The
Abstract
Industrial effluents from tanneries and electroplating industries from small‐ and large‐scale sector industrial plants contain substantial amount of toxic heavy metal, which pollutes rivers and lakes, land, air and sea leading to imbalance of ecosystem and certain health issues to humans, animals as well as plants. The worldwide environmental regulations stipulate the reduction of heavy metals in the effluents to permissible levels before discharging into water bodies. Enzyme‐mediated precipitation of heavy metals affords a novel eco‐friendly method for remediation of toxic heavy metals from various industrial effluents like tannery, electroplating and dye industries. This chapter has paid attention to bacterial alkaline phosphatase (BAP) from Escherichia coli C90 and calf‐intestinal alkaline phosphatase (CIAP), which catalyses phospho mono‐ and diesters and produces inorganic phosphate (Pi). The Pi thus generated precipitates the heavy metals as metal‐phosphate complexes. The kinetic behaviour of both the enzymes with para‐nitrophenyl phosphate, ascorbic acid 2‐phosphate and α‐naphthyl phosphate was investigated at various pH regimes from 8 to 11. The chapter also explains in detail the descriptive information on the capability of BAP‐ and CIAP‐mediated precipitation of heavy metals, which is desirable and convenient method for the toxic heavy metals such as chromium, cadmium, nickel and cobalt.
Keywords
- bacterial alkaline phosphatase
- calf‐intestinal alkaline phosphatase
- heavy metals
- enzyme kinetics
- bioremediation
1. Introduction
Phosphatase‐mediated bioremediation of heavy metals plays an important role in the bioremediation of industrial, municipal and nuclear wastewater. Heavy metals, such as Li3+, Mn2+, Cu2+, Zn2+, Cd2+, Ba2+, Hg2+, Pb2+, Hg2+, Ag2+, Cd2+, A13+, Ni2+, Cu2+ and Pb2+, are found in various industrial effluents. High quantity of Cd2+, Ni2+, Co2+ and Cr3+/6+ was found in tannery and electroplating effluents which creates high risk in the environment which is one of the important issues in Asian countries. The use of alkaline phosphatase offers great promises in vast research field and could be a model to study metal ion‐dependent catalysis to address such a kind of problems in the application field, viz., environmental biotechnology, molecular biology and immunodetection. Alkaline phosphatases are commonly found from bacteria to higher mammals, which efficiently hydrolyze several mono‐ and diesters. They are nonspecific and react with a variety of substrates from natural to synthetic compounds. There has been a considerable effort in recent years towards the application of alkaline phosphatases for bioremediation of heavy metals and radionuclides from industrial and nuclear wastes. In order to extend bioremediation possibilities to heavy metals contaminated wastes, this present research work explores the advantages of using bacterial (
2. Phosphatase enzymes
Phosphatase is a hydrolase enzyme. Phosphatases play a very important role in the phosphate metabolism of the organism by hydrolysis of polyphosphates and organic phosphates [1]. The essential feature of phosphatase is that the enzymes have wide specificity. Phosphatases cleave phosphate ester bonds, which play an important role in the hydrolysis of polyphosphates and organic phosphates. They are also responsible for phosphate transport within the cells [2]. Phosphatase enzymes are used by many soil microorganisms to access organically bound phosphate nutrients.
Two types of phosphatase enzymes are as follows:
Acid phosphatases
Alkaline phosphatases
2.1. Alkaline phosphatase
Alkaline phosphatases (APase or AP or ALP; orthophosphoric monoester phosphohydrolase, EC 3.1.3.1) are common in a wide variety of bacteria and mammals which efficiently hydrolyse several mono‐ and diesters [3]. Alkaline phosphatase, which hydrolyses organic phosphate esters and releases inorganic orthophosphate, is a dimeric metallic enzyme [4]. In bacteria, alkaline phosphatase is present in the periplasmic space. The three‐dimensional structure of
It can hydrolyse a wide variety of substrates, that is, synthetic as well as natural substrates. The substrates include ATP, ADP, AMP, PPi, glucose‐1‐phosphate, glucose‐6‐phosphate, fructose‐6‐phosphate, b‐glycerophosphate, bis‐(p‐nitrophenyl)‐phosphate, and so on. [12]. Based on the X‐ray crystallography study, a revised mechanism of hydrolysis of a substrate by alkaline phosphatase has been proposed by Stec et al. [14] (Figure 3).
