International Journal of Biological Macromolecules
Aspartic protease from Aspergillus niger: Molecular characterization and interaction with pepstatin A
Kavya Purushothaman, Sagar Krishna Bhat, Sridevi Annapurna Singh, Gopal Kedihithlu Marathe, Appu Rao G. Appu Rao
Aspartic protease from Aspergillus niger: Molecular characterization and interaction with pepstatin A
Kavya Purushothaman a, Sagar Krishna Bhat a, Sridevi Annapurna Singh c, Gopal Kedihithlu Marathe b, Appu Rao G Appu Rao a *
a Kaypeeyes Biotech Pvt Ltd, R&D centre, Food industrial area, Metagalli post, Mysuru, 570016, Karnataka, India
b Department of Studies in Biochemistry, University of Mysore, Manasagangothri,
Mysuru, 570006, Karnataka, India
c Department of protein chemistry and technology, CSIR-CFTRI, Mysuru, 570020, Karnataka, India
Abstract:
In the pursuit of industrial aspartic proteases, aspergillopepsin A-like endopeptidase from the fungi Aspergillus niger, was identified and cultured by solid state fermentation. Conventional chromatographic techniques were employed to purify the extracellular aspartic protease to apparent homogeneity.. The enzyme was found to have single polypeptide chain with a molecular mass of 50±0.5 kDa. The optimum pH and temperature for the purified aspartic protease was found to be 3.5 and 60 oC respectively. The enzyme was stable for 60 minutes at 50 oC. The purified enzyme had specific activity of 40,000 ± 1800 U/mg. The enzyme had 85% homology with the reported aspergillopepsin A-like aspartic endopeptidase from Aspergillus niger CBS 513.88, based on tryptic digestion and peptide analysis. Pepstatin A reversibly inhibited the enzyme with a Ki value of 0.045 µM. Based on homology modelling and predicted secondary structure, it was inferred that the aspartic protease is rich in β-structures, which was also confirmed by CD measurements. Interaction of pepstatin A with the enzyme did not affect the conformation of the enzyme as evidenced by CD and fluorescence measurements. Degree of hydrolysis of commercial substrates indicated the order of cleaving ability of the enzyme to be hemoglobin > defatted soya flour > gluten > gelatin > skim milk powder. The enzyme also improved the functional characteristics of defatted soya flour. This aspartic protease was found to be an excellent
candidate for genetic manipulation for biotechnological application in food and feed industries, due to its high catalytic turn over number and thermostability.
Key words: Aspartic protease, structure and stability, pepstatin A interaction, commercial application.
1. Introduction
Proteases are ubiquitous enzymes produced by many organisms such as plants, animals, and microorganisms like bacteria, yeast, fungi and viruses. Nevertheless, microbes are the commercial source of enzymes by virtue of their biochemical diversity and susceptibility to genetic modifications [1].Filamentous fungi- Aspergillus is one of them and have been commercially exploited vigorously in the production of enzymes, mainly because of their ability to produce a variety of hydrolytic enzymes and their ability to grow on agro-waste substrates in large volumes. Just as fungi like Aspergillus, Penicillium and Rhizopus are routinely used for the production of aspartic protease, Endothia, Mucor and other species are employed for the production of rennin- like proteases [2]. Moreover, these species have acquired a Generally Recognized As Safe (GRAS) status, implying the nontoxicity of products of these cultures to humans or animals.
Aspartic proteases, also known as acid proteases are the group of proteases (EC 3.4.23) that contain aspartate residues at the active site. These proteases have characteristic low pI, with optimum pH around 3to5 and working between pH 2 to 6. Most of the aspartic proteases, with the exception of retroviral aspartic protease, are single polypeptide chain with molecular weight around 30- 45 kDa. Two lobes with two-fold symmetry are the identifying feature of the aspartic proteases. The bi-lobed structure is formed by the tertiary structure that creates a catalytic cleft between the lobes. Each lobe contributes an aspartate
residue which takes part in the catalysis. Aspartic protease has a cleavage preference towards hydrophobic/bulky/ aromatic amino acids on either side of peptide bond. The sequence of the catalytic triad namely Asp- Thr (Ser)-Gly is preserved through the evolution. Aspartic protease from various sources share similarities in sequence and tertiary fold. Essentially, these are β- rich proteins evolved to assimilate diversified nitrogen sources. Aspartic proteases from mammals are produced as zymogens and are activated by cleavage. Fungal aspartic proteases are also known to be produced as zymogens [3].
Aspartic proteases could play a major role in modifying the proteins to meet the required functional characteristics due to their specific cleaving sites. Proteases are used in food industry to improve functional properties and nutritional value of food proteins [4]. Aspartic proteases are used in the preparation of protein hydrolysates of high nutritional value from soya beans and other food proteins used in infant foods, medicine and dietary products. One of the limitations in the preparation of protein hydrolysates is, the bitter taste caused by the exposed hydrophobic residues. Aspartic proteases are used in debittering protein hydrolysates [2]. Specific application of proteases can be of help in making available the new generation of protein products such as protein hydrolysates and bio-active peptides [5]. It is possible to generate bio-active peptides from the proteins by identifying the cleavage sites by proteomics and peptidomics approaches. Apart from these, there are applications in dairy/ cheese industry in cheese making wherein, acid proteases from microbial origin are replacing expensive calf rennets [6]. In bakery industry, aspartic proteases are used to modify gluten to improve the properties of dough. This also enhances the flavour and texture of bread [4]. During the manufacture of certain alcoholic beverages and fruit juices, trace amounts of proteins cause turbidity / haziness in the product. Aspartic proteases are used in clarification of beer and wine by degradation of the residual proteins. Aspartic protease from Aspergillus saitoi is used to clarify the juice and beverages [7]. Recently, aspartic proteases
have gained attention for their involvement in serious human diseases like malaria, AIDS and Candida infection [6, 8]. HIV protease, which belongs to aspartic protease family, has a critical role in replication of virus in infected human cells. The enzyme cleaves the polypeptide chain encoded by viral genome generating essential proteins and enzymes needed for viral maturation. Specific inhibitors to HIV aspartic protease may be an answer to this disease [2].
We have been able to isolate and purify an aspartic protease from Aspergillus niger species, which finds application in various food and beverage industries. In this context, we have looked at the molecular characteristics of the enzyme and its interaction with the inhibitor – pepstatin A. We have identified the application of the enzyme for various protein modifications. Based on the comparative evaluation of specific activities of various aspartic proteases from fungal sources, the activity level found by us is among one of the highest reported with greater thermostability and appears to be a good candidate for industrial manipulation.
2. Materials and methods
2.1. Materials
Aspergillus niger culture was from Kaypeeyes biotech pvt Ltd, Hebbal industrial area, Mysuru, Karnataka, India. Haemoglobin (acid denatured) was purchased from MP Biomedicals, Santa Ana, California, USA. Biogel-P-100 was from Biorad, Hercules, California, USA. Pepstatin A, DEAE-sepharose CL 6B, Trypsin, Trypsinogen, oxidised Insulin B-chain (bovine) and Gelatin were obtained from Sigma Aldrich, St. Louis, Missouri, USA. Molecular weight marker was procured from GeNie, Bengaluru, India.
Gluten was gifted by P D Navkar biochem pvt. Ltd, Bengaluru, India. Defatted soya flour was from Kriti nutrients ltd, Indore, Madhya Pradesh, India. All other chemicals were of analytical grade.
Methods
2.2. Identification of the culture by morphological and molecular characteristics
For morphological characterization, pure culture was inoculated on potato-dextrose-agar (PDA) plates and incubated at 30 oC for 7 days. Colony features like colony diameter, pattern and color (both front and reverse of the colony) and microscopic features like morphology of hyphae, conidiophore and spores were recorded. Fungal preparation was mounted on trinocular light microscope and photographed using inbuilt Miacam 6 MP Live Image HD camera.
