Heterologous expression, secretion and characterization of the Geobacillus thermoleovorans US105 type I pullulanase

Abstract

Pullulanase type I of Geobacillus thermoleovorans US105 strain (PUL US105) was produced and secreted efficiently in the E. coli periplasmic or extracellular fraction using two different signal peptides. Hence, the open reading frame was connected downstream of the lipase A signal peptide of Bacillus subtilis strain leading to an efficient secretion of an active form enzyme on the periplasmic fraction. In addition, pul US105 was fused to the alpha-amylase signal sequence of the Bacillus stearothermophilus US100 strain. The monitoring of the pullulanase activity and Western blot analysis for this last construction showed that the most activity was found in the supernatant culture, proving the efficient secretion of this natively cytoplasmic enzyme as an active form. The PUL US105 was purified to homogeneity from the periplasmic fraction, using heat treatment, size exclusion, and anion-exchange chromatography. The native pullulanase has a molecular mass of 160 kDa and is composed of two identical subunits of 80 kDa each. It was independent for metallic ions for its activity, while its thermostability was obviously improved in presence of only 0.1 mM CaCl2.

High-Level Expression of Bacillus naganoensis Pullulanase from Recombinant Escherichia coli with Auto-Induction: Effect of lac Operator

“…So far, pullulanases has been discovered and identified from various microorganisms, such as Anaerobranca gottschalkii [10], Clostridium thermosulfurogenes [11], Geobacillus thermoleovorans [12], Klebsiella variicola [13], Raoultella planticola [14], Rhodothermus marinus [15], Thermotoga neapolitana [16], and species of the genus Bacillus [17-19], and even the uncultured environment [20,21]. However, the employment of those wide-type strains for the production of pullulanase for industrial application is still limited due to low enzymatic activity [14,16].…”

“…Among many systems available for protein expression, the thoroughly characterized Escherichia coli remains one of the most attractive hosts with a fast growth rate at a high density in inexpensive media and the ability to over-synthesize the protein of interest [24-26]. Although a number of genes coding pullulanases have been cloned and expressed, the pullulanase expression level remains somewhat low and mostly reported as specific activity but not extracellular activity in broth [10,12,27]. In addition, in spite of the extensive knowledge on the genetics and molecular biology of E. coli , foreign gene could not always be expressed efficiently as a routine matter in E. coli [12,25,28,29].…”

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

“…Meanwhile, no enzyme activity was detected from uninduced transformants or from transformants harboring the empty pHT43 plasmid (negative control). The high specific activity of PUL US105 from Geobacillus thermoleovorans , obtained in the supernatant of the BL21/pAD21 strain, was 12 U/mg (pullulan substrate) . The specific activity of thermoactive pullulanase from Desulfurococcus mucosus expressed in Bacillus subtilis was 24 U/mg .…”

“…The enzymes from Geobacillus sp. have been classified as thermophilic and employed for several industrial uses, including amylopullulanases from Geobacillus thermoleovorans NP33, type I pullulanase from Geobacillus thermoleovorans US105 and type II pullulanase from Bacillus stearothermophilus G‐82 . Although, the Geobacillus species is well‐known for their ability to produce thermophilic enzymes, only a few reports about pullulanases from G eobacillus species, and no report about pullulanases from Geobacillus kaustophilus (including the wild and recombinant strain) exists.…”

Improving the Thermostability and Catalytic Efficiency of Bacillus deramificans Pullulanase by Site-Directed Mutagenesis

“…To date, many microorganisms, including mesophiles, thermophiles, and hyperthermophiles, have been shown to produce pullulanase, and a large number of pullulanases have been identified and characterized (3)(4)(5)(6). In addition, several pullulanase-encoding genes have been cloned and characterized (7)(8)(9), and their crystal structures have been determined (10)(11)(12).…”