This causes a second inversion of configuration at the phosphorus centre. Nucleophilic Ser102 gets regenerated and reprotonation of Ser102 by the Mg‐coordinated water molecule may facilitate the departure of the inorganic phosphate product from the non‐covalent E.Pi complex. Alternatively, the phosphate group, for its release, may directly get protonated by the Mg‐coordinated water molecule [14].
According to this proposed mechanism, the active site of the free enzyme (E) is occupied by three water molecules and the hydroxyl group of S102 forms hydrogen bond with a hydroxide ion which is coordinated by Mg. As a result of the enzyme‐substrate complex (E.ROP) formation, the ester oxygen atom coordinates with Zn1 and Zn2 in addition to the guanidinium group of Arg166 [9]. The site opposite to the leaving group gets occupied by Ser102. As a result of binding of the phosphomonoester (ROP) to form the enzyme‐substrate complex (E.ROP), the Ser102 OƔ becomes fully deprotonated for nucleophilic attack with the consequent transfer of the proton to the Mg‐coordinated hydroxide group to form Mg‐coordinated water molecule. Coordination of Zn2 stabilizes Ser102 OƔ in its nucleophilic state. The activated hydroxyl group of Ser102 attacks the phosphorus centre of the substrate in the enzyme‐substrate complex (E‐ROP) to form a covalent serine‐phosphate intermediate (E‐P) resulting in inversion of the phosphorus centre and the loss of the leaving group (RO¯). A nucleophilic hydroxide ion coordinated to Zn1 attacks the covalent serine‐phosphate intermediate (E‐P), resulting in the formation of non‐covalent enzyme‐phosphate complex [14].
Alkaline phosphatase catalyses the cleavage of phosphate group from
When reacting with heavy metal:
3. Kinetic behaviour of calf‐intestinal and bacterial alkaline phosphatase with pNPP
3.1. Effect of pH and substrate concentration on hydrolysis of CIAP with pNPP
The effects of pH on the kinetics of CIAP‐catalysed pNPP hydrolysis reaction were determined in Tris‐HCl buffer. CIAP activity was highest at pH 11 in Tris‐HCl buffer, whereas in glycine‐NaOH buffer the highest activity was recorded at pH 9.5. The rate‐determining step at pH 8 was attributed to the non‐dissociation of phosphate from the non‐covalent enzyme‐phosphate complex. At pH 8, the rate of hydrolysis of 4‐nitrophenyl phosphate was increased, in the presence of Tris‐Cl [15] and also with the substrate 4‐methylumbelliferyl [16]. Experimental data show that even at pH 11, Tris buffer was not inhibited. Despite the increase in substrate concentration at pH 11, no reduction in the enzyme activity was observed; by contrast, the activity observed was much higher than that of the activity observed between pH 8.5 and 11 (Figure 4). This states that the enzyme exhibits functionally stable conformations at pH 8–8.5 and at pH 11. In the mean time, at a wide substrate concentration between 0.2 and 4 mM the enzyme showed a relatively stable catalytic activity. Similar increase in the pH to 9 was reported previously with ALPs from dog’s intestine, kidney and liver [17]. Moreover, at pH 11, the enzyme also showed an optimum temperature at 45°C instead of 37°C. Meanwhile, the enzyme ceased its activity upon incubation at 45°C beyond 60 min, whereas at 37°C the activity was recorded till 2h. This states that CIAP at alkaline pH from 8 to 11 undergoes slight conformational changes. The enzyme might be attaining kinetically stable conformations at pH 8.5 and 11, as evident from the experimental findings [18].
The effect of substrate concentration on the CIAP activity across a wide range of pH values between 8 and 11 was observed. The observations were in sharp contrast to the observations made by Fernley and Walker [19], who reported inhibition of the enzyme at 0.2–4 mM concentrations of the substrate. From the enzyme‐substrate saturation kinetics, it was noted that the saturation of the enzyme for substrate binding occurred at 1.5 mM, regardless of the pH 8.5–11 in Tris‐HCl buffer. Inhibition in enzyme activity was observed at increasing substrate concentrations above1.5 mM pNPP at all pH range except pH 11 where stable activity was noticed. At pH 11, dissociation of pNP from pNPP or transfer of the enzyme to another quaternary state might be highly facilitated [18].
