Cloning, Identification And Molecular Modification Of Gibberella Intermedia Nitrilase

Nitrilases are an important biocatalyst in the nitrilase superfamily, which can directlyconvert a wide range of nitrile compounds into corresponding carboxylic acids. Thus theenzyme is well applied in organic acid biosynthesis, surface modification of polyacrylonitrileand bioremediation of high toxic nitrile wastes. However, its application might be hindered byseveral limitations, including less variety, low catalytic activity, poor operational stability, andso on. On the other hand, conventional studies mainly focused on the bacterial nitrilase andthe potential of fungal nitrilase has been far from being fully explored. These factors becamethe barrier to the utilization of nitrilase in industrial biocatalysis. In this study, fungal nitrilaseis selected as the research object and a series of relevant works are carried out as follows.Isolation of nitrilase-producing fungus from environmental samples was firstly performedwith3-cyanopyridine and glycinonitrile as the sole nitrogen source in the present study. Afungus CA3-1displaying high3-cyanopyridine hydrolyzing activity and good thermostabilitywas isolated from81strains through two rounds of enrichment culture, subsequent primaryscreening with phenol-hypochlorite method and final screening with HPLC method. Thisfungal strain was designated as Gibberella intermedia CA3-1using molecular identification,and phylogenetic analysis of this strain was also performed. The strain was deposited in theChina General Microbiological Culture Collection Center and the accession number isCGMCC4903. The catalytic properties of G. intermedia resting cells were determined.Results showed that this fungus showed a wide substrate spectrum with high specificity forheterocyclic and aliphatic nitriles. It also displayed good thermostability and the half-lives at30°C,40°C, and50°C were231.1h,72.9h, and6.4h, respectively. It remained extremelyactive in5%(v/v) propanol.3-Cyanopyridine (50mmol·L-1) was hydrolyzed into nicotinicacid within30min, whereas only less than5%of nicotinamide was detected.Therefore, cloning and heterologous expression of fungal nitrilase gene was carried outusing recombinant DNA technology due to the catalytic potential of this strain for nitrilecompounds. Partial cDNA sequence was amplified from the total RNA of wild Gibberellaintermedia through reverse transcription-PCR. A coding gene of fungal nitrilase wassubsequently cloned through further PCR from cDNA. The open reading frame consisted of963bp and potentially encoded a protein of320amino acid residues with a theoreticalmolecular mass of35.94kDa. The fungal nitrilase from G. intermedia CA3-1showed97%identity with the putative nitrilase from G. moniliformis (ABF83489). The identities of G.intermedia CA3-1nitrilase gene to the other reported nitrilases were lower than40%. Theencoding gene was transformed into E. coli Rosetta-gami (DE3) and recombinant strain E.coli Rosetta-gami (DE3)/pET28a(+)-Nit was successfully constructed. The recombinant straindisplayed good nitrile converting activity and specific activity of resting cells could reach upto0.5U·mg-1with3-cyanopyridine as the substrate. The half-lives of resting cells at30°C,40°C, and50°C were24.75h,2.55h, and1.34h, respectively.Purification and characterization of recombinant nitrilase was studied. The recombinantprotein was purified to electrophoretic homogeneity and the actual molecular mass was about 37.0kDa. The purified enzyme exhibited optimal activity at45°C and pH7.8. This nitrilasewas specific towards aliphatic and aromatic nitriles, especially3-and4-cyanopyridine. Thekinetic parameters Vmaxand Kmfor3-cyanopyridine were determined to be0.81μmol·min-1·mg-1and12.11mmol·L-1through Hanes-Woolf plot, respectively. On the otherhand, the catalytic triad (Glu-45, Lys-127, and Cys-162) was proposed and confirmed byoverlap extension PCR.G. intermedia nitrilase was modified at the gene level through gene engineering approachesin order to improve its enzymatic properties in catalytic application. Site saturationmutagenesis of128-Ile and161-Asn near the active site was conducted to improve thespecific activity and reducing the amide formation of G. intermedia CA3-1nitrilase. The twomutant libraries were constructed and several positive mutants were obtained. Two mostsignificant mutants I128V and N161Q were selected for further study. And double mutationswere subsequently introduced to construct mutant I128L-N161Q, which supported higherspecific activity and lower amide formation. The theremostability of all three mutants wasobviously improved compared with wild-type nitrilase, especially at30°C and40°C. However,double mutation did not further improve thermostability on the basis of single mutation.I128V and N161Q still exhibited wide reaction pH range, while I128L-N161Q becamerelatively narrow. The reaction temperature of I128V and N161Q was increased to50°Csimultaneously and I128L-N161Q remained the same. Mutations led to obvious changes ofsubstrate spectrum, especially for4-cyanopyridine, which resulted in an increase of relativeactivity by more that40%. Also, the catalytic efficiency of mutants was improved.The application studies of G. intermedia nitrilase mutant were performed to fully explore itsapplication potential. Complex immobilization of resting cells harboring nitrilase was utilizedto evaluate the application potential of immobilized nitrilase. Chitosan and PVA were selectedfor entrapment experiment, and acceptable residual activity and mechanical strength wereobserved. The immobilization conditions were8%of PVA and4%of chitosan, and theentrapped beads were maintained in saturated borate solution containing6%of sodiumtripolyphosphate. Interestingly, the thermostability of immobilized cells at30°C and40°Cwas improved by over100%. Also, immobilization led to obvious improvement of storagestability at4°C and-20°C. Conversion of3-cyanopyridine with different concentrationsdemonstrated that100mM3-cyanopyridine in the reaction mixture was the optimumconcentration of substrate. Fed-batch reaction using immobilized cells as the catalyst wascarried out for bioconversion of3-cyanopyridine. Finally,208g·L-1nicotinic acid wasobtained after18feedings (100mmol·L-1) within525min. Repetitive batch reaction modecould further improve nicotinic acid production through22feedings.