Identification Of The Strain JMU-TS529 And Characterization Of The ?-L-Rhamnosidase

?-L-rhamnosidase?EC 3.2.1.40?is one kind of important glucoside hydrolase with potential value in biotechnology research field.It could specifically cleave the terminal?-L-rhamnose from numbers of natural products?naringin,hesperidin,rutin,quercitrin,terpene glucosides and many other natural glycosides containing terminal?-L-rhamnose?.?-L-rhamnosidases from different sources have huge differences in terms of enzymatic property and amino acid sequence,and the enzymes with different physicochemical properties have different application value in fields of medicine and food industry.Based on the previous studies of laboratory,one strain which could produce?-L-rhamnosidase was characterized.The?-L-rhamnosidase in this strain was cloned,heterologous expressed and purificated,and its enzymatic property was analyzed.The main experimental results were as follows:?1?The strain JMU-TS529,was characterized as Aspergillus tubingensis;through morphological observation,SEM observation of conidium and construction of phylogenetic trees by ITS-5.8S rDNA and calmodulin sequences analysis.Do liquid state fermentation and solid state fermentation respectively to this strain,with pomelo peel as the basic medium,JMU-TS529was confirmed that both ways of fermentation could produce?-L-rhamnosidase.?2?The?-L-rhamnosidase gene was inserted into the expression vector pPIC9K,construct recombinant plasmid,and transformed to Pichia pastoris GS115 cell by electroporation,and then analyze of the crude enzyme activity after inducible expression,it is found that it has?-L-rhamnosidase activity.By using precipitation with ammonium sulfate,DEAE-Sepharose chromatographic column,HiTrapTM Bllue HP affinity column as well as SephacrylS-200 HR gel column,the objective protein with high purity,a single band,and with molecular weight 110kDa could be obtained.After MALDI TOF/TOF analyses,it could be confirmed that the protein after purification was the?-L-rhamnosidase from heterologous expression.?3?Analyze the enzymatic property of the recombinant?-L-rhamnosidase,it was found that the optimum pH was 4.0,the optimum temperature was 50-60oC,and when the temperature was60oC and 65oC,the half-life time was 29.09 h and 33.34 min,respectively;when Cu²⁺,Mg²⁺,Fe³⁺and Al³⁺were added 100 mM,the effect on the enzymatic was inhibited;Hg²⁺could also inhibit the enzyme activity when the concentration were 10 mM and 100 mM;effectors could slightly inhibit the enzyme activity;the recombinant?-L-rhamnosidase could only hydrolyze naringin with high efficiency,while for other substrates,the hydrolysis rate is lower,and it could only hydrolyze the naringin of pomelo juice;consider the naringin as the substrate,the kinetic constants were calculated as:K_m:1.27?mol/mL,V_max:3445?M?min^–1,k_cat:2.7×10⁴s^-1,k_cat/K_m:21.5 s^-1??M^-1.This study discovered a new strain A.tubingensis which produces the?-L-rhamnosidase,and a novel property?-L-rhamnosidase.It enriches the theoretical data for the diverse properties of this enzyme,and moreover,it has great reference value for deeply analyzing the structure and function relationship of this enzyme,and also for optimizing the enzymatic property.