3.2. Determination of V max, K m and K cat at different pH for CIAP activity with pNPP
Over a pH range of 8–11,
3.3. Effect of pH and substrate concentration on BAP activity with pNPP as substrate in Tris‐HCl buffer
Buffers play a vital role in the catalytic activity of alkaline phosphatase [23]. In the presence of Tris during the hydrolysis of 4‐nitrophenyl phosphate by alkaline phosphatase at pH 8.0, the rate of 4‐nitrophenol liberation was increased with concomitant phosphorylation of Tris [24]. On the contrary, it has been discussed that the dephosphorylation of the enzyme would take place at a slower pace under alkaline pH than the catalysis of the substrate. It has also been noted that the activity of BAP, like other enzymes, is pH dependent. BAP upon hydrolysis in Tris‐HCl buffer resulted in higher activity at pH 8.5 till pH 10 at 37°C. The activity of enzyme increased linearly with the increase in substrate concentration [18] (Figure 5).
3.4. Determination of V max, K m and K cat at different pH for BAP activity with pNPP
The Michaelis‐Menten (
pH | ||
---|---|---|
8 | 0.73 | 7.4 × 10−3 |
8.5 | 0.82 | 1.5 × 10−3 |
9 | 1.48 | 3.5 × 10−3 |
9.5 | 0.33 | 4.66 × 10−3 |
10 | 1.33 | 1.67 × 10−3 |
10.5 | 0.83 | 1.75 × 10−3 |
11 | 0.73 | 7.5 × 10−3 |
4. Bioprecipitation studies using calf‐intestine alkaline phosphatase
Kinetic parameters obtained for BAP were analysed under standard conditions which showed comparable
4.1. Average pattern of precipitation of Cr6+, Cr3+, Ni2+, Cd2+ and Co2+ from single‐ion solutions
A comparison of the average graphs for the precipitation of each heavy metal across the two pH regimes 8 and 11 and the concentrations employed, viz., 250 and 1000 ppm, shows that the efficiency of the enzyme‐derived reactions is in the order of Cd2+
4.2. Bioprecipitation of Cr3+ and Cr6+ from tannery effluent
The enzyme CIAP was able to precipitate 35.10% of Cr6+ and 38.39% of Cr3+ from the original concentration of 600 and 350 ppm, respectively, present in the tannery effluent. These precipitations were obtained at 120 min and at pH 11. Further, the percentage of precipitation of Cr6+ in these reactions is also comparable to the precipitation observed with Cr6+ in single‐ion solution earlier (250 ppm, pH 11). This is attributed to the fact that only in the presence of CIAP the precipitation of metal ions could take place.
4.3. Bioprecipitation of Ni2+, Cd2+ and Co2+ from electroplating effluent
During experiments with electroplating effluent, it was observed that CIAP was able to precipitate 52.28% of Cd2+ from initial concentration of 934 ppm, 31.34% of Co2+ from 878 ppm and 30.34% of Ni2+ from750 ppm at 120 min at pH 11. The percentage of precipitation of each heavy metal was quite comparable to the observations made with single‐ion solutions. However, values obtained were somewhat lesser than what was seen in the former reactions. It could be attributed to the fact that the Pi generated was competed by all the three metal ions simultaneously for the formation of respective metal‐phosphate precipitate.
5. Bioprecipitation studies using bacterial alkaline phosphatase
Bacterial alkaline phosphatase precipitated Cr6+ and Cr3+ from the two different concentrations such as 250 and 1000 ppm at various pH from 8.5 to 10. At 250 ppm, 42 and 52.81% of precipitation for Cr6+ and Cr3+ were obtained at pH 8.5 during 300 min of incubation. Analysis of the precipitation obtained with BAP under the pH 8.5 and 10, at 250 and 1000 ppm, resulted in differences in the pattern of precipitation. Higher precipitation could be achieved with 250 ppm of Cr6+ with 1000 ppm, since the pH seems to play a less important role in determining the precipitation levels. Whereas with Cr3+, the pH influences the precipitation patterns and at both concentrations higher precipitation was obtained at pH 8.5 and 10 [18]. This is because of the dynamics of Cr3+ and Cr6+ ionization and dissociation as pH dependent rather than the inherent capability of the enzyme in releasing product from pNPP. By determining the statistical data, a significant difference (
5.1. Average pattern of precipitation of Ni2+, Cd2+, Cr6+, Cr3+ and Co2+ from ion solutions
The average precipitation for every heavy metal between pH 8.5 and 10 at two concentrations 250 and 1000 ppm was compared and is shown in Figure 7. The efficiency of the enzyme for the precipitation of different metal ions is in the order of Cd2+>Ni2+>Cr3+>Cr6+>Co2+. No scavenging of inorganic phosphate is expected since the precipitations were carried out in ionic solutions, and in addition the amount of inorganic phosphate available for metal phosphate formation should remain constant. Hence, the variation in the precipitation pattern with different ions might be due to the difference in kinetics of metal phosphate complex formation and also because of the radical effect of metal ions present in the backbone of the enzyme [18].
