Biosorption Of Cu(Ⅱ) And Flocculation By γ-PGA-conjugated Engineered Pseudomonas Putida Cells Surface-displaying GlnBP And SmtA

Heavy metal pollution is becoming a global issue with the rapid development of industry. Heavy metal pollutant not only damages the environment, but also does harm to human body. It gained public concern to remediate heavy metal pollutant. Microbe is a promising material to prevent heavy metal pollution due to its economical cost, high efficiency and mild reaction condition.Pseudomonas putida utilizes diverse carbon sources to grow. It can degrade most of the pollutants in the environment. SmtA, due to its low molecular weight and the ability to bind with heavy metals, plays an important role in remediation of heavy metal pollutants. Poly-gamma-glutamic acid(γ-PGA) is a high molecular polymer, which is biodegradable, water soluble, edible and environmentally friendly. It’s reported to be used as flocculating agent and an ideal material to absorb heavy metals. Conjugating PGA with recombinant bacterium which possesses absorbing heavy metal ability is a feasible way to alleviate heavy metal pollution.In this study, E. coli and P. putida AB 92019 were used as hosts to surface display(InaQ-N)2-GlnBP-GFP and(InaQ-N)2-Gln BP-(SmtA)2 respectively. The recombinant E. coli which displayed(InaQ-N)2-Gln BP-GFP was named as MB543. The fusion protein(InaQ-N)2-GlnBP-GFP was anchored to the surface of AB92019. PGA was conjugated to MB543 and the flocculation property of the composite MB543-PGA was studied. The addition of bivalent cation led to flocculation of the composite. After adding 10 mM Cu2+ to the composite, the flocculation effect was observed by fluorescence microscope through the GFP MB543 carried. Fluorescence microscopy showed MB543 was precipitated and gathered while PGA flocculating. The recombinant P. putida which displayed(InaQ-N)2-GlnBP-(SmtA)2 was named as MB546. The fusion protein(InaQ-N)2-GlnBP-(SmtA)2 was anchored to the cell surface of MB546 by the display ability of InaQ-N. Surface location analyses were then taken. The results of western blot, immune-fluorescence microscopy and flow cytometry analysis showed that the fusion protein was correctly displayed on the cell surface. γ-PGA was activated by NHS/EDC to bind with the GlnBP on the fusion protein, thus MB546 was conjugated with γ-PGA, and formed a composite material. This composite was subsequently used to absorb Cu2+ and gained excellent absorbing effect. The absorbing rate reached 98% when the initial Cu2+ concentration was 32 mg/L. Kinetics and isotherm analysis indicated that the adsorption fitted well with pseudo second-order equation and Freundlich model. The maximum biosorption capacity of MB546-PGA was 127.73 mg g-1 based on the Langmuir model. 0.04 M CaCl2 was added to the solution containing γ-PGA conjugated MB546 to flocculate the composite. The composite was efficiently flocculated after being deposited for 1 h and the flocculation efficiency was 96%. The results of SEM indicated that MB546 was successfully immobilized with γ-PGA, and flocculated MB546-PGA showed a densified structure. γ-PGA was probably immobilized to MB546 due to the carbonyl groups and amino groups on the surface of MB546, while Cu2+ was absorbed by MB546-PGA mainly due to the hydroxyl groups and amino groups based on the FTIR data.