Screening,Optimization Of Conditions,heterologous Expression And Enzymatic Properties Studies Of Highly Efficient Feather-degrading Strains From Marine Source

In recent years,the degradation of feather by microorganisms has attracted wide attention.The reported feather-degrading strains mainly originate from the terrestrial environment,and the diversity and degradation effect of feather-degrading strains in the marine environment have rarely explored.Marine microorganisms have been in extreme environments such as low temperature,high salt and high pressure,and gradually forming a variety of unique metabolic mechanisms,which have potential development and utilization value.Therefore,in this study,an effective feather-degrading strain was screened from the marine environment,and the enzyme-producing conditions,keratinase heterologous expression and enzymatic properties of the strain were studied.The main research results are as following:(1)Screening of the feather-degrading strains.17 feather-degrading strains were screened from the sludge of a marine duck farm in the Beibu Gulf,Guangxi by casein plate preliminary screening and keratinase activity rescreening.Among them,the strain Gxun-7 had the best degradation effect,and was identified as Pseudomonas aeruginosa Gxun-7 by morphological,physiological and biochemical characteristics and 16S r DNA sequence analysis.(2)Optimization of keratinase production conditions of P.aeruginosa Gxun-7.The optimal conditions for enzyme production of the strain were obtained by single factor and orthogonal experiments as following:feather 25 g/L,Zn SO₄ 0.1 g/L,Na Cl 5 g/L,K₂HPO₄ 1.4 g/L,KH₂PO₄ 0.7 g/L,initial p H 8.0,fermentation temperature 32.5?,fermentation time 48 h,and the enzyme activity was 124.03 U/m L,which was 2.3 times as high as before optimization.The amino acids of the feather degradation products were analyzed by the amino acid automatic analyzer,and a total of 16 kinds amino acids were detected,of which 7 were essential amino acids,and the total amino acid content reached2329.80 mg/L.(3)Study on enzymatic properties of keratinase crude enzyme produced by P.aeruginosa Gxun-7.The results showed that the optimal temperature and p H of the keratinase crude enzyme were 70?and 8.0,respectively,and it had good stability when the temperature was lower than 60?and the p H was 7.0-9.5.The metal ions Mn²⁺,Mg²⁺and K⁺promoted the enzyme activity,while Cu²⁺inhibited the enzyme activity.The chemical reagents?-mercaptoethanol and DTT increased the enzyme activity by more than 3 times,while PMSF significantly decreased the enzyme activity,indicating that the enzyme might be a serine protease.The results of substrate specificity showed that the crude enzyme had better degradation effect on keratin and casein.The enzyme had good salt tolerance,the relative enzyme activity was 77.34%in 12.5%Na Cl.(4)Study on the heterologous expression of P.aeruginosa Gxun-7 keratinase gene and enzymatic properties.The keratinase gene kp2 was obtained by PCR amplification,and the recombinant expression plasmid p ET22b-kp2 was constructed by using p ET-22b(+)plasmid as a vector,and transformed into Escherichia coli for induced expression.The optimal induction conditions for protein expression were obtained by optimization as following:IPTG concentration of 0.3 mmol/L,OD₆₀₀ of 0.8,induction temperature of 30�C,induction time of 8 h.The recombinant keratinase was separated and purified using the nickel column.The molecular weight of the recombinant keratinase was about 33 k Da,the specific activity was 427.48 U/mg and the recovery was35.14%.The recombinant keratinase was inhibited by EDTA and PMSF and indicated as the serine metalloproteinase.The optimal temperature and p H of the enzyme are 40?and 8.0,respectively,and it had good stability in the range of30-60?and p H 6.5-8.0.Co²⁺,Cu²⁺and SDS inhibited the enzyme activity,while Mg²⁺,K⁺,?-mercaptoethanol and DTT promoted the enzyme activity.The relative enzyme activity was 87.55%under the action of 12.5%Na Cl,which was better than wild enzyme.When casein was used as substrate,the K_m value and V_maxvalue of the enzyme were 60.92 mg/m L and 9.70 U/m L,respectively.(5)Preliminary study on the degradation mechanism of P.aeruginosa Gxun-7.The activities of extracellular keratinase and disulfide bond reductase increased during feather degradation,suggesting that these two enzymes were involved in feather degradation.Further analysis of transcriptome data showed that P.aeruginosa Gxun-7 had a total of 1403 genes significantly differently expressed under the two treatment conditions of non-feather and feather culture mediums,595 genes were significantly down-regulated and 808 genes were significantly up-regulated,and the highest up-regulated expression was keratinase gene kp2.GO enrichment analysis of differentially expressed genes revealed that a large number of differentially expressed genes were enriched in a series of processes related to ribosomal activity,such as ribonucleoprotein complex assembly,ribosomal subunit composition and ribosomal composition.The KEGG enrichment analysis showed that 45 differentially expressed genes were involved in biofilm formation of P.aeruginosa,44 genes were involved in quorum sensing and 42 genes were involved in ribosomal composition.Among them,all genes involved in ribosomal composition were up-regulated,as well as the keratinase gene kp2 involved in quorum sensing.According to the experimental measurement and transcriptome data analysis,it was speculated that feather as inducer could stimulate the expression of keratinase gene kp2 and ribosomal gene in P.aeroginosa Gxun-7,and promote the synthesis of keratinase to hydrolyze feather.