For molecular characterization, the fungal culture was grown on potato-dextrose broth and the mycelia were harvested. The genomic DNA was isolated from the fungal mycelia using fungal genomic DNA isolation kit. The internal transcribed spacer (ITS) region (~600 base pair) of the genomic DNA was amplified by PCR using high-fidelity PCR polymerase. The PCR product was sequenced bidirectionally using ABI 3500 XL Genetic Analyzer. The sequence data was analyzed to identify the culture and its closest neighbors. The phylogenetic tree construction and evolutionary history was inferred by using maximum likelihood method and Tamura and Nei model [9]. The initial tree for the heuristic search was obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pair wise distances estimated using the Maximum Composite Likelihood (MCL) approach and then selecting the topology with superior log likelihood value. Evolutionary analysis was conducted in MEGA X [10].
2.3. Production of aspartic protease from Aspergillus niger by solid-state fermentation
The culture was grown by solid-state fermentation using wheat bran as substrate. To 100 g of wheat bran, 60 mL of 0.2 N HCl containing trace minerals (70 mg each of CuSO4, ZnSO4 and FeSO4 per 100 mL of 0.2 N HCl) was added. Sterile wheat bran was inoculated with fungal spore suspension and incubated at 30 oC for 7 days. The extracellular aspartic protease secreted into the fermented material was extracted in 0.1 M NaCl, centrifuged at 7000 x g for 10 min at 4 ⁰C and filtered through Whatman filter paper no. 1. The clear supernatant was used as enzyme source.
2.4. Assay of aspartic protease
Aspartic protease was assayed as described by Yin et al [11] with minor modifications. Briefly, 1 mL of 2% haemoglobin (acid denatured) prepared in 0.1 M acetate buffer pH 4.0, was used as substrate. To this, 0.4 mL of enzyme (The enzyme was prepared in 0.2 M acetate buffer, pH 4.0 containing 0.5 M NaCl) was added and incubated at 60 oC for 10 min. The reaction was arrested by the addition of 2 mL of 5% trichloro acetic acid (TCA) and the tubes were incubated at room temperature for 20 min. After 20 min, the reaction mixture was filtered through Whatman filter paper no.1 and the absorbance of the filtrate containing released tyrosine was measured at 280 nm using Eppendorf BioSpectrophotmeter. The amount of tyrosine released was calculated using a tyrosine standard (0 – 60 µg/mL). One unit of protease activity is defined as the amount of enzyme required to liberate 1 µg of tyrosine per minute, under standard assay conditions.
Protein estimation was carried out as described by Lowry et al. [12] using bovine serum albumin as standard.
2.5. Purification of aspartic protease
Protein salting-out was carried out using ammonium sulphate. To the culture supernatant, 60% ammonium sulphate was added with constant stirring at 4 oC for 6 h. The precipitate was collected by centrifugation at 7000 x g for 30 min at 4 oC. Ammonium sulphate pellet was re-suspended in 50 mM acetate buffer pH 5.0 containing 0.1 M NaCl. This sample (2 mL containing 95 mg protein) was loaded on to a 1.5 cm x 78 cm column packed with 137 mL of Biogel P100 polyacrylamide beads, equilibrated with 50 mM acetate buffer, pH 5.0 containing 0.1 M NaCl and eluted with the same buffer. Fractions were collected at a flow rate of 20 mL/h and monitored for absorbance at 280 nm. Each fraction was assessed for protease activity. Active fractions from the Biogel column were pooled and loaded to DEAE-sepharose column (2.5 cm X 8 cm, 39 mL). 6 mL sample containing 13.2 mg protein was loaded on to the column previously equilibrated with 50 mM acetate buffer, pH 5.0 containing 0.1 M NaCl. Fraction size was set to 2 mL with a flow rate 2 mL/min. Elution was carried out in 0 to 1 M NaCl step-gradient. Fractions were assayed for protease activity and active fractions were pooled.
2.6. SDS-PAGE and zymogram
2.6.1. SDS-PAGE
SDS-PAGE was carried out with 10% polyacrylamide gel as described by Lammeli [13]. Silver staining was used to visualize the protein band pattern. The molecular weight of the purified aspartic protease was calculated by plotting the relative migration distance (Rf) values against log molecular weight. The protein standards used were Phosphorylase B (97.4 kDa), BSA (66.0 kDa), ovalbumin (43.0 kDa), carbonic anhydrase (29.0 kDa), soyabean trypsin inhibitor (20.1 kDa) and lysozyme (14.3 kDa).The molecular weight was also
confirmed by measurement using gel reader software (Image Lab, Gel Doc EZ Imager, Bio- Rad).
2.6.2. Zymogram
Activity staining was carried out by native acid electrophoresis as mentioned by Diaz-Lopez et al. [14] with modification in the buffers and pH. The buffer system used was as follows: Stacking buffer: 0.127 M acetic acid and 0.12 M KOH buffer, pH 6.0; Resolving buffer:
0.376 M acetic acid and 0.06 M KOH buffer, pH 4.3; Electrode buffer: 0.14 M acetic acid and 0.35 M β alanine buffer, pH 4.5. The electrophoresis was carried out for2 hours. After electrophoresis, gel was soaked in 0.1 M acetate buffer pH 4.0 for 15 min, then in 0.25% hemoglobin prepared in 0.1 M acetate buffer pH 4.0 at 4 oC for 30 min, followed by soaking in fresh hemoglobin solution for 90 min at 40 oC. The gel was fixed in 12% TCA solution for 15 min, and stained with Coomassie brilliant blue. A control native acid page was also run and bands were visualized by silver staining.
2.7. Estimation of molecular weight by gel filtration chromatography
The molecular weight was calculated by plotting log molecular weight versus Ve/Vo obtained from the gel filtration chromatography using standards: bovine serum albumin (BSA) (66 kDa), DNase I (31 kDa), trypsin (23.3 kDa) and lysozyme (14.6 kDa). The buffer used for running and elution was 50 mM acetate buffer pH 5.0 containing 0.1 M NaCl.
2.8. Characterization of aspartic protease
2.8.1. Effect of pH and temperature on activity and stability
Optimum pH was checked by assaying at different pH from 2.0 to 8.0. Buffers used were 100 mM of glycine-HCl for pH 2 to 2.7, citrate for pH 3 to 3.5, acetate for pH 4 to 5.5 and phosphate for pH 6 to 8. Assay was carried out at 60 oC.
Stability of the protease at different pH was determined by incubating the enzyme in various buffers with pH ranging from 2.0 to 8.0 for 24 h at 30 oC. The residual activity was checked at 60 oC and compared with control, which was taken as 100% activity.
The optimum temperature for protease activity was determined by estimating the activity at different temperatures ranging from 35 to 75 oC. Assay was carried out at 4.0 pH. Highest activity obtained was taken as 100% and expressed as % residual activity.
Thermostability was checked in presence of 0.5 M NaCl, by incubating the enzyme at different temperatures ranging from 40 to 70 oC for 15 min, immediately cooling on ice bath for 5 min and assaying the samples at 60 oC, pH 4.0 to determine the residual activity. Control was taken as 100% activity and test samples expressed as % residual activity.
Thermal inactivation kinetics of the enzyme was checked in presence of sodium chloride. The enzyme was incubated with and without 0.5 M sodium chloride in 0.2 M acetate buffer pH 4.0, at temperatures 50, 55, 58 and 60 oC for 120 min. An aliquot was drawn every 15 min and cooled in ice bath for 5 min. The samples were checked for residual activity at pH 4.0, 60 oC and compared with control. % Residual activity was plotted against time.
