title: A novel bifunctional pectinase from Penicillium oxalicum SX6 with separate pectin methylesterase and polygalacturonase catalytic domains.
abstract: A multimodular pectinase of glycoside hydrolase family 28, S6A, was identified in Penicillium oxalicum SX6 that consists of an N-terminal catalytic domain of pectin methylesterase, a Thr/Ser-rich linker region, and a C-terminal catalytic domain of polygalacturonase. Recombinant S6A and its two derivatives, S6PE (the catalytic domain of pectin methylesterase) and S6PG (the catalytic domain of polygalacturonase), were produced in Pichia pastoris. S6A was a bifunctional protein and had both pectin methylesterase and polygalacturonase activities. Three enzymes showed similar biochemical properties, such as optimal pH and temperature (pH 5.0 and 50 °C) and excellent stability at pH 3.5-6.0 and 40 °C. Most metal ions tested (Na(+), K(+), Ca(2+), Li(+), Co(2+), Cr(3+), Ni(2+), Cu(2+), Mn(2+),Mg(2+), Fe(3+), Zn(2+), and Pb(2+)) enhanced the pectin methylesterase activities of S6PE and S6A, but had little or inhibitory effects on the polygalacturonase activities of S6A and S6PG. In comparison with most fungal pectin methylesterases, S6A had higher specific activity (271.1 U/mg) towards 70 % DM citrus pectin. When S6PE and S6PG were combined at the activity ratio of 1:4, the most significant synergistic effect was observed in citrus pectin degradation and degumming of sisal fiber, which is comparable with the performance of S6A (95 v.s. 100 % and 16.9 v.s. 17.2 %, respectively). To the best of our knowledge, this work represents the first report of gene cloning, heterologous expression, and biochemical characterization of a bifunctional pectinase with separate catalytic domains.
authors.affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing, 100081, People's Republic of China.
authors.forename: Tao
authors.initials: T
authors.lastname: Tu
authors.forename: Yingguo
authors.initials: Y
authors.lastname: Bai
authors.forename: Huiying
authors.initials: H
authors.lastname: Luo
authors.forename: Rui
authors.initials: R
authors.lastname: Ma
authors.forename: Yaru
authors.initials: Y
authors.lastname: Wang
authors.forename: Pengjun
authors.initials: P
authors.lastname: Shi
authors.forename: Peilong
authors.initials: P
authors.lastname: Yang
authors.forename: Kun
authors.initials: K
authors.lastname: Meng
authors.forename: Bin
authors.initials: B
authors.lastname: Yao
mesh_terms: D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D020134:Catalytic Domain; D002957:Citrus / Q000737:chemistry; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D020536:Enzyme Activators / Q000378:metabolism; D004795:Enzyme Stability; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000378:metabolism; D008969:Molecular Sequence Data; D010368:Pectins / Q000378:metabolism; D010407:Penicillium / Q000201:enzymology* / Q000235:genetics; D011096:Polygalacturonase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013696:Temperature
chemical_list: D004271:DNA, Fungal; D020536:Enzyme Activators; D008670:Metals; D011994:Recombinant Proteins; D010368:Pectins; D002265:Carboxylic Ester Hydrolases; C022323:pectinesterase; D011096:Polygalacturonase