ABSTRACT
Cytochrome c oxidase subunit II (COX II) containing a dual core CuA active site is one of the core subunits of mitochondrial Cytochrome c oxidase (Cco), which plays a significant role in the physiological process. In this report, the full-length cDNA of COXII gene was cloned from Sitophilus zeamais, which had an open reading frame (ORF) of 684 bp encoding 227 amino acids residues. The predicted COXII protein had a molecular mass of 26.2 kDa with pI value of 6.37. multiple sequence alignment and phylogenetic analysis indicated that Sitophilus zeamais COXII had high sequence identity with the COXII of other insect species. The gene was subcloned into the expression vector pET-32a, and induced by isopropyl ß-d-thiogalactopyranoside (IPTG) in E. coli Transetta (DE3) expression system. Finally the recombinant COXII with 6-His tag was purified using affinity chromatography with Ni(2+)-NTA agarose. Western Blotting (WB) showed the recombinant protein was about 44 kD, and the concentration of fusion protein was 50 µg/mL. UV-spectrophotometer and infrared spectrometer analysis showed that recombinant COXII could catalyze the oxidation of substrate Cytochrome C (Cyt c), and influenced by allyl isothiocyanate (AITC). By using molecular docking method, It was found that a sulfur atom of AITC structure could form a length of 2.9 Å hydrogen bond with Leu-31. These results suggested that tag-free COXII was functional and one of the action sites of AITC, which will be helpful to carry out a point mutation in binding sites for the future research.
Subject(s)
Electron Transport Complex IV/genetics , Gene Expression Regulation, Enzymologic , Insect Proteins/genetics , Weevils/genetics , Amino Acid Sequence , Animals , Biocatalysis/drug effects , Blotting, Western , Cloning, Molecular , Cytochromes c/metabolism , Electron Transport Complex IV/classification , Electron Transport Complex IV/metabolism , Escherichia coli/genetics , Insect Proteins/chemistry , Insect Proteins/metabolism , Isothiocyanates/metabolism , Isothiocyanates/pharmacology , Molecular Docking Simulation , Molecular Weight , Oxidation-Reduction , Phylogeny , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid , Spectrophotometry, Infrared , Spectrophotometry, Ultraviolet , Substrate Specificity , Weevils/enzymologyABSTRACT
Studies have shown that AITC can effectively control pathogenic fungi, which cause fruits and vegetables decay and rotting during storage. However, because of its strong irritant, AITC has not been conveniently used in fruits and vegetables preservation. Microencapsulation techniques may solve this problem. Up to 2% (w/v) gelatin and 2% (w/v) gum arabic (as wall material and materials), as well as AITC (as core material) were prepared used to form microcapsules with a ratio of 1:2 (the core material: to wall material). On the basis of the microcapsule option conditions, the AITC microcapsule encapsulation efficiency is above 90%, which can effectively control AITC release decrease irritant. Compared with control group, the storage time of the tomato of AITC microcapsule preservation was prolonged significantly, and the sensory quality of the tomato was better. Thus, the AITC microcapsule preservation has broad application prospects and development value prospects.
