb to the resin. Conversely, the resin of cation exchange chromatography can be derivatised with carboxymethyl (CM), which contains negative charge groups, and can therefore attract positively charged proteins (as shown in Figure 3.1).
DEAE-Sepharose fast flow (GE Healthcare Life Sciences, New Zealand) was used to purification the tamarillo proteases. The resin was packed into a 22 × 5 cm column and equilibrated overnight with 0.05 M sodium citrate buffer pH 5.5. Tamarillo crude extract (40 mL), with a protein concentration of 1.17 ±0.03 mg/mL, was applied to the column and the flow rate was set at 1 mL/min. The separation of proteins was achieved using a 200 mL 0.0 – 1.0 M linear gradient of sodium chloride in citrate buffer. Proteins with the weakest negative charges were eluted first from the column at low concentrations of NaCl. A higher concentration of salt was required to elute proteins which have a stronger ionic interaction with the resin (Figure 3.1). 3 mL fractions of eluate solution were collected in 15 mL centrifuge tubes. The absorbance of each fraction was measured at 280 nm using an UVmini-1240 spectrophotometer (Shimadzu Corporation, Australia). The protease activity was assayed as in Section 3.8.1. Fractions corresponding to high activities were pooled and dialysed against Milli-Q water for 24 h at 4°C with at least three changes of the dialysis solution. The purified enzymes were lyophilised by freeze-drying and stored at -80°C until further use.
Determination of protein content
The concentration of protein was determined by the Bradford method (Bradford, 1976). The method relied on the binding of the Coomassie brilliant blue G-250 to the proteins, and the measurement of the dye-protein complex at 595 nm by an UVmini-1240 spectrophotometer (Shimadzu Corporation, Australia). Protein solution (0.1 mL) was added to test tubes containing 3 mL of Bradford reagent (Sigma-Aldrich, New Zealand) then vortexed for 30 seconds. The mixture was incubated at room temperature for 20 min. A blank was prepared by mixing 0.1 mL Milli-Q water with 3 mL Bradford reagent. Bovine serum albumin (BSA) was used to obtain a calibration curve with concentrations of 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 mg/mL protein.
Determination of bovine milk hydrolysed by rennet and tamarillo extract
Bovine milk sample preparation for electrophoresis was adapted from Jin and Park (1996) with minor modifications. Calf rennet (6 μL, 10 times diluted) and tamarillo crude extract (0.7 mL of a 1.17 ±0.03 mg/mL protein extract) were added to 10% (w/w) skim milk (6 g) and 11.25% (w/w) skim milk (6 g) respectively. Both were mixed using a magnetic stirring for 2 min to make a final concentration of 10% (w/w) skim milk samples. Aliquots of the mixed solutions (200 μL) were transferred into glass vials then the lids were closed. The glass vials were incubated at 35°C in an oven (Function Line incubator, Heraeus, Langenselbold, Germany) and removed at different times (0 min, 15 min, 1 h and 24 h). The glass vials containing hydrolysed milk were then heating in a water bath at 100°C to inactivate the enzymes. To each glass vial 400 μL 8 M urea buffer pH 8 was added to disrupt any non-covalent protein bonds, and to solubilise the proteins. Then the samples were kept at 4°C until electrophoresis analysis.
Determination of protease activity
Standard protease assay
Protease activity was measured using the method of Kunitz (1947) with some modifications. Bovine casein was used as the substrate. Enzyme (100 μL) was mixed with 1.1 mL of 0.1 M Tris-HCl buffer (pH 7) containing 1% (w/v) casein, and vortexed for 30 seconds to ensure full dispersion. The reaction was performed at 35°C for 20 min in a water bath. It was then stopped by adding 1.8 mL 10% (w/v) trichloroacetic acid (TCA), and the mixture was allowed to stand for 30 min at room temperature. Mixtures were centrifuged at 10,300 × g for 20 min in a Multifuge 3S-R centrifuge (Thermo Electron Corporation, Germany), then the absorbance of the supernatants was measured at 280 nm against a blank using a UV-Vis spectrophotometer (UV mini-1240, Shimadzu, Japan). The blank was prepared by adding TCA with enzyme solution first to inactive enzyme, then casein solution was added. One unit of protease activity was defined as the amount of enzyme that liberated 1 μg of tyrosine per mL in 1 min under the assay conditions. A standard curve was made using 0-100 mg/L tyrosine (Hadj-Ali et al., 2007). Enzyme activity was defined as units/mg of protein.
Temperature optimum and stability
The effect of temperature on the enzyme activity was studied using casein as a substrate. The optimal temperature of the protease was determined using the standard enzyme assay (Section 3.8.1) over a temperature range from 0 to 100°C for 20 min. To investigate the protease temperature stability, enzyme was pre-incubated at temperatures of 0 to 100°C (at 10°C intervals) for 20 min, then the 1% (w/v) casein solution (same as in Section 3.8.1) was added and the standard enzyme assay performed under optimal pH and temperature conditions. The residual activity of enzyme after pre-incubation were express as a percentage of the control, which was the enzyme activity measured under optimal condition.
pH optimum and stability
The optimal pH value for the protease activity was studied using 50 mM buffers of different pH ranging from pH 6 to 14 at the optimal assay temperature. Preparation of buffers (Table 3.2) was based on the method of Attri et al. (2015) and the pH was measured using an Orion 320 PerpHecT pH meter connected to a model Orion 9106BNWP pH electrode (Massachusetts, USA).
