Kinetic behaviour and stability of Escherichia coli ATCC27257 alkaline phosphatase immobilised in soil humates

Abstract

Alkaline phosphatase (EC 3.1.3.1) extracted from Escherichia coli ATCC27257 was immobilised by co-flocculation with soil humates in the presence of Ca 2þ . The effects of time, temperature, pH and concentration of enzyme and support on immobilisation were studied. Between 58 and 92% of the added phosphatase was strongly bound to the humates, depending on the conditions of immobilisation used. Some characteristics of the humate-phosphatase complexes and of the free enzyme were compared. The enzymatic complexes showed values of K m (2.22 mM) and activation energy (33.4 kJ mol À1 ) similar to those of the free enzyme (2.00 mM and 27.6 kJ mol À1 ). The pH/activity profiles revealed no change in terms of shape or optimum pH (10.5) upon immobilisation of alkaline phosphatase. However, the immobilised enzyme showed maximal activity in the range of 80-100°C, while the free enzyme had its highest activity at 60°C. The thermal stability of alkaline phosphatase was enhanced by complexation to the soil humates.

Barley Seeds Encapsulated in Calcium‐Alginate Gels with Phosphatase and Humate‐Phosphatase Complexes for Improving Phosphorus Bioavailability

“…Barley seeds were coated by encapsulation in Ca‐alginate gels‐containing free or immobilized alkaline phosphatase (EC 3.1.3.1). The enzymatic solution was obtained, as described by Pilar et al (2003), from Escherichia coli ATCC27257, a phosphatase hyperproductive strain obtained from the Spanish Official Culture Collection (Valencia, Spain). Phosphatase was immobilized by co‐flocculation with soil humates.…”

“…Alkaline phosphatase was immobilized by co‐flocculation with soil humates in the presence of Ca 2+ . The method used to prepare the HPC and the biochemical properties of the resultant complexes have been fully described by Pilar et al (2003). According to their protocol, 1 mL of the enzyme solution was mixed at 4°C during 120 min by orbital shaking (150 rpm) with 2 mL of soil humates, obtained following the method described by Busto et al (1997), and 4 mL of 1 mM 2‐amine‐2‐methyl‐1‐propanol (2A2M1P) buffer (pH 10.5).…”

Synthesis and characterization of a stable humic–urease complex: application to barley seed encapsulation for improving N uptake

“…Busto et al also observed immobilization yields higher than 100% in humate–β‐glucosidase and humate–carboxymethyl cellulase complexes by 180% and 350%, respectively. In contrast, Pilar et al reported a loss of activity in phosphatase–humate complexes with respect to the initial enzyme concentration. The enzymes attached to the humic matrix have a microenvironment quite different from that of free enzymes in solution, because they are affected by different diffusion, charge and steric effects .…”

“…Urease was immobilized by co‐flocculation with humic acids (HA) and Ca 2+ . The HA were obtained according to Busto et al The HUC were obtained as described by Pilar et al , with some modifications. Briefly, 2 mL HA (ranging from 2 to 6 mg mL −1 ) was added to 2 mL urease solution (0.25 mg mL −1 ) and 3 mL phosphate–citrate–borate buffer (PCBB: 100 mmol L −1 phosphate, 57 mmol L −1 borate, 36 mmol L −1 citrate) at different pH values (6.0–10.0).…”

Barley seed coating with urease and phosphatase to improve N and P uptake

“…Urease (urea amidohydrolase, EC 3.5.1.5) was obtained from jack bean (Canavalia ensiformis). Alkaline phosphatase (EC 3.1.3.1) was obtained from Escherichia coli ATCC27257, a phosphatase hyperproductive strain supplied by the Spanish Official Culture Collection (Valencia, Spain), according to Pilar et al (2003). Carboxymethylcellulose (CMC), p-nitrophenylphosphate (pNPP) and all other reagents were of analytical grade.…”