| Using ?-L-rhamnosidase to hydrolyze substrate rutin to produce isoquercetin by biotransformation method has become a hot spot in today's research.However,the microorganisms that can produce ?-L-rhamnosidase so far always have the activity of ?-D-glucosidase or rutinosidase,which cause its poor specificity.Therefore,the hydrolysates besides the target product isoquercetin also contain quercetin and other by-products.The presence of by-products not only causes the diffic ulty of separation and purification,but also reduces the yield of isoquercetin.In this study,Bacillus litoralis C44 which can hydrolyze rutin to produce isoquercetin efficiently and specifically was used to explore its potential ?-L-rhamnosidase genes by sequencing its whole genome and analyzing the results.C loning and expression of these genes within E.coli BL21(DE3)were realized.Then,the specific mechanism of ?-L-rhamnosidases encoded by strain C44 was revealed by isolation and purification of recombinant proteins,detection of activity,determination o f enzymatic properties and other methods.All above is ready for promoting application of the enzyme in the biotransformation and reducing production costs of isoquercetin.According to the analysis results of genomic sequencing of strain C44,there were 4 genes of ?-L-rhamnosidase(rha1,rha2,rha3 and rha4),were identified from the genomic DNA,and the open reading frames of the genes were 2859 bp,2910 bp,2712 bp and 1572 bp long,respectively.At the same time,there were 3 ?-D-glucosidase genes were encoded and none rutinosidase gene was found.No specific or non-specific ability to hydrolyze rutin was founded after the 3 ?-D-glucosidase genes were cloned,expressed and detected.The 4 ?-L-rhamnosidase genes were cloned and the recombinant expression vectors pET-28a(+)– rha1 to p ET-28a(+)– rha 4 were constructed.The molecular weight of recombinant proteins were 112.468 KD,110.67 KD,106.764 KD and 64.595 KD,respectively.When the recombinant organisms were induced by 0.2 mmol/L of IPTG for 5h at 37? after the cell densitiy reached OD600 of 0.6,the recombinant proteins accounted for 10.13%,18.016%,19.36% and 35.258% of the total protein,respectively.In the pH 7.0,0.1 mol/L PBS buffer containing 6% methanol(V/V),3 mg/mL rutin was transformed with four recombinant proteins,respectively.TLC dection result shows that only the protein encoded by rha 4 has ?-L-rhamnosidase activity.The recovery concentration was 0.335 mg / mL and the yield was 15.59% when N i-NTA affinity purification was carried out.RHA4 had the best effect in hydrolyzing rutin in Atkins-Pantin buffer at 50 ? and pH 8.0.Under this condition,20 mg recombinant cell lysate could convert about 1.8 mg of rutin in 3h.The enzymatic properties of the RHA4 were determined by using pNP-?-Lrhamnoside as substrate.The optimum temperature of the recombinase was 60?,stable in the range of 20-55 ?,the optimum pH value was 6.0,and stable only around p H 6.0;the activity of purified enzyme was 372.76 U / mL,the specific activity was 1112.7 U / mg,the Vm was 0.31 mmol/L·h,and the K m was 0.6933 mmol/L in Mcilvaine's buffer at 60 ?and pH 6.0;10 mmol/L metal ions Mn2 +,Cd2 + had a positive effect on the recombinant enzyme activity. |
PDF Full Text Request