2.8.2. Kinetic parameters of the enzyme
The Km and Vmax values of aspartic protease were determined using hemoglobin as substrate in the range of 0 to 2% concentration. Assay was carried out for 10 min at pH 4.0 and 60 oC. Lineweaver- Burk plot was used to determine the Km and Vmax values.
2.9. Inhibition studies
The enzyme was treated with various class-specific protease inhibitors for 30 min at 30 oC. The inhibitors and their concentrations used were: pepstatin 20 µM (aspartic protease inhibitor), PMSF 2 mM (serine protease inhibitor), Iodoacetamide 2 mM (cysteine protease inhibitor) and EDTA 10 mM (metalloprotease inhibitor) [15]. The residual activity was assayed under standard conditions. The activity measured in the absence of inhibitors or chelator was taken as 100%.
2.9.1. Inhibition by pepstatin A
The purified enzyme was treated with pepstatin A (prepared in 10% acetic acid in methanol) and incubated at 30 oC for 30 min. After the reaction, the residual activity was checked under optimal assay conditions and compared with the control enzyme incubated without pepstatin A. Lineweaver-Burk plot was constructed by assaying protease activity using different concentrations of hemoglobin (0 to 20 mg/mL) and pepstatin A (0.035 to 0.14 µM). The type of inhibition of pepstatin towards aspartic protease was established. Dixon plot (1/V versus pepstatin concentration) was used to calculate the Ki value of purified protease using acid denatured hemoglobin as substrate in presence of increasing concentrations of pepstatin A (0.035 to 0.14 µM) .
2.10. Identification of the protease by peptide characterization using LC-MS/MS and homology modeling of protein
The peptide characterization and identification of the protein was carried out at Centre for Cellular And Molecular Platforms (C-CAMP), Bengaluru, India. Briefly, the purified protease was resolved on SDS-PAGE and stained using Coomassie brilliant blue. Protease band was excised from the gel and destained by incubating in 100 mM ammonium bicarbonate/ acetonitrile (1:1) solution for 30 min followed by washing in acetonitrile.
Trypsin and chymotrypsin digestion of the destained protein was carried out overnight at 37 oC. Digested peptide mixture was resolved on nano-HPLC column (EASY SPRAY PEPMAP RSLC C18 2µm; 50cm x75µm) using acetonitrile/ formic acid gradient. Ions were generated by an automated Electron Spray Ionisation (ESI) source. An LC coupler connected the flow from LC to the ESI-chip (Nanomate Triversa, Advion) and the nano-ESI generated ions were transferred to mass spectrometer (LTQ- Orbitrap Discovery, Thermo Scientific). The peptide data obtained from MS was used to establish the identity of the proteins. MASCOT and PEAKS software were used to search databases. The peptide sequences were aligned with the protein sequence with highest homology. Using the reported sequence, homology model was generated using Phyre 2 web server [16]. 3-D model was visualized using Jmol. The apparent active site residues were identified.
2.11. Fluorescence and Circular dichroism (CD) measurements
2.11.1. Fluorescence measurements
Fluorescence measurements were carried out using CARY ECLIPSE Fluorescence spectrophotometer with temperature control system. Aspartic protease was prepared in 50 mM acetate buffer pH 4.0. Fluorescence excitation was carried out at 280 nm and emission was observed between 300 to 400 nm at 25 oC. Fluorescence was monitored in the presence and absence of the inhibitor pepstatin A in the concentration range of 0 to 20 µM. Average of three accumulations was taken.
2.11.2. Circular dichroism (CD) measurements
CD measurements were carried out using JASCO J-810 spectropolarimeter. For far UV CD,
0.25 mg/mL protein sample prepared in 50 mM acetate buffer pH 4.0 was used and the spectrum was recorded between 200 to 240 nm using 1 mm cuvette at 25 oC. For near UV
CD, 1.04 mg/mL protein solution prepared in 50 mM acetate buffer pH 4.0 was used. The spectrum of the protease was recorded between 240 to 320 nm at 25 oC in presence and absence of pepstatin A in the concentration range of 0 to 20 µM. The secondary structures were calculated as described by Yang et.al
2.12. Identification of cleavage sites:
The cleavage specificities of aspartic protease was determined by examining the conversion of inactive trypsinogen to active trypsin, which requires the cleavage of a single, specific K-I bond and also by analysing the peptides formed by the hydrolysis of insulin B-chain.
2.12.1. Activation of trypsinogen:
Trypsinogen activation was carried out as described by Vishwanatha et.al [18] with modifications. Trypsinogen was prepared at the concentration of 1mg/mL in 0.1 M acetate buffer, pH 4.0. Purified aspartic protease was added to the trypsinogen preparation (Enzyme to substrate ratio was 0.2% based on protein concentration) and incubated at 60 oC for 1 min. The formation of trypsin was immediately examined by checking the activity using casein as substrate. Casein (1%) prepared in 0.1 M phosphate buffer pH 7.5, was incubated with activated trypsin mixture at 37 oC for 10 min. Reaction with inactive trypsinogen blank and enzyme blank were also carried out. After the incubation, reaction was arrested by the addition of 5% TCA solution. Absorbance of the filtered supernatant was checked at 280 nm and the activity was calculated using tyrosine standard. Activated trypsin mixture was also checked for the formation of clear zone in zymogram. A non-denaturing SDS-PAGE was carried out with copolymerised gelatin, as described by Heussen et.al [19]. Resolving gel prepared with 10% polyacrylamide was incorporated with 0.1% gelatin. After electrophoresis, the gel was washed in 2.5% triton X-100 solution, followed by washing in
0.1 M phosphate buffer, pH 7.5. Further, the gel was incubated in 0.1 M phosphate buffer,
pH 7.5, at 37 oC for 16 hrs. The gel was stained with Coomassie brilliant blue and destained to visualize the clear bands. A control non-denaturing SDS-PAGE without gelatin was also carried out.
2.12.2. Cleavage specificity using oxidised insulin B-chain:
Cleavage specificity using insulin B-chain was determined as mentioned by Sreedhar et.al
[20] and Vishwanatha et.al [18]. Oxidised insulin B-chain (1 mg/mL) prepared in 0.1 M acetate buffer, pH 4.0, was incubated with purified aspartic protease with an enzyme to substrate ratio of 1%, based on protein concentration. Reaction was carried out for 10 min at 60 oC, after which reaction was terminated by the addition of 0.01% trifluroacetic acid (TFA). The digested mixture was subjected to fractionation using Shimadzu reverse-phase HPLC system with C 18 column (25 cm x 4.6 mm, 5 µm, Ascentis). The solvent system used was 0.1% TFA and acetonitrile, with a flow rate of 1mL/min. Elution was carried out with 0- 50% acetonitrile gradient and the eluted peptides were monitored at 215 nm. Each peptide peak was collected; evaporated using a vacuum evaporator (Eppendorf) and mass of the peptide sequences were deduced using Triple TOF 5600-1 mass spectrometer (AB Sciex instruments, model-5016230/K) with positive polarity. Fragmentation was performed using CID (collision- induced dissociation).The ‘y’ and ‘b’ values were derived and the peptide sequences were deduced. By comparing the amino acid sequence of each peptide peak with the intact insulin B-chain sequence, the specific cleavage sites of aspartic protease were determined.
2.13.. Identification of substrate preferences
Various commercial proteins like defatted soya flour, gelatin, gluten, skim milk powder and hemoglobin were used to check the effect of hydrolysis by aspartic protease.
Protein substrates were prepared in the range of 2 to 10% in 50 mM acetate buffer pH 4.5 containing 50 mM NaCl. Protease enzyme (ammonium sulfate pellet) was added at the rate of 1000 units per mL of protein preparation and the reaction was carried out at 50 oC for 16
h. After the reaction, the enzyme was denatured by heating at 75 oC for 10 min and the samples were centrifuged at 10,000 g for 10 min. The supernatant was used to check the following parameters:
2.13.1.. Banding pattern on SDS-PAGE
The supernatant after enzyme reaction using each substrate was subjected to SDS-PAGE using 10% polyacrylamide gel and bands were visualised by silver staining as mentioned earlier.
