Premium
Expression and Characterization of Rice (Oryza sativa) Monolignol β‐Glucosidase
Author(s) -
baiya supaporn,
Okita Thomas W,
Cairns James Ketudat
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.lb188
Subject(s) - monolignol , salicin , glucoside , beta glucosidase , biochemistry , glucosidases , chemistry , biology , enteropeptidase , enzyme , recombinant dna , cellulase , gene , fusion protein , biosynthesis , medicine , alternative medicine , pathology
Phylogenetic analysis of Oryza sativa GH1 β‐glucosidases indicated that Os4BGlu14, Os4BGlu16, and Os4BGlu18 are closely related to known monolignol β‐glucosidases, leading to the hypothesis that they may be involved in lignification by hydrolysing monolignol glucosides. The cDNAs for each of these rice GH1 β‐glucosidase genes, Os4BGlu14 , Os4BGlu16 , and Os4bglu18 were cloned, sequenced, and ligated into pET32a, and the resulting recombinant plasmids were used to express the putative β‐glucosidases as fusion proteins with N‐terminal thioredoxin and His 6 tags in E. coli Origami (DE3). No activity could be detected for proteins expressed from Os4BGlu14 and Os4BGlu16 in this system. Optimal expression of soluble Os4BGlu18 β‐glucosidase activity was obtained with induction at 18°C, 0.1 mM IPTG for 16 to 18 h. The recombinant protein was extracted and purified by immobilized metal affinity chromatography (IMAC). The optimum pH for Os4BGlu18 was found to be 5 and the enzyme was stable from pH 4 to pH 8. The temperature optimum of Os4BGlu18 was 55°C, but the enzyme was most stable at 20°C‐40°C. Os4BGlu18 hydrolyzed p‐ nitrophenol ( pNP )‐β‐D‐fucoside best, followed by p NP‐β‐D‐glucoside, p NP‐α‐L‐arabinoside, p NP‐β‐D‐galactoside and p NP‐β‐D‐xyloside, respectively. Moreover, Os4BGlu18 hydrozyed n‐octyl‐ β‐glucoside, n‐heptyl‐β‐glucoside, methyl‐β‐D‐glucopyranoside and daidzin. Among possible natural substrates, Os4BGlu18 slowly hydrolyzed the monolignol glycoside p‐ coumaryl alcohol glucoside, along with salicin and arbutin. No activity was detected with coniferin and sinigrin, two other monolignol glucosides, suggesting another isoenzyme may be responsible for their hydrolysis in the plant. Financial support was provided by The Royal Golden Jubilee Ph.D Program of the Thailand Research Fund.