Electronic Supplementary Information
Green Synthesis of Silver Nanoparticles from Purple Acid Phosphatase apo-enzyme
isolated from a new source Limonia acidissima
Omkar Pawar, Neelima Deshpande, Sharda Dagade, Preeti Nigam-Joshi, Shobha Waghmode
1. Purification, characterization and physical properties of Wood Apple Purple Acid
Phosphatase (waPAP)
2. Figures
Figure S1: Calibration curve for D-glucose by phenol-sulphuric acid.
Figure S2: Gel filtration profile of PAP on sephadex G-100 (a) Activity units; (b) Protein
concentration
Figure S3: SDS PAGE of PAP.
Figure S4: Calibration curve for molecular weight estimation of PAP by using gel filtration
column.
1. Purification, characterization and physical properties of Wood Apple Purple Acid
Phosphatase (waPAP
Wood apple is a novel source for purple acid phosphatase and the enzyme was purified by using
ammonium sulphate fractionation method followed by gel filtration column. We have replaced
DEAE anion exchanger column separation step by gel filtration on Sephadex G-100 in between
affinity chromatography step and is worked out for better separation and concentration of
glycoprotein PAP. These changes result in increase of protein concentration in purified enzyme.
As affinity column of Con A seralose possesses carbohydrate binding activity, total carbohydrate
of glycoprotein PAP is estimated from standard D-Glucose plot shown in fig.S1. It is Phenol
Sulphuric Acid method in which hot acidic solution of glucose is dehydrated to Hydroxymethyl
furfural which forms green colored product with phenol at λmax 490 nm.
Gel filtration profile of waPAP on sephadex G-100 (purified fraction from step) is shown in
fig.S2 (a, b). Electrophorogram of polyacrylamide gel electrophoresis at pH 8.0 shows
characteristic single blue spot in lane one for PAP purified fraction from last step 7 [Fig.S3].
Single band is criterion of homogeneity compared to inhomogeneous protein seen from multiple
bands in fractions of former steps. Here separation based on shape and size of protein where as it
depends only on size in SDS-PAGE.
Molecular mass determination:
Gel filtration on sephadex G-100 gel was followed to determine molecular mass of isolated
purified waPAP. Gel filtration was used for series of standard proteins with known molecular
weights and compared to waPAP. From plot of logatithm of molecular mass vs. elution volume
as shown in fig.S4, molecular mass of waPAP was estimated as 110kD.
Biological Activity Assay:
As the enzyme is non-specific and it will catalyze phosphate ester hydrolysis on many different
substrates. So it is possible to use artificial phosphate ester p-nitro-ф-phosphate (p-NPP). It is a
good substrate to use since the product formed after ester hydrolysis is p-nitrophenol which can
be easily measured.
In first part of reaction, the catalyst is the phosphate and the products are p-nitrophenol and
inorganic phosphate or Pi. Second part is the way to detect p-nitrophenol formed as a product in
the phosphatase catalyzed reaction. In this part NaOH is added to end the phosphatase assay after
a given reaction time of 10 mins. The hydroxide reacts with p-nitrophenol to remove the
phenolic proton and p-nitrophenolate is formed which a yellow color compound is absorbing at
420nm. Since nothing else in the reaction mixture absorbs light at this wavelength it is easy to
measure in a quantitative way the amount of p-nitrophenol formed in the enzyme catalyzed
reaction. This type of enzyme assay is called as an ‘end-point’ assay since the activity of the
enzyme is evaluated after a given time of reaction. End point enzyme assays are often used in
clinical and other analytical, bio-chemical assays since they are easy to do and useful for
detecting the presence of an enzyme in a sample in quantitative manner.
2. Figures
Figure S1: Calibration curve for D-glucose by phenol-sulphuric acid
Figure S2: Gel filtration profile of PAP on sephadex G-100 (a) Activity units; (b) Protein
concentration
Figure S3: SDS PAGE of PAP
Figure S4: Calibration curve for molecular weight estimation of PAP by using gel filtration
column.