2.13.2.. Degree of hydrolysis
Ninhydrin test was performed to determine the degree of hydrolysis by aspartic protease by estimating the amount of free amino groups in the reaction mixture before and after reaction [21]. To 1 mL of sample prepared in 0.1 M citrate buffer pH 5.0, 1 mL of 2% ninhydrin reagent (prepared in 75% methyl cellosolve containing 1 N sodium acetate buffer pH 5.5) was added. Tubes were covered with foil and boiled for exactly 15 min. Tubes were added with 1 mL of 1:1 diluted isopropanol and the absorbance of the solutions were checked at 570 nm. The amount of free amino acids in the reaction mixture was determined using the Leucine standard graph prepared in the range of 0 to 0.5 µmoles/mL concentration. The degree of hydrolysis was expressed as µmoles of amino nitrogen formed per mL of reaction mixture.
2.13.3.. Protein estimation in the supernatant of the reaction mixture
Protein estimation was carried out by Lowry’s method [12] to check the amount of protein released into the supernatant by the action of aspartic protease.
2.13.4.. Absorbance of the TCA supernatant at 280 nm
The amount of protein in the TCA soluble fraction of the reaction mixture was determined as per Zhang et al. [22] with slight modifications. After the reaction, 10% TCA solution was added to the reaction supernatant in the ratio of 1:1 to remove undigested protein. Tubes were allowed to stand at 30 oC for 30 min followed by centrifugation at 10,000 x g for 15 min. The supernatant was diluted 2 to 20 times with distilled water and absorbance was recorded at 280 nm.
2.14.. Hydrolysis of defatted soya flour
Commercial defatted soya flour was subjected to hydrolysis by aspartic protease and the amount of protein in the supernatant and the TCA soluble proteins were checked as mentioned earlier. The reaction supernatant was also subjected to SDS PAGE using 10% gel and bands were visualised by silver staining.
2.15.. Functional properties of defatted soya flour before and after protease treatment 2.15.1.. Preparation of soya protein hydrolysate
The experiments were carried out using defatted soya flour. Defatted soya flour (10%) suspension was prepared in 50 mM acetate buffer pH 4.5 containing 50 mM NaCl. Protease enzyme (1000 U/ mL) was added to soya preparation and the reaction was carried out at 50 oC for 16 h with shaking. A control was prepared without enzyme. After the reaction, the enzyme was denatured by heating at 75 oC for 10 min. Samples were dried at 60 oC for 2 h. Dried samples were cooled in a desiccator and stored at 4 oC. Functional properties like protein solubility, emulsifying activity, water holding capacity, oil binding capacity and foaming properties were checked.
2.15.2.. Protein solubility
Protein solubility was checked at pH 4.0 and pH 7.0. 20 mg of sample was dispersed in 20 mL distilled water and the pH was set to 4.0 and 7.0 using 0.1 N HCl or 0.1 N NaOH. Samples were incubated at 30 oC for 30 min in a shaker and were centrifuged at 7000 x g for 20 min. The supernatant was used for protein estimation by Lowry’s method. A control sample was treated with 0.5 N NaOH and the protein content in this sample was taken as total protein content.
Solubility is calculated as follows:
Solubility (%) = Protein content in the supernatant X 100 / Total protein content in the sample.
2.15.3.. Emulsifying property
Emulsifying activity was checked according to Yousif et.al [23], wherein, 15 mL of 0.5% protein solution was added with 5 mL of refined sunflower oil and homogenised for 1 min. From the emulsion formed, 100 µL sample was drawn from the bottom of the container and added to 5 mL of 0.1% SDS solution. The absorbance of this solution was measured at 500 nm. Emulsifying activity index (EAI) was determined using the following formula:
EAI (m2/g) = 2 X 2.303 X A 500 X dilution/ Sample mass in g/m3 X (1-Ø) Where, A 500 is the absorbance at 500 nm, Ø is the oil volume fraction.
2.15.4.. Water holding capacity (WHC) and oil binding capacity (OBC)
WHC and OBC were determined as per the Zhang et.al [22]. 1 g sample was added to a pre- weighed centrifuge tube to which 10 mL distilled water was added and mixed well. The tubes were allowed to stand at 30 oC for 30 min and centrifuged at 7000 x g for 30 min. The
weight of the residue along with the tube, after decanting the supernatant was weighed. WHC was determined using the formula:
WHC (g/g sample) = Weight of the (tube+ sediment) [g] – weight of the (tube + dry sample) [g]/ Weight of the dry sample [g]
Oil binding capacity was determined by weighing 1 g sample in to a pre-weighed centrifuge tube to which 10 mL of refined sunflower oil was added and mixed thoroughly. The mixture was allowed to stand at 30 oC for 30 min and centrifuged at 7000 g for 30 min. The volume of the supernatant was measured after decanting carefully into a measuring cylinder.
OBC (mL /g sample) = Initial volume of oil taken [mL] – volume of oil after decanting [mL]/ Weight of the dry sample [g].
2.15.5.. Foaming properties
Foaming capacity was determined as described by Yousif et.al [23]. 1 g sample was dispersed in 100 mL of distilled water, homogenised for 1 min, transferred to measuring cylinder and the total volume of the solution (including foam) in cm3 was measured after 30 sec. Foaming capacity was determined as follows:
Foaming capacity (%) =
Total volume after homogenisation [mL] – volume before homogenisation [mL] X100 / Volume before homogenisation [mL]
2.16. Statistical analysis:All the experiments were performed at least three times, independently. In each experiment,
values are presented as average of three replicates. Error bars represent standard deviation from the mean value. The enzyme kinetic parameters were plotted using Graphpad prism
V5.0. The best-fit data points were determined by standard errors, 95% confidence intervals and regression coefficients (R2).
Results
3.1. Identification of fungal strain by morphological and molecular characteristics and phylogenetic analysis
The identification of the fungal culture was carried out by studying the morphology of the culture by macroscopic and microscopic methods. The colony inoculated on PDA media was observed after 7 days. Colony was profusely sporulated and was typically black coloured with a diameter of 60 to 70 mm (Fig. 1a). Reverse of the colony was colourless to pale yellow (Fig. 1b). Mycelia were filamentous and grew radially. Microscopic observations revealed that the hyphae were septate. Conidiophore appeared globose and biseriate, bearing small spherical spores which were dark-brown to black in colour (Fig. 1c). The spore surface appeared smooth (Fig. 1d). The morphological features described here are typical of fungus belonging to the species Aspergillus niger [24] . The culture was identified to be Aspergillus niger by amplification and analysis of the DNA sequence of the ITS region of the genomic region. The Phylogenetic analysis revealed the nearest neighbor to be Aspergillus niger strain IR3_11 with accession number MK461093.1 (Fig. 1e). The identity of the fungus based on morphological characteristics was in good agreement with that of molecular characterization.
3.2. Purification of aspartic protease from Aspergillus niger
Under solid-state fermentation, A.niger expressed aspartic protease to the level of 1,50,000±15000 U/g of fermented material, after 7 days of fermentation. The extracellular aspartic protease enzyme from solid-state fermentation was eluted with 0.1 N NaCl.The
protein was precipitated using 60% ammonium sulfate. The pellet was dissolved in 50 mM acetate buffer pH 5.0 and resolved on Biogel P 100 gel filtration column followed by DEAE-Sepharose ion exchange chromatography. The elution profile of the enzyme on Biogel P 100 is shown in Fig. 2a. The active fractions eluting between 25 and 30 were pooled. At this stage, the enzyme had a specific activity of 19,743 U/mg with 5.35 fold purity and a recovery of 65%. The pooled fraction from Biogel P 100 column, following resolution on DEAE sepharose column was eluted using a gradient of 0 to 1 M NaCl concentration. Active protease enzyme was eluted in 0.4 M NaCl (Fig. 2b). The purification steps, fold purity, percent yield and specific activity are presented in Table 1. In the final purification , specific activity of 38,362 U/mg with 10.4 fold purity and 56% yield was obtained. For comparison purposes, the enzyme activity was expressed in the same units as reported earlier [11, 18, 25, and 26] (Table 2).
