| ?-L-rhamnosidase specifically hydrolyze terminal ?-L-rhamnose from a large number of natural products.As a natural flavonoid,Rutin is catalysed under the action of ?-L-rhamnosidase to produce L-rhamnose and isoquercitrin,the latter of which is further cleaved to form glucose and quercetin using ?-D-glucosidase as biocatalyst.Here,three glycosidases ?-L-1,2-rhamnosidase,?-L-1,6-rhamnosidase and?-D-glucosidase were purified from Aspergillus niger WZ001.Both?-L-1,6-rhamnosidase and ?-D-glucosidase were subsequently characterized in details and their applications on rutin hydrolysis were investigated.To further explore the structural characteristics of ?-L-1,6-rhamnosidase and its industrial potential,the study over-expressing the gene encoding ?-L-1,6-rhamnosidase in Pichia pastoris was carried out.1.Three glycosidases from A.niger WZ001 were purified using a set of purification steps:salting out,hydrophobic chromatography,anion exchange chromatography and gel filtration,and each N-terminal amino acid sequence of the purified enzymes were determined.Firstly,the purification folds and yield of?-L-1,2-rhamnosidase were 27.2 and 9.4%,respectively.The enzyme had a subunit size of 106 kDa and the specific activity of 49 U/mg,which amino terminal was blocked.Secondly,the purification folds and yield of ?-L-1,6-rhamnosidase were 36.0 and 9.2%,respectively.The enzyme had a subunit size of 66 kDa and the specific activity of 79.1 U/mg,which amino terminal was determined to be ETISLPQTDKWWTNY.In addition,the purification folds and yield of ?-D-glucosidase were 40.4 and 2.7%,respectively.The enzyme had a subunit size of 120 kDa and the specific activity of 113.1 U/mg,which amino terminal was determined to be DELAYSPPYYPSPWA.2.Characterization of both ?-L-1,6-rhamnosidase and and ?-D-glucosidase was carried out in order to establish the process of enzymatic hydrolysis of rutin.The optimal temperature and pH of ?-L-1,6-rhamnosidase were 65? and pH 4.0-5.0,respectively.a-L-1,6-rhamnosidase could tolerate organic co-solvents and retained 66.9%of the original activity in the presence of 40%(v/v)methanol.Except EDTA,various metal ions at a concentration of 2 mM decreased the enzyme activity.The enzyme exhibited good thermostability at 55 and 60?.In respect to ?-D-glucosidase,its optimal temperature and pH values were 60?and pH 3.0,respectively.The enzyme retained 27.8%of the original activity in the presence of 40%(v/v)methanol.The metal ion Al3+at a concentration of 2 mM increased the enzyme activity by 132%,whereas the enzyme activity was decreased in the presence of 2 mM EDTA.The conditions on the enzymatic hydrolysis of rutin were optimized:the rutin concentration,15%;temperature,60?;pH 4.5;rotation stirring speed,200 rpm;no requirement of co-solvent.After 48 h,the rutin at a concentration of 15%was completely hydrolyzed to isoquercitrin by the sole ?-L-1,6-rhamnosidase,while the complete hydrolysis of rutin to quercetin by the action of a-L-1,6-rhamnosidase and?-D-glucosidase would take 72 h.In addition,a bioseparation process was established to obtained the products isoquercitrin,quercetin and L-rhamnose,from which the product purity could reach up to 98%.3.The gene encoding ?-L-1,6-rhamnosidase from A.niger WZ001 had a length of 1317 bp,which corresponded to 438 amino acids.In comparison with the homologue gene with a GenBank accession number of AM269977.1,the gene was totally identical to the counterpart in the gene with a GenBank accession number of AM269977.1 except the missing N-terminal 201 amino acids.The genes rhaY(a sequence include 201 missing amino acid residues)and rhaNY(a sequence without 201 missing amino acid residues)were codon-optimized,and then the recombinant plasmids were constructed using the shuttle plasmid pPICZa.Both recombinant plasmids were linearized and transformed into P.pastoris GS115 as the host.Two genetically engineered P.pastoris strains harboring the gene rhaY or rhaNY were successfully obtained,which provide a solid basis to explore the role of the missing N-terminal seuqence and industrial application of ?-L-1,6-rhamnosidase. |
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