Cloning, expression of Aspergillus niger JL-15 endo-polygalacturonase A gene in Pichia pastoris and oligo-galacturonates production.
The endo-galacturonase A gene (pgaA) was cloned using the cDNAs synthesized from total RNA of Aspergillus niger JL-15 by reverse transcription as template. The open reading frame (ORF) of pgaA was 1113bp, encoding a peptide of 370 amino acids with the predicted molecular mass of 38.8kDa. The pgaA was successfully expressed in Pichia pastoris GS115 under the control of AOX1 promoter. After induction by methanol for 96h, the activity of the recombinant endo-galacturonase A (rePgaA) in culture supernatant was 2091.0U/mg. SDS-PAGE analysis showed that the molecular mass of rePgaA was about 40.0kDa. Enzymatic properties assays showed that the optimum temperature and pH for rePgaA were 50°C and pH 5.0, respectively. The Michaelis-Menten constant (Km) and maximal velocity (Vmax) of rePgaA for citrus pectin were 3.20mgml(-1) and 40.97μmolmin(-1)ml(-1), respectively. The rePgaA mediated a rapid decrease in viscosity of pectin solution with release of small amount of reducing sugar. High performance liquid chromatography (HPLC) analysis revealed that digalacturonate (G2) and trigalalcturonate (G3) were the main hydrolysis products released from pectin by rePgaA. The rePgaA showed very low activity on G2 and G3, which suggested it was a typical endo-acting enzyme.
Zhejiang Provincial Engineering Laboratory of Quality Controlling Technology and Instrumentation for Marine Food, College of Life Science, China JiLiang University, Hangzhou 310018, China; Zhejiang Provincial Key Laboratory of Biometrology and Inspection and Quarantine, China JiLiang University, Hangzhou 310018, China. Electronic address: mqliu524@163.com.
Ming-Qi
MQ
Liu
Zhejiang Provincial Engineering Laboratory of Quality Controlling Technology and Instrumentation for Marine Food, College of Life Science, China JiLiang University, Hangzhou 310018, China; Zhejiang Provincial Key Laboratory of Biometrology and Inspection and Quarantine, China JiLiang University, Hangzhou 310018, China.
Xian-Jun
XJ
Dai
Zhejiang Provincial Engineering Laboratory of Quality Controlling Technology and Instrumentation for Marine Food, College of Life Science, China JiLiang University, Hangzhou 310018, China.
Lan-Fang
LF
Bai
Zhejiang Provincial Engineering Laboratory of Quality Controlling Technology and Instrumentation for Marine Food, College of Life Science, China JiLiang University, Hangzhou 310018, China.
Xin
X
Xu
D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D015870:Gene Expression; D006603:Hexuronic Acids / Q000737:chemistry / Q000302:isolation & purification; D010843:Pichia / Q000235:genetics*; D011096:Polygalacturonase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D013696:Temperature
D006603:Hexuronic Acids; C007819:galacturonic acid; D011096:Polygalacturonase
Endo-galacturonase; Enzymatic properties; Expression; Hydrolysis; Oligo-galacturonates