3.3. Gel electrophoresis
3.3.1. SDS-PAGE
The enzyme was purified to apparent homogeneity as evidenced by the single band in the SDS-PAGE (Fig. 2c). The molecular weights of enzyme obtained by measuring the Rf value and by using gel reader software were 50.54±0.75 kDa and 51.06±0.37 kDa, respectively.
3.3.2. Zymogram
The zymogram was carried out using native acidic PAGE, since the enzyme lost activity when run on basic PAGE. Clear activity zone was observed with hemoglobin substrate at pH
4.0 (Fig. 2d).
3.4. Estimation of molecular weight
Gel filtration chromatography was utilised to determine the molecular weight of the aspartic protease. By using the graph with log molecular weight plotted against Ve/Vo, the protease was estimated to have a molecular weight of 50±1 kDa (Fig. 2e)
3.5. Characterization of aspartic protease:
3.5.1. Effect of temperature and pH on the activity and stability of purified aspartic protease
The purified protease was active between the pH 3.0 to 4.5 with optimum activity at pH 3.5. Activity decreased drastically below pH 3.0 and above pH 5.0. The enzyme was stable between pH 2.7 and pH 6.5 for 24 h at 30 oC (Fig. 3a). The enzyme activity as a function of temperature was checked between 35 to 75 oC (Fig. 3b). Optimum temperature for the enzyme activity was between 60 and 65 oC at pH 4.0. Enzyme activity was found to rapidly fall to zero at 75 oC. The thermal stability of the enzyme was measured between 40 to 70 oC. The enzyme retains its thermal stability up to 60 oC and the activity falls-off subsequently to zero at 65 oC. The thermal stability measurements in the presence of buffer and added NaCl as a function of time and temperature between 50 and 60 oC were carried out (Fig. 3c). The enzyme retains 80% activity at 50 oC up to 120 min. At 55 oC, the enzyme lost 50% activity at 120 min. However, at higher temperatures, the activity loss was rapid. As evidenced by the graph, the thermostability of the enzyme is better in the presence of 0.5 M NaCl. NaCl was found to stabilise the enzyme against thermal inactivation
3.5.2. Determination of Km and Vmax
The Km and Vmax values of aspartic protease were determined using Lineweaver- Burk plot. The Km and Vmax values were 6.3 mg/mL (98.52 µM) and 50 µmol/min with hemoglobin as substrate (Fig. 3d).
3.6. Inhibition studies
Studies on inhibition of protease using class-specific inhibitors such as iodoacetamide, PMSF and EDTA indicated that the enzyme activity is not inhibited by the above inhibitors at the prescribed effective concentrations (Results not shown).
3.6.1. Inhibition by pepstatin A
Pepstatin A, a specific inhibitor of aspartic protease, was used at different concentrations to check the inhibition level. To understand the nature of inhibition, Line weaver-Burk plot was constructed by carrying out the reaction by varying substrate and inhibitor concentrations. It was inferred that pepstatin A competitively inhibits aspartic protease (Fig. 3e). To determine the inhibition constant, Dixon plot was constructed by plotting pepstatin A concentration versus 1/V (Fig. 3f). The derived Ki value was 0.045 µM. Pepstatin A- treated protease on washing, regained its activity when assayed under standard conditions. This established that the pepstatin A reversibly inhibits aspartic protease (Results not shown).
3.7. Identification of the protease by peptide characterization and homology modeling
The peptide characterization of the protease was carried out by LC-MS/MS as described in the methods. Peptides (223 numbers) thus obtained were blasted and aligned. The peptides shared 85% identity with the reported aspergillopepsin A-like aspartic endopeptidase from Aspergillus niger CBS 513.88 with accession number: XP_001401093.1. The apparent active site residues were identified to be Asp 101 and Asp 283 (Fig. 4a). Based on the sequence identity, a homology model was constructed (Fig. 4b). The homology modeling with the above protease suggested the dominance of β structures in the molecule.
3.8. Fluorescence and CD measurements:
3.8.1. Fluorescence:
The protein emission spectrum, after being excited at 280 nm, is shown in Fig. 5a. The fluorescence emission is characterised by emission at 325 nm and is typical of tryptophan fluorescence in hydrophobic environment. With the addition of pepstatin A, there was no change in the emission maxima. The interaction of pepstatin A with aspartic protease did not bring about any change in the tertiary structure or in the vicinity of tryptophan residues of aspartic protease.
3.8.2. Circular dichroism
The secondary and tertiary structure of the aspartic protease was followed by CD spectra in the far and near ultra-violet region. The protease spectra in the near UV region (Fig. 5b) had characteristic troughs at 287, 277 and 244 nm and small peaks at 291 and 283 nm followed by a broad peak between 270 and 250 nm. The negative peak at 287 and the peak at 291 nm could be attributed to tryptophan residues of the protein and the peaks at 283 nm and 277 nm could be attributed to tyrosine residues, while the broad peak between 270 to 250 nm could be due to the cysteine residues in the protein molecule. The addition of pepstatin A causes very little observable changes in the near UV CD spectra suggesting the non- involvement of aromatic residues with pepstatin A. This result is also supported by fluorescence measurements. The far UV CD spectra (Fig. 5c) was characterised by a positive peak around 201 and 232 nm followed by a broad minima between 203-223 nm. The far UV spectra characterised by the above peaks was typical to aspartic protease family [18]. The secondary structure was estimated to be around 44% β structure, 3% α structure, 27% turns and 26% random structure. The secondary structure analysis of the protein suggests that the protease is rich in β structures and β turns. The addition of pepstatin A to the protein did not bring about any change in the secondary structures as evidenced by CD spectra.
3.9. Identification of cleavage sites:
Cleavage site of aspartic protease was deduced by following the activation of trypsinogen to trypsin. Trypsin activation was evidenced by enzymatic activity using casein as substrate at pH 7.5 (Result not presented). The activation of trypsin was also demonstrated by the formation of clear band in gelatin-embedded non-denaturing SDS-PAGE (Fig. 6a). The cleavage sites were also identified by analysing the peptides formed by the hydrolysis of oxidised insulin B-chain by aspartic protease. The peptides formed by the hydrolysis were separated by RP-HPLC. Six major peaks were observed in comparison with control insulin peak (Fig. 6b). The peaks from mass spectrometer were fragmented, ‘y’ and ‘b’ values were evaluated and the peptide sequences were deduced (Fig. 6c). The list of peptides formed is shown in supplementary Table 1. From the deduced peptide sequences, the cleavage points were identified as H-L, L-V, F-F, L-Y, A-L and E-A (Fig. 6d).
3.10.. Hydrolysis of commercial substrates:
Industrial substrates like defatted soya flour, gluten, gelatin, skim milk powder and hemoglobin were tested for the hydrolysis by protease.