5.2. Enzymatic precipitation of Cr6+, Ni2+ and Cd2+ from tannery and electroplating industry effluents
The applicability of the enzymatic precipitation of heavy metals from actual industrial effluent was validated by processing the effluent with BAP and pNPP. About 621 ppm of Cr6+ was present in tannery effluent. Upon treatment with the enzyme and substrate pNPP for 300 min, the amount of Cr6+ present in the supernatant was 403 ppm, resulting in 35.1% of precipitation. On the other hand, 97 ppm of Ni2+ and 122 ppm of Cd2+ were also present in the electroplating effluent. Final concentration of 21.43 and 51.95 ppm of Ni2+ and Cd2+ was present in the supernatant after treating with the enzyme, representing a 77.8 and 57.42% precipitation of the metals, respectively. From the results, it is evident that the two metal ions competed simultaneously for the inorganic phosphate generated by the enzyme. Moreover, Cr6+ precipitation from tannery effluent (35.1%) was compared with the values observed in ion solution (42.3%) [18].
The application of BAP and CIAP for the precipitation of heavy metals such as Ni2+, Cd2+, Cr3+/6+ and Co2+ (from single‐ion solutions as well as tannery and electroplating industrial effluents) under alkaline pH was studied using ascorbic acid 2‐phosphate, a natural substrate. To determine the potential of the enzyme to remain stable at certain environmental conditions, the kinetic characteristics of BAP and CIAP at various pH 8–11 were also studied. Higher activity of the enzyme was obtained at pH 9.5 and 10 for BAP and CIAP, respectively. The average precipitation pattern of metal ions from single‐ion solutions by BAP and CIAP occurred in the order of Cd2+ > Ni2+ > Co2+ > Cr3+ > Cr6+ and Co2+ > Cd2+ > Ni2+ > Cr6+ > Cr3+, respectively. Results state that the precipitation of Cr6+ from tannery effluent by BAP was 15.57% after 300 min of incubation while CIAP resulted in 71.47% at 120 min incubation. By contrast, BAP precipitated Cd2+ at much higher percentage than CIAP from electroplating effluents with the percentage of 94.6 and 66, respectively. Since ascorbic acid 2‐phosphate is a natural and biodegradable substrate, this study offers an eco‐friendly approach for a sustainable environment [25].
6. Kinetic behaviour of BAP (E. coli C90) and CIAP with α‐naphthyl phosphate
6.1. Effect of pH and substrate concentration on BAP (E. coli C90) and CIAP activity with α‐naphthyl phosphate as substrate
The investigation revealed that there is an immense effect of pH as well as the substrate α‐naphthyl phosphate concentration on the kinetics of BAP‐ and CIAP‐catalysed α‐naphthyl phosphate hydrolysis reaction. The maximum activity of CIAP was found to be at pH 11 (Figure 8a) followed by pH 9.5. The second highest pH value after pH 11 was found to be pH 9.5. Experimental data showed that among all pH studied, the highest activity for BAP was found at pH 8.5 (Figure 8b). The second highest pH value after pH 8.5 was found to be pH 9. It was observed that with an increase in the substrate concentration, the activity was also being increased. Kinetic parameters such as
6.2. Average pattern of precipitation of Cr6+, Cr3+, Ni2+, Cd2+ and Co2+ from single‐ion solutions by CIAP and BAP with α‐naphthyl phosphate
A comparative analysis of the average graph, obtained on CIAP‐ and α‐naphthyl phosphate‐mediated precipitation, was done in order to get a clear picture of the pattern of precipitation. The average precipitation of metals was found to be in the following order: Co2+ > Ni2+> Cd2+ > Cr3+ >Cr6+ (Figure 9a).
