Study On The Enzymatic Properties Of Intracellular Esterase In Aspergillus Niger GZUF36

Esterases come from a wide range of sources,its types,structures and catalytic reaction of esterases are varied.Therefore,it is widely used in food,medicine,cosmetics and new energy development and other fields,and is one of the important industrial enzymes.The catalytic properties of GDSL family esterases from different sources are also quite different.The study on the protein structure is very important to explain the relationship between the structure and catalytic properties and guide the enzyme engineering transformation.In this study,a GDSL family esterase gene was selected from the transcriptase of Aspergillus Niger GZUF36 wild strain.The recombinant esterase was expressed by gene cloning,its enzymatic characterization,crystal structure prediction,molecular docking and immobilized characterization of crude enzymes were performed,that provides data support for the catalytic mechanism and function of Aspergillus Niger GDSL family esterase,and provides reference and guidance for further crystal structure analysis and molecular modification.The main research contents and results are as follows:(1)High activity expression and enzymatic properties of Pichia pastorisBy sequencing transcriptome genes,the gene sequence of GDSL family esterase was selected from NCBI database.It was translated into amino acid sequence,which encodes 293 amino acids,which were cloned by gene,expressed in Pichia,purified and then bioinformatics analysis was performed,with an isoelectric point of 4.62,a molecular weight of 32.3 kDa,no signal peptide,belonging to intracellular protein.The gene encoding mature peptide was cloned and expressed into Pichia coli,and the expression was induced by methanol for 72 h.The enzyme activity(1.06±0.06U/mL)was obtained from the fermentation supernatant.The recombinant esterase was purified from the fermentation supernatant by combining ammonium sulfate precipitation with a nickel column.The enzymatic properties of the recombinant esterase showed that the optimum temperature was 35℃,the optimum pH was 8.0,and the p-nitrophenol acetate was preferred.When p-nitrophenol acetate was used as substrate,the Km value and Vmax value of the recombinant esterase were 10.64 m M and 2.70 m M/min.It has higher stability at pH 8.0 and temperature below 35℃.And has a good tolerance to hydrophobic organic solvents,and generally has no tolerance to hydrophilic organic solvents,Most of the metal ions had no significant inhibition on it,but only the ferric ions had strong inhibition on it.Some surfactants(Tween-80,Tween-20,Tritone X-100)were well tolerated,but ionic surfactants(SDS,CTAB)had a strong inhibitory effect on their activity.(2)Study on crude enzyme immobilization(cross-linked enzyme aggregation)and its enzymatic propertiesThe optimum conditions for the preparation of recombinant esterase crosslinked enzyme aggregates were as follows: the product volume of acetone and crude enzyme liquid was 4:1,the amount of crosslinking agent(glutaraldehyde)was 60 μL,the reaction time was 1 hour,and the centrifugation was 6000 rpm.Under this condition,the relative enzyme activity of the recombinant esterase crosslinked enzyme aggregate was 115±3%,and the enzyme activity was about 1.22 U/mL.The results of enzymatic properties showed that the optimum temperature was 40℃ and the optimum pH was9.0.P-nitrophenol acetate is preferred as substrate.When p-nitrophenol acetate was the substrate,the Km value and Vmax value of the recombinant esterase CLEAs were13.53 m M and 2.81 m M/min.Compared with free enzyme,the substrate affinity and maximum reaction speed of recombinant esterase CLEAs were increased.The enzyme activity was maintained at 100% under pH 3.0-10.0.It has higher stability under temperature lower than 30℃.It has no tolerance to hydrophilic organic solvent,but it can retain about 40% of enzyme activity.It has good tolerance to hydrophobic organic solvent and can realize superenzyme activity.Most metal ions have no significant effect on it,only ferric iron ions have inhibitory effect on it,but they can retain more than 90% enzyme activity.The surfactant can inhibit its activity,but can retain more than 50% of the enzyme activity.(3)Homologous modeling and molecular docking testsThe recombinant esterase prediction was modeled by ab initio calculation,and the model quality test result was: Verify 3D score 97.67%,ERRAT score 75.427,and Z-scores-6.95.Regional statistics of dihedral angles in the recombinant ester enzyme crystal structure in the Ramachandran Map showed that 70.5% of the amino acid residues were in the most reasonable region,26.7% of the amino acid residues were in the additional allowed region,and only 2.8% of the residues were in the unreasonable position.Based on the above evaluation results,the recombinant esterase model constructed by homologous modeling has high rationality.The overall verification results show that the structure is suitable for further structural analysis.Molecular junction showed that the recombinant esterase formed van der Waals forces with Leu100,Ile96,Asn53,Gly92,Pro54 and Asn269 of P-NPA,firmly bonded with Gln39,Ser43 and His272 by conventional hydrogen bonds,formed HYDROCARBON bonds with Ser52,and formed Pi bonds with Ala94 and Thr65.