According to ninhydrin method, amount of free amino nitrogen released was seen to be the highest in hemoglobin and least in skim milk powder. Gelatin, gluten and soya had similar values (Fig. 7a). Protein content in the supernatant, after the reaction, estimated by Lowry’s method was found to be the highest in hemoglobin followed by soya, gluten and skim milk powder (Fig. 7b). Since gelatin was already in solution, amount of protein in the supernatant was the same in blank and test. Degradation of protein could not be quantified by the method employed. After hydrolysis by aspartic protease, the unhydrolysed protein was
precipitated by TCA and the soluble proteins quantified by reading the absorbance at 280 nm. The absorbance of the TCA supernatant was also the highest in hemoglobin followed by soya, gluten and skim milk powder (Fig. 7c). Skim milk powder dispersed in the aqueous solution. Some proteins apparently solubilised in the acidic conditions maintained for the reaction and hence were available for hydrolysis by protease. At pH 4.5, due to low pH the untreated sample got coagulated, but absorbance of the TCA supernatant revealed hydrolysis of the solubilised protein. In case of gelatin, absorbance of the TCA supernatant gave same values for test and blank. Gelatin is completely soluble in the reaction buffer (pH 4.5); unlike in the case of soya and gluten where most of the proteins insoluble under the reaction conditions are solubilised by the enzyme..
3.10.1.. SDS-PAGE pattern of hydrolyzed substrates
Soya protein solubilisation/degradation by aspartic protease was evident from the electrogram (Fig. 7d). A number of low molecular weight proteins (< 30 kDa) absent in untreated soya, were seen in enzyme treated samples. . High molecular weight bands were not seen in any lanes, apparently due to the insolubility of the proteins into the buffer. The band intensity increased with the increase in soya concentration. SDS PAGE analysis of different substrates revealed that gelatin had high molecular weight proteins in control and practically no bands in test, indicating complete hydrolysis. Gluten lane showed number of bands below 50 kDa in control sample; in the enzyme treated sample, almost all the bands were degraded. Skim milk powder showed practically no visible bands in either control or test, indicating that the enzyme has solubilised very little milk proteins into the reaction buffer. Hemoglobin PAGE analysis revealed nearly complete hydrolysis of all proteins seen in control lane (Fig. 7e).
3.11.. Functional properties of defatted soya flour before and after protease treatment
3.11.1.. Protein solubility
Soya proteins were more soluble at pH 7.0 than at 4.0. After enzyme treatment, solubility at pH 4.0 was increased from 6% to 30% and at pH 7.0, solubility increased from 18% to 56% (Fig. 8a).
3.11.2.. Emulsifying property
The EAI values of untreated soya and protease treated soya were 12.83 m2/g and 20.20 m2/g respectively (Fig. 8b). There was an increase in the emulsifying property of soya by 57%, when treated with aspartic protease.
3.11.3.. WHC/OBC
After enzyme treatment, the WHC of soya decreased from 1.865 to 1.365 g/g sample, OBC did not change after hydrolysis by enzyme.
3.11.4.. Foaming properties
Foaming capacity of the protease-treated soya improved by 24%.
4. Discussion
Aspartic proteases have wide applications in food and feed industries. Although a number of authors have reported purification and characterisation of aspartic proteases from various fungal origins, the demand for proteases with industrial qualities such as greater specific activity and thermostability, is high. The aspartic protease in the current study appears to be a potential candidate for industrial applications.
The fungus used to isolate the aspartic protease is identified to be Aspergillus niger based on the macroscopic and microscopic studies and by the analysis of phylogenetic tree
constructed by comparing the amplified ITS DNA sequences. The colony morphology presented by the fungus complimented with the ITS method in identifying the genus and species of the fungus. Aspergillus species such as A. niger, A. oryzae and A. sojae fall under the Generally Recognized as Safe (GRAS) status of US Food and Drug Administration [27]. The apparent purity of the protease from Aspergillus niger has been achieved by 3 step purification. The specific activities of various aspartic proteases have been reported in the literature using different activity expressions, namely, OD units, HUT units, µg tyrosine units and µmol tyrosine units. For comparative purposes, the results are expressed using common activity units (Table 2). Aspartic protease from Aspergillus niger in this investigation has second highest reported specific activity compared to different fungal species. The highest specific activity was reported for aspartic protease from Aspergillus oryzae BCRC 30118 [11]. However, the protease reported here has greater thermostability than the aspartic protease reported with greater specific activity. Although aspartic protease Peptidase R from Rhizopus oryzae has an optimum temperature at 75 oC, it is reportedly stable below 40 oC and prone to inactivation above 40 oC [25]. The aspartic protease in the current study scores over the other fungal aspartic proteases in terms of thermostability (Table 2).
Earlier, Yin et al. [26] have reported the characterisation of aspartic protease from Aspergillus niger BCRC 32720. They have reported the enzyme with similar molecular weight of 47.5 kDa, compared to the one reported here. Based on the N-terminal sequence, they have established the identity of their aspartic protease with Aspergillopepsin A-like Aspartic Endopeptidase from A. niger CBS 513.88. Aspartic protease reported in this study has high similarity towards Aspergillopepsin A-like Aspartic Endopeptidase from A. niger CBS 513.88, based on the peptide sequences obtained upon trypsin and chymotrypsin digestion. Their approach to protein purification is slightly different with cation exchange
chromatography followed by size exclusion chromatography. We have achieved better yield and fold purity with enhanced specific activity of 38.36 X 103 U/mg compared to their specific activity of 23.29 X 103 U/mg. There are differences in pH and temperature profiles and stabilities. The optimum pH and temperatures reported are pH 2.5 and 50 oC respectively, while ours are pH 3.5 and 60- 65 oC, respectively. There are reports of aspartic proteases with optimum pH at 3.0 [11, 18, 28] but the activities at pH 4.0 are low except for protease from Rhizopus oryzae [25]. pH stability (pH 2.7 and pH 6.5) is similar to the reported aspartic protease from Aspergillus oryzae with pH stability between pH 2.5 to 6.0 [18].
The temperature optimum was close to the reported aspartic proteases from Aspergillus species with an optimum at 60 oC for Aspergillus oryzae BCRC 30118 [11], 55 oC for Aspergillus oryzae MTCC 5341 [18] and 60 oC for Aspergillus niger l1 [28]. The protease under the current study retained the activity in the form of ammonium sulfate pellet at 4 oC for more than 12 months (Data not presented). It was also found that NaCl at the concentration of 0.5 M, stabilizes aspartic protease enzyme against temperature (Fig. 3c). The stability at higher temperatures constituted an attractive feature for an enzyme for industrial applications [29].
The purified enzyme has a low Km value of 6.3 mg/mL (98.5 µM) indicating high affinity towards hemoglobin. Similar values are reported by Vishwanatha et al. [18] with Km of 0.8% (8 mg/mL) for acid protease from Aspergillus oryzae and Yin et al. [11] with a Km of
0.12 mM for acid protease from Aspergillus oryzae, both using hemoglobin as substrate.
Higher Km values has been reported by Siala et al. [28] with Km value of 1.02 mM for acid protease from Aspergillus niger using casein as substrate. Much lower Km value of 0.8 mg/mL is observed by Devi et al. [29], for alkaline protease from Aspergillus niger using casein as substrate and 1.28 mg/mL by Ire et al. [30] for acid protease from Aspergillus
carbonarius using gelatin as substrate, indicating higher affinity towards the respective substrates.
The nature of the aspartic protease was established by enzyme inhibition studies using classical protease inhibitors. Inhibitors like PMSF, iodoacetamide and EDTA did not affect the protease activity. However, pepstatin A inhibited the activity reversibly with a Ki value of 0.045 µM. Pepstatin A, a known potent inhibitor specifically and reversibly inhibits most of the known aspartic proteases [31]. The protease enzyme under current study can be considered to be an aspartic protease with very high affinity towards pepstatin A. Earlier reports observe that the pepstatin A inhibits porcine pepsin with a very low Ki value of about 1 X 10 -10 M [32] and human pepsin with a Ki value of 3 X 10 -9 M [33]. Vishwanatha et al.
[18] have reported Ki value for aspartic protease from Aspergillus oryzae as 0.37 µM, using hemoglobin as substrate.