Average graphs for the precipitation of Cr3+, Ni2+, Cd2+,Co2+ and Cr6+ across the two concentrations (250 and 1000 ppm) and the two pH regimes (pH 8.5 and pH 9) are represented in Figure 9b. It can be inferred from the comparative analysis that BAP‐ and α‐naphthyl phosphate‐mediated heavy‐metal precipitation were in the following order: Ni2+ > Co2+ > Cr6+ > Cr3+ > Cd2+.
6.3. Bioprecipitation of heavy metals from electroplating industrial effluents by CIAP and BAP with α‐naphthyl phosphate
CIAP‐mediated investigation was performed at pH 11. In electroplating effluent, heavy metals present were Ni2+ (initial concentration was 134 ppm), Cd2+ (initial concentration was 734 ppm) and Co2+ (initial concentration was 278 ppm). The precipitations of Ni2+, Cd2+ and Co2+ were 66.17, 66.93 and 49.07%, respectively.
On the other hand, the observations showed that BAP‐mediated precipitation of Cd2+, Ni2+ and Co2+ from electroplating effluent was 20.37% (initial concentration was 734 ppm), 57.93% (initial concentration was 134 ppm) and 34.4% (initial concentration was 278 ppm). CIAP with α‐naphthyl phosphate‐mediated precipitation was greater than that of BAP and α‐naphthyl phosphate. This could be attributed to the fact that more
7. Kinetic study of BAP (E. coli C90) and CIAP with L‐ascorbic acid 2‐phosphate
7.1. Effect of pH and substrate concentration on BAP (E. coli C90) and CIAP activity with L‐ascorbic acid 2‐phosphate as substrate
The capacity of BAP and CIAP along with ascorbic acid 2‐phosphate is being reported as new heavy‐metal‐remediating tool, the effect of pH and substrate concentration on BAP and CIAP activity was studied at the range of pH values from 8 to 11 and at a range of substrate concentrations from 1 to 17 mM. Generally, tannery and electroplating effluents will have pH of alkaline range. The maximum activity of BAP was observed at pH 9.5 (Figure 10a), whereas for CIAP it was at pH 10 (Figure 10b).
7.2. Average precipitation of heavy metals from single‐ion solution with BAP (E. coli C90) and CIAP with ascorbic acid 2‐phosphate
A comparative analysis of the average graphs for the precipitation of each heavy metal (Cd2+, Co2+, Ni2+, Cr3+ and Cr6+) across the two pH values (pH 9.5 and 10.5 in case of BAP; pH 8 and pH 10 in case of CIAP) and the two concentrations, that is, 250 and 1000 ppm, was employed. The comparative analysis reveals the effect of BAP (
7.3. Bioprecipitation of heavy metals from real‐time effluent
The efficiency of BAP and CIAP in precipitating heavy metals from real‐time effluents was studied by treating the effluents along with the substrate. The concentration of Cd2+ in electroplating effluent initially was found to be 734 ppm. After enzymatic treatment with BAP for 60, 120 and 300 min at pH 9.5, the percentage precipitations obtained were 81.94, 91.39 and 94.62%, respectively, for each time. Likewise, treatment using CIAP for 30, 60 and 120 min at pH 10 resulted in 32.75, 53.75 and 65.89% of precipitation, respectively. The removal of Cr6+ from tannery effluent at an initial concentration of 560 ppm was analysed by treating with BAP and CIAP using ascorbic acid 2‐phosphate as substrate. When compared to CIAP, BAP showed reduced levels of precipitation. Moreover, at pH 9.5 for 60, 120 and 300 min, the percentages of precipitation of Cr6+ by BAP were 0, 5.61 and 15.57%, respectively. Whereas at pH 10 for 30, 60 and 120 min, the percentage of removal of Cr6+ were 38.32, 64.55 and 71.47%, respectively [25].
8. Conclusion
Bioprecipitation study with BAP and CIAP along with the substrate ascorbic acid 2‐phosphate is a much safe and convenient method for the removal of heavy metals such as cobalt, nickel, chromium, cadmium, and so on. The comparative studies have given a clear picture of both the enzymes’ kinetic behaviour and bioprecipitation efficiency at different pH, different metal concentration and at different time. Based on this information, further studies can be conducted with other metals which have not been studied in the present work. Immobilization of these enzymes also can be done for large‐scale application in industries.
Acknowledgments
The authors are thankful to SRM University, Kattankulathur, Tamil Nadu, India, for providing the facilities to carry out the research work.
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