Enzymatic Synthesis of Rhamnose Containing Chemicals by Reverse Hydrolysis.
Rhamnose containing chemicals (RCCs) are widely occurred in plants and bacteria and are known to possess important bioactivities. However, few of them were available using the enzymatic synthesis method because of the scarcity of the α-L-rhamnosidases with wide acceptor specificity. In this work, an α-L-rhamnosidase from Alternaria sp. L1 was expressed in Pichia pastroris strain GS115. The recombinant enzyme was purified and used to synthesize novel RCCs through reverse hydrolysis in the presence of rhamnose as donor and mannitol, fructose or esculin as acceptors. The effects of initial substrate concentrations, reaction time, and temperature on RCC yields were investigated in detail when using mannitol as the acceptor. The mannitol derivative achieved a maximal yield of 36.1% by incubation of the enzyme with 0.4 M L-rhamnose and 0.2 M mannitol in pH 6.5 buffers at 55°C for 48 h. In identical conditions except for the initial acceptor concentrations, the maximal yields of fructose and esculin derivatives reached 11.9% and 17.9% respectively. The structures of the three derivatives were identified to be α-L-rhamnopyranosyl-(1→6')-D-mannitol, α-L-rhamnopyranosyl-(1→1')-β-D-fructopyranose, and 6,7-dihydroxycoumarin α-L-rhamnopyranosyl-(1→6')-β-D-glucopyranoside by ESI-MS and NMR spectroscopy. The high glycosylation efficiency as well as the broad acceptor specificity of this enzyme makes it a powerful tool for the synthesis of novel rhamnosyl glycosides.
State Key Lab of Microbial Technology and National Glycoengineering Research Center, Shandong Provincial Key Laboratory of Carbohydrate Chemistry and Glycobiology, Shandong University, Jinan 250100, PR China.
Lili
L
Lu
State Key Lab of Microbial Technology and National Glycoengineering Research Center, Shandong Provincial Key Laboratory of Carbohydrate Chemistry and Glycobiology, Shandong University, Jinan 250100, PR China; Academy of State Administration of Grain, Beijing 100037, PR China.
Qian
Q
Liu
State Key Lab of Microbial Technology and National Glycoengineering Research Center, Shandong Provincial Key Laboratory of Carbohydrate Chemistry and Glycobiology, Shandong University, Jinan 250100, PR China.
Lan
L
Jin
State Key Lab of Microbial Technology and National Glycoengineering Research Center, Shandong Provincial Key Laboratory of Carbohydrate Chemistry and Glycobiology, Shandong University, Jinan 250100, PR China.
Zhenhao
Z
Yin
State Key Lab of Microbial Technology and National Glycoengineering Research Center, Shandong Provincial Key Laboratory of Carbohydrate Chemistry and Glycobiology, Shandong University, Jinan 250100, PR China.
Li
L
Xu
State Key Lab of Microbial Technology and National Glycoengineering Research Center, Shandong Provincial Key Laboratory of Carbohydrate Chemistry and Glycobiology, Shandong University, Jinan 250100, PR China.
Min
M
Xiao
D000528:Alternaria / Q000201:enzymology*; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics*; D006027:Glycosides / Q000737:chemistry; D006031:Glycosylation; D006868:Hydrolysis; D019906:Nuclear Magnetic Resonance, Biomolecular; D010843:Pichia / Q000235:genetics*; D012210:Rhamnose / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D013379:Substrate Specificity
D006027:Glycosides; D006026:Glycoside Hydrolases; C022525:alpha-L-rhamnosidase; D012210:Rhamnose