| Ester hydrolases have high catalytic activity,wide substrate spectrum,and strong organic solvent tolerance,making them of great industrial value in the fields of food,medicine,and animal husbandry.Sinapine in rapeseed meal can be degraded by ester hydrolase,thus improving its feed value.The rumen microbiota contains abundant untapped microbial and enzyme resources,making it valuable to explore ester hydrolases.This study was conducted by using the functional screening medium from rumen Fosmid library of goat,to screen clones with ester hydrolase activity and conduct Illumina sequencing,identify potential ester hydrolase genes,and further screen ester hydrolase genes by inducing expression in Escherichia coli,studying its enzymatic properties and degradation of sinapine,Subsequently,the screened genes were inserted into the genome of Lactococcus lactis using CRISPR/Cas9technology,and the obtained engineering bacteria laid a theoretical foundation for the development and application of ester hydrolases in rumen microorganisms.The main results were as follows:1.Six ester hydrolase genes were identified from the rumen Fosmid library of Tibetan goats,namely Estg1,Estg2,Esty1,Estp,Estk1 and Estk2.Sequence comparisons revealed a high degree of homology（99-100%similarity）with known amino acid sequences,but none were functionally validated.Bioinformatics analysis showed that Estg1 and Estg2 belong to the GDPD superfamily with two highly conserved His catalytic residues,while Esty1,Estp,Estk1 and Estk2 all belong to the esterase superfamily with the typical catalytic triad Ser-Asp-His,and phylogenetic analysis showed that they belong to esterase families V,III,VI and VI,respectively.2.After the six ester hydrolase genes were successfully induced and expressed in E.coli BL21（DE3）,the obtained ester hydrolase activities were significantly higher than that of the empty control（P<0.05）.Among them,Estp and Estk1 had the highest enzyme activities,205.33 and 212.67 U/m L,respectively,with molecular weights of 33 and 28 k Da,and could degrade sinapine,with degradation rates of 13.26%and 11.66%,respectively.Esterases Estp and Estk1 have the highest activity at 40℃and p H 11,and are relatively stable at 40-50℃and p H 8-11 conditions;Metal ions Mn2+and Ni2+significantly promoted the activity of esterase Estp and Estk1（P<0.01）,while organic solvent n-hexane significantly enhanced its activity（P<0.05）;EDTA,metal ions Cu2+,Zn2+,Co2+,and Fe3+,organic solvent acetonitrile,and surfactants Tween-80 and Triton X-100 significantly inhibited the activity of Estp and Estk1（P<0.01）.3.Using the previously constructed CRISPR-Cas9 Lactococcus lactis genome editing system,the ester hydrolase gene Estk1 was successfully inserted into L.lactis NZ900 genome,and the obtained mutant strain achieved secretory expression of esterase Estk1,with an enzyme activity of 11.48 U/m L,which was significantly higher than that of the wild strain control（P<0.01）.In conclusion,the heterologous expression of ester hydrolase genes identified from the goat rumen metagenome Fosmid library has the characteristics of mesophilic,alkali tolerance,organic solvent tolerance,etc.,which makes them have great value in industrial applications,enrich the resource base of ester hydrolase genes,and they can degrade sinapine,which provide a basis for improving the feeding value of rapeseed meal. |
