The Study Of Recombinant ?-L-Rhamnosidase For Microfluidic Biocatalysis

Miniaturizing biocatalysis has become an efficient approach to sugar-based directional transformation of rare natural products,which could significantly increase the glycosidic enzyme reaction rate and selectivity.However,the natural glycosidase with low content was tedious to purify.The free enzyme was difficult to immobilize and easy to agglomerate in the microchannel reactor.The industrialization of miniaturizing biocatalysis was limited to some degree.In the present study,the directional hydrolyze rutin biotransformation synthetic isoquercitrin was carried out.The heterologous expression of RHA in different host strains was implemented by genetic engineering,and its catalytic performances were explored.The major research content of this study as follows:?1?The codon preference and adaptation of ?-L-rhamnosidase gene?rhaA?,which derived from Aspergillus nige,was modified by codon optimization techniques to improve heterologous expression quantity.Saccharomyces cerevisiae EBY100 cell surface display system was used for the heterologous expression of codon-optimized?-L-rhamnosidase gene?corhaA?.The results indicated that,comparing with the expression of original genetic strain?rhaA?,under the identical expression conditions,the expression of coRha A?the protein expressed by codon-optimized gene??320±10mg/L?was increased by 2.9 times,and the enzyme activity of coRhaA?56.9±2.9 U/g?was increased by 2.7 times under the same enzyme content after 60 h induced.The optimal pH and temperature for coRhaA were 5.0 and 45 °C respectively,with pNPR as substrate.A yield of 79.8±3.1% was achieved by coRhaA under optimum conditions?pH 5.0,60 °C?.?2?A strain of E.coli BL21-pET21a-rhaB1 was constructed.The metagenomic library DNA derived from elephant feces led to the identification of a novel bacterial?-L-rhamnosidase belonging to glycoside hydrolase family 78,and rhaB1 now is the first biochemically characterized enzyme of this subclass.The results of RhaB1 activity assay indicated that,the most RhaB1 exist in intracellular.Comparsing with the crude RhaB1,the purified RhaB1 activity was lost 37.7%.With pNPR as substrate,the optimum conditions of crude RhaB1 hydrolysis was 45 °C and pH 6.0-6.5,and Km and vmax was 2.175 g/L and 0.335 [g/?L×min?],respectively.The yield of isoquercitrin was 98.3±3.8% by crude RhaB1 from rutin hydrolysis under optimum conditions?0.02 g/L rutin,35 °C,pH 5.0-6.0?.?3?A cosolvent system was established in the microchannel reactor for recombinant RhaB1 directional hydrolysis rutin biotransformation synthetic isoquercitrin.In the reaction medium with 0.02 g/mL [Toma][Tf₂N],the yield of isoquercitrin reached to 99.3±5.1%?35 °C,pH 5.0-6.0,0.02 g/L rutin,2 ?L/min?.Compared with in a batch reactor,the enzymatic reaction time was shortened by98.3%,Km reduced to 1/3,kcat improved from 0.29 s^-1 to 5.57 s^-1,v_max/K_m improved65.7 times and space-time yield improved 61.4 times.Besides,the results by circular dichroism?CD?displayed the presence of 0.02 g/mL [Toma][Tf₂N] not only do not changed the activity of recombinant RhaB1 but also was used as cosolvent to overcome the jam in the microchannel.In conclusion,the heterologous expression of genes encoding coRhaA on the surface of yeast cells was effective improved by codon modification.Compared with the batch reactor,crude RhaB1 was directly used to hydrolyze rutin and established a cosolvent system in the microchannel reactor with the presence 0.02 g/m L[Toma][Tf₂N],which showed incomparable advantages of process intensification and space-time yield was greatly promoted.It has a favourable application prospect for effective biomanufacturing the rare and high value-added natural medicine.