Direct electrochemistry of bacterial surface displayed cytokinin oxidase and its application in the sensitive electrochemical detection of cytokinins

Highlights

• Cytokinin oxidase from Nipponbare is successfully surface-displayed on E. coli.

• Direct electrochemistry of surface-displayed cytokinin oxidase is achieved.

• The cytokinin oxidase modified electrode is developed to detect isopentenyladenine.

Abstract

Cytokinin oxidase from Nipponbare (OsCKX4) was successfully displayed on the surface of E. coli cells by an ice nucleation protein from Pseudomonas borealis DL7 as an anchoring motif and a maltodextrin-binding protein(MBP) from E. coli as a solubility enhancer. The OsCKX4-displayed bacteria can be directly immobilized onto an electrode to selectively detect cytokinins, thus eliminating the need for enzyme extraction and purification. Direct electrochemistry of the cofactor FADH₂ in OsCKX4 has been achieved on an edge-plane pyrolytic graphite electrode (PGE) with a formal potential (E^0’) of −0.45 V at pH 7.0 in phosphate buffer. With the addition of isopentenyladenine, the reduction peak current for FADH₂ decreased, and the oxidative peak current increased gradually. Therefore, a bacteria-OsCKX4-modified PGE has been developed for the detection of isopentenyladenine with a linear range of 1.0–11.0 μM and a lower limit of detection of 0.7 μM (S/N = 3). Slight interference was observed in the presence of other phytohormones, including brassinosteroid, abscisic acid, methylene jasminate and gibberellin. The proposed bacterial biosensor is stable, specific and simple and has great potential for applications that require the detection of cytokinins.

Introduction

Cytokinins regulate many aspects of growth and development in plants, including cell division, seed and fruit development, cambial activity, shoot and root growth, flower, and senescence [1]. Therefore, the detection of cytokinins in plants is important. Because the concentration levels of CTKs in plant tissues are extremely low and their matrix is complex, the sensitivity and specificity of current analytical methods for CTK analysis are two important factors that must be considered. Modern instrumental analytical methods for the analysis of CTKs, such as gas chromatography-coupled mass spectrometry [2,3] and high-performance liquid chromatography-coupled mass spectrometry [4,5], have achieved high sensitivity and specificity for samples after pretreatment and purification. Immunoassay methods, such as radioimmunoassay [6] and enzyme-linked immunosorbent assay (ELISA) [7,8], can analyze crude extract solutions directly without purification steps owing to the specific recognition of antibody to antigen. Because of being small molecules, CTKs should be conjugated with a carrier protein such as bovine serum albumin prior to immunization. Furthermore, polyclonal antibodies lead to cross-recognition among the CTKs and result in a decrease in the specificity. Alternatively, enzymes with their naturally occurring selectivity and catalytic ability are attractive alternatives for use in the biosensor development [9].

Cytokinin oxidase (CKX) enzymes, along with their associated FAD cofactors, cleave the N6-side chain of isopentenyladenine, zeatin, and their ribosides directly to produce adenine and a side chain-derived aldehyde; simultaneously, the cofactor FAD is reduced to FADH₂, as depicted in Scheme S1 [10]. The CKX enzymes from maize (ZmCKO1) [11] and from Arabidopsis thaliana (AtCKX2) [12] have been expressed in Yarrowia lipolytica and in Saccharomyces cerevisiae, respectively, and their biochemical characteristics were studied after enzyme purification. In Nipponbare, eleven distinct CKX-encoding genes (OsCKX1-OsCKX11) were identified. Chu et al. demonstrated that OsCKX4 is located exclusively in the cytosol by expression of the fused C terminus of OsCKX4 with GFP in rice protoplasts, and the results indicated that OsCKX4 exist in cytoplasm [13]. OsCKX4 has been expressed in a prokaryotic expression system in our lab, and it was found to exist mainly as an inclusion body. The strategy of using bacterial surface expression of OsCKX4 can not only address this issue but can also avoid the requirement for enzyme purification.

Heterologous proteins displayed on the surface of living cells are widely used in biotechnological applications. P450 BM3, an oxidoreductase with a heme and diflavin cofactor, has been reported to display on the surface of E. coli cells using the Inp system [14]. The surfaced-displayed P450 BM3 can be used directly to develop whole-cell biocatalysts and to screen ligands for the enzyme, such as substrates, inhibitors or activators. Liu's group displayed xylose dehydrogenase [15] and glucose dehydrogenase [16] on the surface of bacteria to detect xylose and glucose, respectively, using electrochemical methods. They found that the prepared microbial biosensor exhibited high selectivity, good reproducibility and long-term stability and it can be applied in biofuel cells. To the best of our knowledge, the display of enzymes related to phytohormone metabolism on the bacterial surface has not yet been reported. In this paper, a recombinant plasmid pMAL-INP/OsCKX4 was constructed in such a manner that the OsCKX4 gene was fused to the C terminus gene of the ice-nucleation protein (INP).The OsCKX4 gene was obtained from the cDNA of Nipponbare, and the N terminus gene of ice-nucleation was from the genome of the strain Pseudomonas borealis DL7. The plasmid could be digested into a 1584 bp strip of OsCKX4 using the XbaI/SalI double enzyme. The plasmid pMAL contains the maltodextrin-binding protein(MBP) and INP was inserted into the C terminus of MBP at the EcoRI/BamHI double enzyme site. With the aid of MBP and INP, OsCKX4 was successfully displayed on the surface of E. coli. Without any enzyme purification, the bacteria were immobilized onto the pyrolysis graphite electrode directly and direct electrochemistry of OsCKX4 was achieved. Due to its selectivity and catalytic activity toward cytokinins, a facile bacterial biosensor for the sensitive electrochemical detection of cytokinins was developed.