Construction Of Recombinant Lactococcus Lactis With Laccase Secreting Ability And Its Application In Silage Of Corn Stalk

Silage is an important method to prepare feed for ruminant animal.Lactic acid bacterium(LAB),the core of silage,plays a determinant role in control the silage quality.Isolating the area adaptive LAB and constructing the efficient and safe LAB with obvious function are the major direction of current silage development.The low efficiency in cellulose conversion leads to the low digestibility of silage,which results in the high cost of breeding.Laccase involves in lignin degradation,which could improve the efficiency of cellulose conversion.Constructing LAB with laccase secreting ability is deemed as an effective solution to enhance utilization efficiency of feed.Bacillus subtilis CotA laccase gene containing Usp45 signal peptide(S-CotA)was optimized according to the codon preference of Lactococcus lactis NZ9000.CotA laccase gene from Bacillus subtilis was expressed in E.coli Transetta(DE3)to verify the laccase activity firstly in this paper.And then the S-CotA laccase gene was connected with pMG36e vector.The recombinant plasmid was transformed into L.lactis NZ9000 by electricity to construct a recombinant L.lactis with laccase secreting ability.The culture conditions were optimized to improve the CotA laccase production.The optimal catalytic conditions of the produced CotA laccase were also characterized.Finally,the silage qualities of corn stalk with recombinant L.lactis NZ9000 were analyzed.The main results were stated as follows:1.The optimized CotA laccase gene was connected to pET-30a(+)expression vector and transformed into E.coli Transetta(DE3).The results of SDS-PAGE and Western Blot showed that CotA laccase could be expressed in E.coli successfully.The optimal intracellular CotA laccase production from the recombinant E.coli could reach 443.5 U/L.2.The S-CotA laccase gene was connected with the lactobacillus expression vector pMG36e and transformed into L.lactis NZ9000 by electricity.The results of Western Blot showed that recombinant L.lactis NZ9000 could secrete CotA laccase.The highest extracellular CotA laccase production(69.6 U/L)was obtained when the fermentation was performed at 16~°C for 20 h.3.The optimal catalytic temperature and pH value of CotA laccase was 70~°C and 4,respectively.The relative activity could maintain higher than 70%,when the enzyme was kept at the temperatures ranging from20 to 60~°C for 2 h.The relative activity could maintain higher than 55%,when the enzyme was kept at pH values ranging from 4 to 10 for 24 h.4.When the sailage with the recombinant L.lactis NZ9000 was performed at room temperature for 45 d,the contents of generated lactic acid,formic acid and acetic acid were 106.17,52.70 and 24.10 mg/g respectively.Furthermore,the pH value was around 3.96,indicating that the silage quality was better than the control.And no content of propionic acid and butyric acid was detected.The recombinant L.lactis NZ9000could modify the lignin in corn stalk during the silage process based on the FTIR results.The reducing sugar yield was obviously increased,which was 0.19 times higher than that of the control.The recombinant L.lactis NZ9000 with the CotA laccase secreting ability was constructed to improve the silage quality successful,which might be potential inoculants that applied in silage.