Biochemical Characterization of a Novel Thermostable Type I Pullulanase Produced Recombinantly in Bacillus subtilis. (30th January 2018)

• Record Type: Journal Article
• Authors: Li, Lingmeng; Dong, Fengying; Lin, Lin; He, Dannong; Chen, Jingwen; Wei, Wei; Wei, Dongzhi
• Is Part Of: Stärke. Volume 70:Number 5/6(2018)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2018-01-30
• Subjects: Bacillus subtilis -- enzymatic properties -- expression -- Geobacillus kaustophilus -- pullulanase -- thermostable -- secretion expression; Starch -- Periodicals; 572.566
• DOI: 10.1002/star.201700179
• Languages: English
• ISSNs: 0038-9056

Abstract

Abstract : The pullulanase gene ( pul GK ), encoding a thermostable type I pullulanase (PulGK ), is obtained from the strain Geobacillus kaustophilus DSM7263. The gene has an open reading frame of 2157 bp that encodes a 718‐amino‐acid pullulanase, and shows the highest identity with the pullulanase from Geobacillus thermoleovorans US105. The pul GK is expressed in Bacillus subtilis WB800N using the plasmid pHT43, and the recombinant protein is secreted using the amyQ signal peptide. The level of PulGK produced in B. subtilis reaches 0.08 mg mL −1 after induction for 40 h at 30 °C. The purified recombinant PulGK can attack the α‐1, 6 linkages specifically in pullulan to generate maltotriose as the major product. Its specific activity is observed to be 64.75 U mg −1 and the K m and V max values of purified PulGK are 11.7 mg mL −1 and 23.6 μmol min −1 . Purified PulGK shows optimal activity at pH 6.0 and 65 °C. It also shows significant thermostability, with a T 1/2 of 60 h at 65 °C. Recombinant PulGK is immobilized and the thermostability of immobilized PulGK (Im‐PulGK ) is significantly improved (55–75 °C). PulGK hydrolyzes pullulan, amylopectin, starch, and glycogen, but not amylose. Substrate specificity and product analysis proves that the purified pullulanase from Geobacillus kaustophilus DSM7263 belongs to a type I pullulanase. This is the first report of pullulanase from Geobacillus kaustophilus (which includes the wild strain and the recombinant production of the enzyme) with detailed enzymatic properties of heterologous expression. The significant thermostability and production of recombinant pullulanase by B. subtilis may also potentially prove to be valuable in industrial applications. Abstract : Type I pullulanase in Geobacillus kaustophilus (including wild strain and recombinant strain) is investigated. Recombinant pullulanase is expressed extracellularly in Bacillus subtilis WB800N and determines the detailed enzyme characterizations, showing significant thermostability, and the stability of immobilized PulGK is further improved by immobilization.