Biochemical Characterization of a Novel Thermostable Type I Pullulanase Produced Recombinantly in Bacillus subtilis

Abstract

The pullulanase gene (pulGK), 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 pulGK 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 Km and Vmax 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 T1/2 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.

N-Terminal Domain Truncation and Domain Insertion-Based Engineering of a Novel Thermostable Type I Pullulanase from Geobacillus thermocatenulatus

“…Ni-NTA affinity chromatography was an appropriate method to purify this protein SI Table S3). The purified Pul GT exhibited the high specific activity of 192.56 U/mg (70 °C, pH 6.5) toward pullulan, which is obviously higher than those of the pullulanases from B. thermoleovorans US105 (3.7 U/mg, 75 °C, pH 6.0), 33 T. neapolitana (28.7 U/mg, 80 °C, pH 6.5), 17 Geobacillus kaustophilus (64.75 U/mg, 65 °C, pH 6.0), 32 and Paenibacillus barengoltzii CAU904 (68.3 U/mg, 50 °C, pH 5.5). 34 These results showed that optimizing the expression conditions and selecting appropriate purification methods could result in higher specific activity.…”

“…thermoleovorans US105 (3.7 U/mg, 75 °C, pH 6.0), T. neapolitana (28.7 U/mg, 80 °C, pH 6.5), Geobacillus kaustophilus (64.75 U/mg, 65 °C, pH 6.0), and Paenibacillus barengoltzii CAU904 (68.3 U/mg, 50 °C, pH 5.5) . These results showed that optimizing the expression conditions and selecting appropriate purification methods could result in higher specific activity.…”

“…Furthermore, Pul GT retained superior enzyme activity (over 80% activity) at pH 5.0–7.0 (Figure c), indicating that it has a wider range of pH adaptability than pullulanase from G. kaustophilus, Anoxybacillus sp. LM18-11, and T.…”

“…Geobacillus species are well-known for their ability to secrete thermophilic enzymes. There are few reports on pullulanases from Geobacillus species, , but there are no reports on pullulanases isolated from G. thermocatenulatus, including wild-type or recombinant strains.…”

Optimizing the DO‐stat protocol for enhanced production of thermostable pullulanase in Escherichia coli by using oxygen control strategies

“…Recently, to meet the multiple demands from the variety industries that makes pullulanase play diverse roles, many attempts have been made to overexpress recombinant pullulanase from alternative enzyme sources using Escherichia coli , Bacillus subtilis , or Pichia pastoris as hosts, obtained many enzymes with improved functional enzymatic properties, such as thermostable, thermo‐active, pH‐stable, detergent and chelator resistant (Kang et al, 2011; Li et al, 2018; Lu et al, 2018; Nisha & Satyanarayana, 2017; Wang et al, 2019). While, there are only a few researchers studied the high cell density fermentation of recombinant pullulanase (Duan et al, 2013; Wang et al, 2019), which is the basis of industrialization.…”