Heterologous Expression And Enzymatic Characteristics Of Keratinase From Feather-degrading Actinomycetes

Keratin is a kind of fibrous structural protein which can be isolated from many tissues.Due to the presence of many hydrophobic residues,hydrogen bonding and disulfide bonds,its structure is very stable,resistant to most of the protease hydrolysis.Keratinase is a serine protease or metalloproteinase that can specifically hydrolyze keratin.It has great potential in organic fertilizer production,feed production,leather production,detergent formulation,cosmetics,medicine and nanotechnology.In order to study and develop keratinase resources better,two keratinase genes amy199 and gm2886 from two feather-degrading Actinomycetes BJA-103 and Fea-10 were heterologously expressed in other hosts.The main results are as follows :1.Try to express two keratin genes in Pichia pastoris.The genes amy199 and gm2886 were constructed into the vector p PICZ? A and successfully transformed into Pichia pastoris SMD1168 H by electroporation.However,the recombinant strain did not show protease activity,could not degrade feathers,and could not detect the target protein in the fermentation supernatant.To further explore whether the existence of signal peptide and precursor peptide of gm2886 affects the expression of the gene.2886 pro without self signaling peptide and 2886 mat without signal peptide and leading peptide were expressed in SMD1168 H again.The results showed that these genes could not be expressed effectively in Pichia pastoris SMD1168 H.2.Try to use Streptomyces heterologous expression of keratin gene amy199.The expression system was changed,and Streptomyces pactum Act12 was selected as the host.The keratinase gene amy199 was constructed into the vector p SET152 with erythromycin promoter and integrated into the genome of Act12 through conjugation and transfer.The recombinant strain showed obvious protease activity and feather degradation ability.The recombinant keratinase with the size of 27.3 k Da was obtained by ammonium sulfate precipitation and Ni column purification.In order to increase the production of recombinant keratinase,the fermentation medium was optimized.After the optimization,the enzyme activity of the fermentation broth of the optimized medium was increased by 3.4 times.3.The enzymatic properties of recombinant keratinase were analyzed.The results showed that its optimum temperature was 60?,and the optimum p H was10-11.Incubating at 60? for 1 h,it can still maintain more than 80% of the enzyme activity.The recombinant keratinase is a heat-resistant basic protein.The results of substrate specificity analysis showed that the protein is active on soluble substrates,including casein,bovine serum albumin,bovine hemoglobin,and keratin;it also has a certain degradation effect on insoluble substrates,including feather meal and azure keratin.