Facile purification and immobilization of organophosphorus hydrolase on protein-inorganic hybrid phosphate nanosheets

Oriented immobilization of enzymes helps to maintain their native structure and proper orientation for high-performance engineering to meet extensive biocatalysis demands.However,the supporting materials used for orientated immobilization are usually costly or complicated in preparation,affecting their practical applications.In this work,a facile purification and immobilization method was proposed for enzyme immobilization based on organic-inorganic hybrid calcium phosphate nanocrystal(Ca Ps)induced by Cu^(2+) modified bovine serum albumin(BSA-Cu).Then,the as-prepared hybrid calcium phosphate nanosheet,BSA-Cu@Ca Ps,was utilized for one-pot purification and immobilization of His-tagged organophosphorus hydrolase(OPH)by metal-affinity binding to the incorporated BSA.BSA-Cu@Ca PsOPH exhibited enhanced p H stability and thermal stability compared to the free enzyme.Moreover,BSA-Cu@Ca Ps-OPH could retain more than 75%and 56%of initial activity after reuse 5 and 10 times,respectively.The results demonstrated that this facile strategy was promising for the effective biodegradation of organophosphorus pesticides with the immobilized enzyme.

Transient expression of organophosphorus hydrolase to enhance the degrading activity of tomato fruit on coumaphos

We constructed an expression cassette of the organophosphorus pesticide degrading （opal） gene under the control of the E8 promoter. Then opd was transformed into tomato fruit using an agroinfiltration transient expression system. β-Glucuronidase （GUS） staining, reverse transcription-polymerase chain reaction （RT-PCR）, wavelength scanning, and fluorescent reaction were performed to examine the expression of the opd gene and the hydrolysis activity on coumaphos of organo- phosphorus hydrolase （OPH） in tomato fruit. The results show that the agroinfiltrated tomato fruit-expressed OPH had the maximum hydrolysis activity of about 11.59 Uhng total soluble protein. These results will allow us to focus on breeding transgenic plants that could not only enhance the degrading capability of fruit and but also hold no negative effects on pest control when spraying organophosphorus pesticides onto the seedlings in fields.

Visible light assisted enzyme-photocatalytic cascade degradation of organophosphorus pesticides

The worldwide application of organophosphorus pesticides(OPs)has promoted agricultural development,but their gradual accumulation in soil and water can seriously affect the central nervous system of humans and other mammals.Organophosphorus hydrolase(OPH)is an effective enzyme that can catalyze the degradation of the residual OPs.However,the degradation products such as p-nitrophenol(p-NP)is still toxic.Thus,it is of great significance to develop a multi-functional support that can be simultaneously used for the immobilization of OPH and the further degradation of p-NP.Herein,a visible light assisted enzyme-photocatalytic integrated catalyst was constructed by immobilizing OPH on hollow structured Au-TiO_(2)(named OPH@H-Au-TiO_(2))for the degradation of OPs.The obtained OPH@H-Au-TiO_(2)can degrade methyl parathion to p-NP by OPH and then degrade p-NP to hydroquinone with low toxicity by using H-Au-TiO_(2)under visible light.OPH molecules were immobilized on HAu-TiO_(2)through adsorption method to prepare OPH@H-Au-TiO_(2).After 2.5 h of reaction,methyl parathion is completely degraded,and about 82.64%of the generated p-NP is further degraded into hydroquinone.After reused for 4 times,the OPH@H-Au-TiO_(2)retains more than 80%of the initial degradation activity.This research presents a new insight in designing and constructing multi-functional biocatalyst,which greatly expands the application scenarios and industrial value of enzyme catalysis.