Selection of LAAO producing strain
The fungal strains were initially grown on Czapek Dox synthetic agar media by spread plating method. The single culture of different types of fungi was further isolated by continuous streaking and spread plating methodology. On the basis of the results obtained after treating isolates with different L-amino acids, it was concluded that the Aspergillus terreusMZ769058 isolate is the potent fungal strain that has given high LAAO activity with L-tyrosine amino acid (among different amino acids).
Purification and biochemical characterization of LAAO from Aspergillus terreusMZ769058
Purification of LAAO
The enzyme precipitation had been carried out using ammonium sulfate with saturation level of 0-30%, 30-60%, 60-90% as shown in Fig 1. The maximal specific activity of purified L-Amino acid oxidase was achieved as 87.9 U/mg in compare to specific activity of crude extract as 51.9 U/mg. The partially purified protein has demonstrated enzyme activity 242.8 U with total protein as 2.76mg. The purification fold of LAAO enzyme was estimated nearby 1.69 folds at 30–60% saturation. The enzyme showed greatest specific activity for saturation level of 30-60%with ammonium sulfate. The purification parameters for production of LAAO enzyme in crude extract, ammonium sulfate precipitation and purified extract had been represented in Table 1. Ion-exchange chromatography was carried on (Diethylamine amino ethyl) DEAE cellulose A-50 column, and the fraction containing LAAO activity had been collected. The content of total protein and enzyme activity had been further determined for all these fractions (Fig 2). The highly purified enzyme showed higher purification fold as 2.55 and higher specific activity as 132.5 (U/mg). SDS-electrophoresis was further carried out for estimation of molecular unit of subunit of enzyme by observing thin and fine band. The wells of a polyacrylamide gel with a 12 percent concentration were filled with the protein samples. Under reducing conditions, the purified LAAO migrated as a single band on the SDS PAGE. The molecular weight of purified LAAO enzyme was estimated at 90 kDa and 180 kDa by SDS electrophoresis and NATIVE PAGE respectively (Fig 3). These data confirmed the homodimeric nature of LAAO enzyme protein. These findings were differ for molecular weights of snake venom LAAO such as Daboia russelliisiamensis LAAO (58kDa) and Agkistrodon contotrixlaticinctus LAAO (60kDa) as well as Rhodococcus opacus LAAO (53.2kDa) by SDS PAGE (Geueke& Hummel, 2002) (Souza et al. 1999). On examination of gel filtration and SDS-PAGE in both reducing and non-reducing conditions, the molecular mass of snake venom was 50-70kDa. Snake venom had typical homodimeric glycoproteins with a molecular mass of about 110-150kDa (Stabeli et al. 2004). Singh et al. (2014) had also isolated LAAO from Aspergillus fumigatus and determined its molecular mass as 55 kDa by SDS-PAGE.
Biochemical characterization of L-Amino acid oxidase
L-Amino acid oxidase sequence analysis
The SDS page band with monomeric LAAO from Aspergillus terreusMZ769058 was digested with trypsin and analyzed using matrix-assisted laser desorption ionization time-of-flight (MALDI TOF). The tryptic digested LAAO enzyme had generated a spectrum of peaks representing the m/z ratio of each peptide fragment as shown in Fig 4. The automated analysis of this spectrum had been performed using software Protein Prospector Auto MS-Fit. Fragmentation reaction will exhibit the formation of immonium ions as substituted and modified protonated α-amino acids as shown in Table 2. Amino acids of β-, γ-, and δ-groups are being not involved in the formation of any immonium ions. The fragmentationof these groups have been subsequently carried out with the loss of H2O and NH3molecules.These fragmentation reactions have been finally shown with the specific indicator peaks in spectrum of MALDI/TOF tandem as shown in Fig 4. The presence of specific amino acids has been analyzed for each peak using the fragmented data given by (Gogichaeva and Alterman, 2019) in Table 2. These peaks indicate the presence of glycine, alanine, serine, proline, valine, threonine, leucine, asparagine, aspartic acid, lysine, glutamine, glutamate, methionine, histidine, phenylalanine, arginine, tyrosine within m/z ratio from 85 to 250 as peptide sequences in structure of LAAO enzyme.
Effect of pH on LAAO activity
The effect of pH on the activity of purified extract of LAAO enzyme had been studied by varying pH values form 3-10 and results were plotted in Fig 5. The activity of LAAO enzyme had been obtained maximum as 193.5 U/l at optimum value of pH 6.0. The activity of the enzyme decreased at pH 8 with decline level of stability of LAAO enzyme. This decline in stability may be caused by inactivation of amino acid residues in three-dimensional structure of LAAO enzyme. L-Amino acid oxidase from Aspergillus fumigatus was stable over a wide pH range of 5.6-9.2, with higher enzyme activity at pH 7.2 (Singh et al. 2014). According to Bohmer et al. (1989), L-glutamate oxidase from S.endus was stable between pH values of 5.5-7.5, but LAAO from R.opacus showed at ability for pH values of 8-9 (Geueke& Hummel, 2002).
