Studies On The Metabolism Of Cyanide And Characterization Of The Recombinant Strain BL21-pET28a-cdE Producing Cyanide-degradation Enzyme

Cyanide dihydratase and cyanide hydratase,belonging to the nitrrilase superfamily,are involved in the hydrolysis of HCN.The products of hydrolysis of HCN by cyanide dihydratase are formate and ammonia,however,the products of hydrolysis of HCN by cyanide hydratase is only formamide.These two enzymes seems to be the most attractive in the cyanide-degrading enzymes:the products are of low toxicity and can be converted further,the enzymes do not require any cofactors,and their specific activities are high.We successfully cloned a recombinant strain BL21-pET28a-cdE from Alcaligenes sp.DN25 in the previous research.We had found the products,which from cdE catalyzes the hydrolysis of KCN,inculded fonnate,formamide,and ammonia.Then we know that the cdE has double-fuction with cyanide dihydratase and cyanide hydratase.So a series of experiments were conducted to further discuss the catalytic action of cdE in this study.Firstly,the stability of the recombinant strain BL21-pET28a-cdE,enzyme purification and the effects of preservation adding the stabilizer for enzyme were studied.The result showed 34%plasmids were lost after 150 generations under no antibiotic selection pressure,but the growth state and the expression of protein did not show differences among the generations.And compared with the the original generation,the 150 generation of cdE still has 99%relative activity.The target protein(38 kDa)was obtained after improving the purification methods by Ni-chelating affinity chromatography.It was confirmed that the optimal acitvity of the purified enzyme cdE is at pH 7.0 and 30?.Glycerinum and n-butyl alcohol could be used as the stabilizer of enzyme for the short time preservation at 4?,and ethanol,n-propyl alcohol could be added for the long time preservation at-20?.Secondly,compared with the amino acids sequence of Pseudomonas stutzeri AK61 of cyanidase,only three points amino acids were other than cdE,and the sequence identity is 99%.But the cdE still has the double-fuction and not be affected after the point mutations experiment.Finally,We studied the metabolism and degradation of the recombinant strain BL21-pET28a-cdE.The optimal reaction conditions were proved as follows:pH 8,35?.The results showed that it could gain high cyanide-degrading enzyme(29.9 U/mL)in a short time.It has the ability to grow on KCN,acetonitrile and mandelonitrile,especially grow on a mixing of potassium ferrocyanide,potassium thiocyanate and benzonitrile as the nitrogen source.And it almost completely degraded 30 mM KCN within 60min.Furthermore,the degradation KCN of the recombinant strain BL21-pET28a-cdE was not influenced by phenol.Above all,this study will be performed to verify BL21-pET28a-cdE is a good candidate in treating cyanide-containing wastewater.