Expression,Identification And Modification Of Keratinase And Its Application In Feather Degradation And Transformation

There is a shortage of forage protein resources in our country,and soybean meal and fish meal are mainly imported.Feather is the main by-product of poultry processing,and its composition is mainly keratin.However,feather keratin is abandoned in large quantities because it is difficult to be digested and utilized,which not only causes serious waste of protein resources,but also causes a series of environmental pollution.Keratinase is an enzyme that can specifically degrade this insoluble protein and widely exists in nature.Although researchers around the world have studied keratinases deeply,the large-scale commercial application of keratinases still faces many problems.For example,in the feed industry,enzymes that can withstand short-term high temperature are needed for pellet feed granulation,and enzymes that can withstand acid are needed for the fermentation process of lactic acid bacteria feed.Therefore,this study aims to develop a keratinase with strong heat resistance and high stability in acidic conditions to promote the large-scale application of unconventional feed resources such as feather keratin in the feed industry.The main research is as follows:（1）KerQ7 is a keratinase from Bacillus tequilensis strain Q7.The synthesized gene of kerQ7 was ligated into the vector pET-22b and expressed in Escherichia coli BL21.The highest specific activity of kerQ7 was 6.8 times higher than that of a commercial keratinase.However,the pH stability and thermal stability of kerQ7 were relatively poor,so kerQ7 was further immobilized and modified.（2）KerQ7 was immobilized by spore surface display.First,kerQ7 was fused with the spore coat proteins cotG and cotB,respectively,and displayed on the surface of Bacillus subtilis spores.The results showed that the pH tolerance and heat resistance of kerQ7 enzyme displayed on the spore surface were improved compared with those of the free kerQ7 enzyme.In particular,the heat resistance of the immobilized enzyme could maintain more than 70%of its residual activity after being kept at 60℃for 1 h.However,the free kerQ7 had only 20%residual activity,and cotG-kerQ7 was more thermostable than cotB-kerQ7.Further optimization of the linker peptide between cotG and kerQ7 showed that the long flexible linker peptide L3 could significantly improve the activity of fusion protein cotG-kerQ7,and its maximum activity increased to 131.2±3.4%,and after being kept at 60℃for 4 h,the residual enzyme activity was still 62.5±2.2%.It was 1.36-fold higher than the activity of cotG-kerQ7 under the same conditions.Then,the effects of common chemical reagents and metal ions on the activity of cotG-L3-kerQ7 were explored.It was found that 1 mM DTT and 1 mM CaCl₂could increase its activity to 172.3±4.2%and 129.8±3.1%,respectively.The cotG-L3-kerQ7 spore solution treated at60℃for 5 minutes could still degrade chicken feathers within 36 hours.This further indicates that kerQ7 after spore display and linker peptide optimization has strong thermal stability（3）Then a high-throughput screening method was established based on spore display,and the acid-resistant kerQ7 fused with cotG was modified by error-prone PCR and directional screening.Five kerQ7 mutants with increased activity were obtained at pH 5,especially the mutant H4（N43K/N143I）showed the most significant increase in enzyme activity,which was 134.8±4.4%of that of cotG-kerQ7.Further analysis of the enzymatic properties of H4 showed that the optimum pH of H4 changed from 7 to 6,and the highest activity was 118.2±3.2%of cotG-kerQ7,and its pH stability shifted to acidic conditions as a whole.For example,after incubating at pH 4-6 for 1 h,the residual enzyme activity could maintain more than 80%of the maximum activity.However,the stability of the control cotG-kerQ7at pH 4 was only 65.5±3.4%,and the thermal stability of H4 was also slightly improved.Furthermore,the molecular mechanism of the improved acid and heat resistance of keratinases was discussed by bioinformatics methods,which provides an important reference for the further molecular modification of keratinases in the future.（4）Finally,common probiotics including lactic acid bacteria and the spore enzyme preparation in this study were used to coferment feather meal feed.After 7days of fermentation,the contents of small peptides and total free amino acids in cotG empty plasmid spore and cotG-kerQ7 control groups were 10.15±0.71%,20.68±0.57 mg/g and 13.44±0.55%,33.97±0.66 mg/g,respectively.The fixed and modified cotG-L3-kerQ7 and cotG-kerQ7-H4 spore enzyme preparations increased the small peptide content of feather meal feed from 3.36±0.53%to 15.42±0.68%and 15.27±0.75%,respectively.The total free amino acid content increased from7.26±0.51 mg/g before fermentation to 39.00±1.24 mg/g and 37.57±0.88 mg/g,respectively,which was significantly higher than that of the control group,but there was no significant difference between the cotG-L3-kerQ7 and cotG-kerQ7-H4 groups.This indicates that cotG-kerQ7 modified by linker peptide optimization and directed evolution has higher application value,which also provides an important reference for the modification of keratinase and its further application in the feed industry in the future.