Based on the CD studies, it has been revealed that pepstatin A did not bring about changes in secondary and tertiary structures. The secondary structures as observed by CD measurements, have confirmed the β- rich nature of the protein. The homology modelling based on sequence similarity indicates the dominance of β- structures in the protein. These observations are in conformity with the other reported aspartic proteases [18].
Proteases, according to their nature, can act both as non-specific aggressors to completely degrade the proteins, as well as sharp catalysts that break very specific peptide bonds [8]. To identify the cleavage points, activation of trypsinogen by aspartic protease was checked by zymogram method. Trypsin has optimum activity between pH 7.0 to 9.0 [34]. Since purified aspartic protease has no activity above pH 7.0 and trypsinogen has no activity as such, the clear band in zymogram carried out at pH 7.5, is due to the activity of trypsin, which is formed by the conversion of trypsinogen by aspartic protease. The ability of the fungal acid proteinases to activate trypsinogen at pH 3-4 has been reported earlier [35]. Acid proteinases
from different species of Penicillium, Aspergillus, Rhizopus and Endothia have been studied for the activation of trypsinogen, which requires the cleavage of K-I bond. It can be concluded that the aspartic protease under study can cleave the peptide bond between K-I. However, at higher concentrations of enzyme and with longer incubation time, trypsinogen was completely degraded (results not shown).
By analysing the peptides of the aspartic protease-digested insulin B-chain, the cleavage points were identified as H-L, L-V, F-F, L-Y, A-L and E-A. Apparently, the aspartic protease has broad specificity. The enzyme appears to have greater preference for hydrophobic amino acid residues. Shintani et.al [36] have described that aspartic proteases generally favour hydrophobic amino acids for cleavage. They also report that aspergillopepsin can cleave lysine, leading to the activation of trypsinogen.
The properties of proteins can be modified, under controlled conditions, by partial hydrolysis by selective proteases. The enzymatic hydrolysis potentially affects the functional properties of the proteins, especially in food industries [37]. Proteolysis of food proteins known to improve nutritional value of food has immense application in various food, pharmaceutical and beverage industries [2, 4, 7].
Since the aspartic protease was found to have multiple cleaving sites, hydrolysis of different industrial substrates were performed to establish the potential applications of the enzyme.
Hydrolysis studies, using aspartic proteaserevealed that gelatin, gluten and hemoglobin get almost completely hydrolyzed. Skim milk powder, since only partially soluble, was not available for complete hydrolysis by the enzyme. In case of soya, treatment with enzyme not only improved the solubility of the protein, but also caused the hydrolysis of the solubilised protein (Fig. 7d). An improvement in solubilization and degradation of wheat gluten was observed by Brijs et al. [38] using barley malt protease at pH 4.0, which has application in beer clarification. They reported a 70% improvement in the solubilization of
gluten by the treatment of protease evidenced by the protein estimation and SDS PAGE pattern analysis.
Among food proteins, soya tops the list of most used plant proteins, by virtue of its high quality proteins with supreme functional properties. It is successfully used in diverse food applications such as baked food, cereal food, infant food, beverages and dairy. Treatment of soya proteins with suitable proteolytic enzymes is known to enhance their functional properties like solubility, emulsification properties, water and oil binding capacities and foaming abilities, while preserving the nutritional value of the proteins [39]. From the current study, it is evident that the treatment of defatted soya flour with protease improved the solubility from 6% to 30% at pH 4.0 and 18% to 56% at pH 7.0 (Fig. 8a) indicating a greater degree of hydrolysis. Soya proteins have low solubility in acidic conditions. After hydrolysis by enzyme, generally the solubility of the soya proteins improves, apparently due to the formation of smaller molecular weight peptides which have more solubility in aqueous conditions. Wu et al. [40] report that the papain hydrolysis of soya protein isolates significantly improves solubility from 56% to 94%. Our findings are very close to the values reported by Meinlschmidt et al. [41], wherein, soya protein isolates are treated with various commercial proteases and the protein solubility has been checked. Flavourzyme is found to solubilise 30% and 55% protein at pH 4.0 and 7.0, respectively, while papain hydrolysis yielded 35% and 56% protein at pH 4.0 and 7,0 respectively. They also report higher yields of > 80% protein by using other commercial enzymes such as Alcalase and pepsin.
Wu et al [40] also showed improvement in EAI by treatment of soya protein isolate using papain. It was explained to be due to the exposed hydrophobic moieties that could interact more with lipids. Treatment of our enzyme did not bring about any major improvement in the OBC of soya proteins, while WHC decreased from 1.865 to 1.365 g/g sample. Several reports pointed out that enzyme treatment decreases the WHC of the soya protein isolate,
which is caused by the disturbance of the protein network [41]. Kempka et al. [39] reported decrease in both WHC (6.33 to 3.15 g/g sample) and OBC (2.4 to 1.00 mL/g sample) of soya protein isolates when treated with a protease ‘Flavourzyme’. Reports also indicate that the high solubility of proteins is inversely proportional to water holding capacity [42]. High solubility of the soya proteins due to proteolysis, finds applications in various liquid preparations of soya proteins.
5. Conclusion
The aspartic protease isolated from Aspergillus niger reported in this paper has a great potential in applications like preparation of protein hydrolysates, bio-active peptides and modification of functional characteristics of proteins like soya. With high catalytic activity and thermostability, the protease seems to be a promising candidate for industrial applications.
Acknowledgment
KP and AGA gratefully acknowledge the financial support and Aspergillus cultures from Kaypeeyes biotech pvt ltd, Mysuru, India. KP thanks Mr. Krishna Bhat Kadappu, Managing Director and staff of Kaypeeyes biotech pvt ltd, Mysuru, India, for their support and guidance while carrying out the research. Authors are grateful to Ms. Prathima M, Kaypeeyes biotech pvt ltd, Mysuru, India, for fungal identification work. Authors thank CCAMP, Bangalore, India, for the protein identification work by LC/MS. Authors thank Mr. Shiva Siddappa and Ms. Chaitra V H, Department of Studies in Biochemistry, University of Mysore, Mysuru, India, for their assistance in preparing the illustrations and for technical help..Authors also thank the Director of CFTRI, Mysuru, India, for providing
the facilities and Dr. Gnanesh Kumar B S, Department of Biochemistry, CFTRI, for assistance in interpreting MS data.
Declaration of interest
All the authors have read the manuscript and have no conflict of interest.Contributors
AGA and GMK conceived the project, designed the experiments and critically evaluated and edited the manuscript, KP performed the experiments and wrote the manuscript, SKB performed few experiments and assisted in the interpretation of the results, SAS assisted in cleavage-site identification experiments and helped in design and interpretation of fluorescence and CD studies.
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Table 1: Purification of aspartic protease from Aspergillus niger
Ammonium sulphate pellet 2 7,61,510 94.8 8,032 2.17 87
Gel filtration (Biogel) 13 5,64,655 28.6 19,743 5.35 65
Ion-exchange (DEAE
sepharose) 80 4,91,040 12.8 38,362 10.41 56
Note: Data represents typical elution profile of three separate experiments.
Table 2: Comparison of activity and thermostability of aspartic protease with other reported aspartic proteases.