Thermal stability and temperature effects on LAAO
The enzyme was active throughout a wide range of temperatures and showed maximum activity (227.08U/l) at optimum temperature 30°C as shown in Fig 6. The enzyme showed active forms for higher temperature ranging from 40°C-50°C. The enzyme activity had started to decrease after 15 minutes of incubation and completely inactivated after 30 minutes.These studies showed stability of Crotalus adamanteus LAAO at 70°C in the presence of L-leucine with decreased activity after 60 minutes of incubation (Wellner& Meister, 1960). The inhibtion of LAAO enzyme from R.opacus was also observed by incubating the enzyme at a temperature above 37°C. This enzyme was completely inhibited for incubation for 5 minutes at 50°C (Geueke& Hummel, 2002).
Enzyme kinetics and substrate specificity of LAAO
By evaluating the LAAO activity using various L-amino acids in the reaction mixture, the substrate specificity of the isolated enzyme was estimated. The reaction mixture was supplemented with the ideal concentration of 20mM substrates, such as L-tryptophan, L-alanine, L-cystine, L-threonine, L-leucine and incubated under assay conditions. The enzyme activity of purified LAAO enzyme has been represented in Fig 7 for using different L-amino acid substrate. The highest activity of LAAO enzyme was observed for L-tyrosine amino acid and least activity was observed for L-cystine amino acid (Table 3). Purified enzyme was incubated with various substrate concentrations from 20mM-200mMto determine the kinetic constant values Michaelis-Menten (Km) and Vmax (maximum velocity). The rectangular hyperbolic and Line Weaver Burk plot was used to calculate the values of Km and Vmax as shown in Fig. 8 and Fig. 9 respectively. The steady-state velocity (V) and substrate concentration (S) of the enzyme-catalyzed reaction exhibit a hyperbolic connection through Michaelis Menten equation as shown below:
Where Km is Michaeli's constant and Vmax is the maximum velocity.
The strong affinity of the enzyme for the substrate was demonstrated by the low value of Kmwith higher value of Vmax. The following equation was used to determine the kinetic parameters using the inverse values of 1/V and 1/S in the Line weaver Burk plot.
The value of Michaelis parameters of Km and Vmax was estimated as 26 mM and 250 µmole/min/mg proteinsrespectively. The enzyme's catalytic efficiency (Kcat) value was determined to be 2.5 µmole/min/mg. Singh et al. (2014) had reported the substrate specificity of LAAO enzyme from Aspergillus fumigatus for the hydrophobic aromatic amino acids like L-tyrosine and L-phenylalanine. The substrate specificity of A. oryzae LAAO was examined in a different study by El-Sayed et al. (2013). It was shown that the enzyme showed a high catalytic specificity and affinity for L-lysine with values of Km 3.3mM and Kcat 0.04s-1.
Metal ions and inhibitors affect LAAO activity
Metal ions play a significant role in the stability of LAAO because cations are necessary cofactors for enzyme activity and results have been shown in Fig. 10. Metal ions such as FeSO4 (85.4 %), Na2MO4 (81.2 %), and CuSO4 have a negative effect on the activity of LAAO enzyme. Metal ions like MgSO4, H3BO3, and ZnSO4 have very little effect on L-Amino acid oxidase activity. The activity of LAAO enzyme was strongly inhibited at a concentration of 10 mM CaCl2.The inhibition of enzyme was observed with α-napthol (34.4%), EDTA (34.2%), Glycine (39%) sodium azide (41.4%), and riboflavin (85.3%) and data were shown in Table 4. El-Sayed et al. (2013) had observed the effect of inhibitors on the enzyme LAAO from Aspergillus oryzae. The enzyme activity was decreased upto 36.8 percent with the component of hydroxylamine.
Study of LAAO using Fourier Transform Infrared (FTIR) spectroscopy
L-amino acids are converted into products containing ammonia, hydrogen peroxide, and α-keto acid during the catalytic reaction. The presence of the amine and aldehyde groups in the purified sample was examined using Fourier transform infrared spectroscopy. The functional group with C-H stretch had been represented by the peak at 2927.95, C=O stretch represented by the peak at 1745.25, and C-O stretch had been illustrated by the peak at 1078.64 as shown in Fig. 11.