Question

Based on the document below,

1. Describe the hypothesis Chaudhuri et al ids attempting to evaluate; in other words, what is the goal of this paper? Why is he writing it?

2. Does the data presented in the paper support the hypothesis stated in the introduction? Explain.

3.According to Chaudhuri, what is the potential role of thew alkaline phosphatase in the cleanup of industrial waste.

CHAUDHURI et al: KINETIC BEHAVIOUR OF CALF INTESTINAL ALP WITH PNPP 8.5, 9, 9.5, 10, 10.5, 11, 11.5 and 12). In a typical reaction, the buffer along with enzyme and substrate in a total volume of 3.0 mL were incubated at 37°C for 30 min. However, the reaction mixtures were held at different temperatures (30, 45, 60, 75 and 90°C) and incubation periods (30, 60, 120, 180 and 240 min) whenever required. The reaction was stopped with the addition of 1.0 mL of 1 M-K HPO4-KOH buffer, pH 10.4. Initial rates of hydrolysis were determined from time-dependent release of p-nitrophenol (€405 = 1.65 x 104 M'cm') Michaelis-Menten parameters were determined using Lineweaver-Burk plots. All hydrolytic reactions were performed in triplicate. essential for the catalytic activity and structural stability of ALP. Mammalian ALPs show 25-35% sequence identity with the E. coli enzyme in those regions of the protein that assume a-helix and B-strand secondary structures and also those regions that are critical for catalytic activity. The catalytic residues i.e., Asp91, Ser92 and Arg166 and ligands coordinating the divalent metal ions (Zn* and Mg +) are all conserved. These structural similarities suggest that mammalian ALPs may catalyze hydrolysis of both pyrophosphate and orthophosphate moieties via a mechanism similar to that of E. coli enzyme The ability of purified human liver and small intestinal ALPs to act on a number of organic di- and tri-phosphates of physiological significance is reported previously. The ALP from calf intestinal mucosa (CIAP) is one of the most active ALPs known. Although the kinetic behaviour of CIAP with 4-methylumbelliferyl phosphate is reported, in the present paper, we report the kinetic behaviour of CIAP with p-nitrophenyl phosphate (PNPP) and the effects of pH, buffer, substrate concentration, temperature and incubation time on the hydrolysis of PNPP by the enzyme, in order to understand the efficiency of CIAP for enhancing bioprecipitation of heavy metals. Further, we also demonstrate precipitation of copper by CIAP-mediated catalysis of PNPP. Bioprecipitation of Cu? Typical reactions containing 50 mM Tris-HCI buffer (pH 11), 15 mM PNPP, 50 ppm or 100 ppm Cu* mono-ion solution (prepared from CuCl) along with the enzyme in a volume of 2.0 mL were incubated for 30, 60 or 120 min at 37°C. After incubation, the reactions were stopped by adding 10 M NaOH (116 uL) and the reaction mixtures were centrifuged (15000 x g) for 15 min to precipitate metal-phosphate complex. The supernatant was collected and used for analysis by atomic absorption flame spectrophotometer (AAS). Quantification of metal precipitated was calculated as the difference between the initial and final concentration of metal present in the supernatant. Appropriate control reactions without enzyme were also carried out. Percentage of precipitation of metal was calculated by the following formula: X = (a-b)/ a x100 where X = % of copper precipitation, a = Initial conc. of copper in the aliquot, and b = Final conc. of copper in the aliquot (i.e., in the supernatant) Materials and Methods Two buffers viz., 50 mM Tris-HCl and 100 mm glycine-NaOH were used during the course of experiments in the range of pH 8 to 12. A purified ALP from calf intestinal mucosa (Invitrogen) was used all throughout. One unit of enzyme hydrolyzed 1 umole of PNPP in 1 min at 37°C. The enzyme was stored at -20°C till use. PNPP (Sigma) was used as substrate for the CIAP. The substrate as well as the working stock was stored at 4°C. Statistical analysis The data were statistically analyzed using SPSS 17.0 software (Mean + SD, n = 3). The values for Cu precipitation were given as mean of triplicates with SEM (standard error of the mean). Results and Discussion Determination of CIAP-catalyzed hydrolysis of pNPP The catalytic activity was followed by monitoring the increase in the absorbance at 405 nm from p-nitrophenol (PNP) generated in an enzyme- catalyzed hydrolysis of PNPP using Shimadzu UV spectrophotometer. All hydrolytic reactions for studying the effects of different substrate concentrations (0.2, 0.5, 1, 1.5, 2, 2.5, 3 and 4 mM), pH and time of incubation were performed in 50 mM Tris-HCl and 100 mM glycine-NaOH buffers (pH 8, Kinetic study of CIAP with PNPP in Tris-HCI buffer (50 mm) and Glycine-NaOH buffer (100 mm) Effect of pH, substrate concentration and buffer The activity of ALP like for any other enzyme is known to be pH-dependent. In this investigation, the enzymatic activity as a function of pH and buffer was

Answer 1

1.

report the kinetic behaviour This paper of CIAP (active ALP) with p-nitrophenyl phosphate and the effects of pH, buffer Substoute concentoülion, tempauturi and encubation time on the hydrolysis of PNPP by the enzyme, to understand the efficiency of CeAP for enhancing nu bioprecipitation of heavy metals. It also demonstrates the prupitation of copper by CIAP-mediated catalysis of PNPP.

2.The study came to the conclusion that the enzyme at different conditions(pH,Buffer,substrate concentration) it undergo some conformational changes leading to the changes in the cataalytic activity.These changes can be analysedand CIAP can precipitate heavy metals used for industrial purposes.Thus the data in the paper supports the hypothesis

3.CIAP can precipitate Cu2+.It has the ability of inorganic phosphate driven precipitation catalysed by CIAP.This effect can be used for the clean up of industrial waste.