Culture Activity expression Thermostability Reference
1 Aspergillus 2,52,266 1,06363 1,17,620 4- 35 oC [11]
oryzae BCRC
30118
2 Aspergillus 43,658 18,555 20,247 40 – 57 oC [18]
oryzae
MTCC 5341
3 Rhizopus oryzae 57,500 24,243 ≤ 40 oC [25]
26,682
4 Aspergillus 50,214 21,172 23,290 ≤ 40 oC [26]
niger
BCRC 32720
5 Aspergillus 80,639 34,000 38,362 40 – 60 oC Current
niger study
Note: OD= optical density; HUT= hemoglobin units of tyrosine
Captions to illustrations
Fig 1: Identification of the fungal culture by morphological features and molecular approach. a) Colony of Aspergillus niger on potato dextrose agar. b) Colony on reverse side of the plate. c) Conidiophore of Aspergillus niger under light microscope at 40X magnification. Note the spores arranged circumferentially covering the vesicle. d) Spores at 40X magnification. Colour of the spores varies from light-brown to black, depending on the age of the spore. Young spores are lighter in colour while older ones tend to grow darker. e) Phylogenetic tree of the fungal culture showing the nearest neighbours. The culture was identified based on the Internal Transcribed Spacer (ITS) region of the genomic DNA. The evolutionary history was inferred using maximum likelihood method. K1 is the culture under study. The culture was identified to be Aspergillus niger and the nearest neighbor was identified to be Aspergillus niger strain IR3_11 with accession number MK461093.1.
Fig 2: Purification of aspartic protease from Aspergillus niger. a) Gel permeation chromatography using Biogel-P100. b) Ion-exchange chromatography using DEAE sepharose. c) SDS-PAGE pattern of purified protease. 10% polyacrylamide gel was used and visualisation was carried out by silver staining. Lanes 1: Marker; 2: Crude extract; 3: Biogel protease fraction; 4: DEAE Sepharose protease fraction. d) Native acid PAGE and zymogram: Lanes 1: Native acid gel pattern; 2: Zymogram with hemoglobin. e) Standard graph for the estimation of molecular weight of the aspartic protease. Molecular weight markers used were (closed circles) Bovine serum albumin (66 kDa), DNase I (31 kDa), Trypsin (23.3 kDa) and Lysozyme (14.6 kDa). Molecular weight of aspartic protease (open circle, dotted line) was found to be around 50 kDa.
Fig 3: Kinetic parameters of purified aspartic protease: a) Optimum pH and pH stability were checked from pH 2.0 to 8.0. Buffers used were 100 mM of glycine-HCl for pH 2-2.7, citrate for pH 3 to 3.5, acetate for pH 4 to 5.5, phosphate for pH 6 to 8. Assay was carried out at 60 oC. b) Optimum temperature and temperature stability were checked at pH 4.0 at temperatures ranging between 30 to 75 oC. c) Effect of NaCl on thermal inactivation kinetics of aspartic protease. The enzyme was incubated at different temperatures in the range of 50 to 60 oC in the presence and absence of 0.5 M NaCl. Residual activity was assayed under standard conditions. d) Km and Vmax of aspartic protease was determined with hemoglobin substrate using Lineweaver- Burk plot. e) Lineweaver- Burk plot with pepstatin A: Reaction was carried out by varying substrate and inhibitor concentrations and 1/V was plotted against 1/[S]; f) Dixon plot: 1/V versus pepstatin A concentration was plotted to calculate the Ki value of purified protease using hemoglobin as substrate in presence of different concentrations of pepstatin A ranging from 0.035 to 0.14 µM.
Fig 4: a) .Alignment of peptides of aspartic protease from Aspergillus niger. (1) Amino acid sequence of apergillopepsin A-like aspartic endopeptidase, (2) Amino acid sequence of aspartic protease from Aspergillus niger(current study). The purified protease was sequenced by trypsin and chymotrypsin digestion, followed by LC-MS/MS. The peptides obtained shared 85% identity with the reported aspergillopepsin A-like aspartic endopeptidase (Aspergillus niger CBS 513.88). Missing amino acids are marked in red colour. The apparent active site residues are boxed (D101 and D283). b) Structural model of aspergillopepsin A-like aspartic endopeptidase. Using the reported sequence, homology model was generated using Phyre 2 web server. 3-D model was visualized using Jmol. β structures are shown in golden yellow colour and α helices are in magenta colour.
Fig 5: Spectral characterisation of aspartic protease in the presence and absence of pepstatin
A. a) Fluorescence spectra of the purified aspartic protease. Aspartic protease sample was
prepared in 50 mM acetate buffer pH 4.0. Fluorescence excitation was carried out at 280 nm and emission was observed between 300 to 420 nm at 25 oC in the presence and absence of pepstatin A (20 µM). Emission maximum of protease was at 325 nm. b) CD spectra. Near UV-CD spectra were generated using 1.04 mg/mL protein in the presence and absence of pepstatin. c) Far UV-CD spectra of aspartic protease with 0.27 mg/mL protein in the presence and absence of pepstatin (20 µM) carried out at 25 oC. Accumulations of three runs are shown.
Fig 6: Cleavage specificity of aspartic protease. a) Activation of trypsinogen. i) Non- denaturing SDS-PAGE. Lanes 1: Protein marker; 2: Trypsinogen; 3. Aspartic protease; 4: Trypsin, activated by aspartic protease. ii) Zymographic display of trypsin, activated by aspartic protease. Lanes 1: Trypsin, activated by aspartic protease. Trypsinogen (1mg/mL) was incubated with 1 µL of purified aspartic protease at 60 oC for 1 min. After the reaction,
10 µL sample was immediately drawn, mixed with SDS buffer and loaded to 10% polyacrylamide gel. For zymogram, 0.1% gelatin was incorporated into the resolving gel and electrophoresis was carried out as described in methods section. b) Cleavage specificity on oxidised insulin B-chain. RP-HPLC profile of i) native oxidised insulin B-chain, ii) oxidised insulin B-chain hydrolysed by aspartic protease. c) Representative electrogram of a peak (726) from mass spectrometer fragmented using CID (collision- induced dissocistion).The ‘y’ and ‘b’ values were evaluated and the peptide sequence was deduced d) Amino acid sequence of insulin B-chain with arrows indicating the cleavage points. Insulin B-chain (1mg/mL) was treated with purified aspartic protease at pH 4.0 for 10 min at 60 oC. 20 µL reaction mixture was loaded to C18 column and resolution of the peptides was carried out using 0.1% TFA- acetonitrile solvent system. Peptide peaks were subjected to mass spectrometry to deduce the sequence.
Fig 7: Hydrolysis of commercial substrates by aspartic protease. a) Degree of hydrolysis by ninhydrin method expressed as µmoles amino nitrogen/mL. b) Protein estimation in the reaction supernatant by Lowry’s method. c) Absorbance of the TCA supernatant at 280 nm using reaction mixture. Substrates were used in the concentration of 2 to 10 % and enzyme dosage was kept constant at 1000 U/mL. d) SDS-PAGE pattern of defatted soya flour with and without protease treatment. Lanes. 1: Marker; 2: Soya 2% (C); 3: Soya 2% (T); 4:
Soya 4% (C); 5: Soya 4% (T) ; 6: Soya 6% (C) ; Lane 7: Soya 6% (T) ; 8: Soya 8% (C);
9: Soya 8% (T) ; 10: Enzyme control. e) SDS PAGE pattern of commercial protein substrates (2% suspension) treated with protease at the dosage of 1000 U/mL. Lanes; 1: Marker; 2: Gelatin (C); 3: Gelatin (T); 4: Gluten (C); 5: Gluten (T); 6: Skim milk powder (C); 7: Skim milk powder (T); 8: Hemoglobin (C); 9: Hemoglobin (T); 10: Enzyme control. Note: C= no enzyme treatment; T= enzyme treated.
Fig 8: a) Protein solubility of defatted soya flour before and after enzyme treatment. Enzyme dosage was at 1000 U/mL. Samples were dispersed in distilled water and pH was set to pH 4.0 and 7.0. The protein content in the supernatant was estimated by Lowry’s method. A control sample was treated with 0.5 N NaOH and the protein content in this sample was taken as 100%. b) Emulsifying activity Pepstatin A index (EAI) of soya before (control) and after enzyme treatment (test). EAI, expressed in m2/g, of the soya before and after treatment with aspartic protease was checked. Emulsion was generated using refined sunflower oil at 30 oC.