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

Abstract

A novel thermostable type I pullulanase gene ( pul ) from Geobacillus thermocatenulatus DSMZ730 was cloned. It has an open reading frame of 2154 bp encoding 718 amino acids. G. thermocatenulatus pullulanase (Pul) was found to be optimally active at pH 6.5 and 70 °C. It exhibited stable activity in the pH range of 5.5-7.0. Pul lacked three domains (CBM41 domain, X25 domain, and X45 domain) compared with the pullulanase from Bacillus acidopullulyticus ( 2WAN ). Different N-terminally domain truncated (730T) or spliced (730T-U1 and 730T-U2) mutants were constructed. Truncating the N-terminal 85 amino acids decreased the K value and did not change its optimum pH, an advantageous biochemical property in some applications. Compared with 2WAN , Pul can be used directly for maize starch saccharification without adjusting the pH, which reduces cost and improves efficiency.

Roles of the N-terminal domain and remote substrate binding subsites in activity of the debranching barley limit dextrinase

“…Notably, HvLD-Ser14Arg/His108Arg and HvLD-His108Arg were both also significantly more prone to substrate inhibition by pullulan (Table 1). Overall, the enzymatic properties of the mutants in the CBM21-like domain indicate an involvement in maintaining HvLD activity, as generally envisaged for a multidomain enzyme [56][57][58]. The larger losses of activity towards pullulan, amylopectin and -limit dextrin compared to the oligosaccharide substrate is in agreement with polysaccharides more readily perceiving changes at a remote area of the protein surface.…”

Synergistic Effect of Metagenome‐Derived Starch‐Degrading Enzymes on Quality of Functional Bread with Antioxidant Activity

“…These results are in agreement with the reported type I pullulanases that hydrolyzed the 𝛼-1,6-glycosidic bonds of substrates. [45,46]…”

“…Combination of enzymes and synergistic interactions between them could develop the bread characteristics and dough rheology. [10] Geobacillus thermocatenulatus DSMZ730 70 °C-pH 6.5 <50% activity after 120 min in 70 °C 7-8 [46] Bacillus methanolicus PB1 50 °C-pH 5.5 <20% activity after 120 min in 60 °C 5.5-6.5 [49] Anoxybacillus SK3-4 60 °C-pH 6 <10% activity after 60 min in 70 °C 5-7 [45] Paenibacillus polymyxa Nws-pp2 35 °C-pH 6 <10% activity after 50 min in 50 °C 5-6.5 [50] Exiguobacterium SH3 45 °C-pH 8.5 <10% activity after 90 min in 70 °C 5-11 [51] Shewanella arctica 35 °C-pH 6-7 <10% activity after 60 min in 40 °C 5-8 [52] Bacillus pseudofirmus 703 45 °C-pH 7-8 <20% activity after 10 min in 50 °C 6.5-8.5 [53] Metagenome-derived 40 °C-pH 6 -6-8 [18] Metagenome-derived (PersiPul1) 60 °C-pH 7 58.70% activity after 120 min in 80 °C 4-9 This study The same findings were reported after the usage of thermostable enzyme cocktail in the wheat bread. [9] 3.3.…”

Production of a Thermostable Pullulanase in Bacillus subtilis by Optimization of the Expression Elements

“…SH3, [9] mesophilic Bacillus cereus, [10] thermophilic Geobacillus thermocatenulatus, and Anoxybacillus sp. WB42, [11,12] hyperthermophilic Pyrococcus yayanosii CH1, [13] etc. However, wild-type strains generally produce pullulanase at low level, leading to limited potential for industrial application.…”