Measurement of the pH stability of the protease was performed by pre-incubating 100 µL purified enzyme with 150 µL of the different pH buffers at 25°C for 20 min, then the remaining activity of the enzyme was detected under optimum assay condition. The results of residual enzyme activities were reported as a percentage of the control.
Effect of organic solvents on protease activity
Protease (100 μL) was mixed with different organic solvents (25% (v/v)) including isopropanol, methanol, ethanol, glycerol, dimethyl sulfoxide (DMSO), and chloroform. The mixtures were incubated at 25°C for 20 min. The residual activity of protease was then determined and compared to that of the control without organic solvent.
Inhibitors and effect of metal ions on protease activity
The enzyme inhibitors phenylmethylsulphonyl fluoride (PMSF, a serine protease inhibitor), p chloromercuribenzoic acid (PCMB, a cysteine protease inhibitor) and ethylenediaminetetraacetic acid (EDTA, a metalloprotease inhibitor) were aded to the standard assay at concentrations of 1 mM and 5 mM. The enzyme was pre-mixed with the inhibitors and incubated at room temperature for 20 min prior to the enzyme assay (Section 3.8.1).
The effect of different chloride salts on protease activity, which included Na+, Hg2+, Zn2+, Co2+, Ca2+, Mn2+, Mg2+ (1 mM and 5 mM), was studied. The enzyme was pre-incubated with the individual metal ions for 20 min at room temperature, followed by standard enzyme assay (Section 3.8.1). The results were expressed as the percentage of the protease activity compared to the absence of the inhibitors or the metal ions.
Preparation of casein hydrolysate
The method to prepare casein hydrolysate was based on Egito et al. (2007). Sodium caseinate was dissolved in 100 mM sodium phosphate buffer, pH 6.5 to make 10% and 11.25% (w/v) casein solutions. Rennet (0.1 mL calf rennet) was diluted with 0.9 mL Milli-Q water and vortexed for 1 min. Rennet (3 µL, diluted 10-fold) and tamarillo crude extract (350 µL, 1.17 ± 0.03 mg/mL) were added to 3 mL of 10% (w/v) and 11.25 % (w/v) casein solution, respectively, to make a final casein concentration of 10% (w/v). The casein solutions containing tamarillo protease or rennet were incubated at 35°C in a Function Line incubator (Heraeus, Langenselbold, Germany), and hydrolysate aliquots (200 µL) were removed at 15 min, 30 min,1 h, 4 h and 24 h. The enzymatic reaction was stopped by heating the hydrolysates at 100°C for 5 min. Casein solution (10% w/v) was used as a standard. The hydrolysate solutions were stored at -80°C until analysed.
To prepare individual casein protein solutions, each of α-, β- and -casein were dissolved in 10 mM sodium phosphate buffer at pH 6.5. The final concentration of the casein solution (5 mL) was 10% (w/v). Purified tamarillo protease (1 mg) and rennet (10 µL, diluted 200-fold) were mixed with the casein solutions using a magnetic stirrer for 2 min. The mixtures were incubated at 35°C. Aliquots of hydrolysate (200 µL) were removed at different times: 15 min, 30 min, 1 h, 4 h, 10 h and 24 h. The hydrolysates were heated immediately at 100°C for 5 min to stop the enzymatic reaction. The hydrolysates were lyophilised using a freeze-dryer and kept in -80°C until analysed.
Table of content
Abstract of thesis
Table of content
List of Abbreviations and Symbols
Co-authorship Chapter 4
Co-authorship Chapter 5
Co-authorship Chapter 6
Co-authorship Chapter 7
1. Introduction and overview
1.2 Objective of this project
1.3 Thesis structure
2. Literature review on Milk and Plant proteases
2.2 Milk constituents
3. Materials and methods
3.2 Milk sample preparation
3.3 Sodium caseinate preparation
3.4 Preparation of crude protease extract from tamarillo fruit
3.5 Purification of tamarillo protease
3.6 Determination of protein content
3.7 Determination of bovine milk hydrolysed by rennet and tamarillo extract
3.8 Determination of protease activity
3.9 Preparation of casein hydrolysate
3.10 Chemical methods
3.11 Physical methods
3.12 Statistical analysis
4. Purification and characterisation of a protease (tamarillin) from tamarillo
4.2. Material and methods
4.3 Results and discussion
5. Effect of temperature and pH on the properties of skim milk gels made from a tamarillo protease extract and rennet
5.2 Material and methods
5.3 Results and Discussion
6. Protease activity of enzyme extracts from tamarillo fruit and their specific hydrolysis of bovine caseins
6.2 Materials and methods
6.3 Results and discussion
7. Rheological and structural properties of coagulated milks reconstituted in D2O: Comparison between rennet and tamarillo protease (tamarillin)
7.2 Materials and methods
7.3 Results and discussion
8. Conclusions and future work
8.2 Future works
GET THE COMPLETE PROJECT
Tamarillin – the purification and properties of a novel serine protease from tamarillo fruit (Cyphomandra betacea (Cav.)), and the characterisation of its milk-clotting activity