Improved Stability and Hydrolysates of Hyperthermophilic GH57 Type II Pullulanase from the Deep-Sea Archaeon Thermococcus siculi HJ21 by Truncation

Abstract

Pullulanase (EC 3.2.1.41) belongs to the amylase family and is often used alone or in combination with other amylases in the industrial production of starch-based products. This enzyme is often required in industrial production because of its better stability. We here truncated the pullulanase gene from the deep-sea hydrothermal anaerobic archaeon Thermococcus siculi HJ21 and obtained Pul-HJΔ782, which is a member of the α-amylase family GH57. The results revealed that the optimum temperature for Pul-HJΔ782 was 100 °C, and its thermostability at 100 °C improved after truncation. Less than 15% of its enzyme activity was lost after 1 h of incubation at 100 °C, and 57% activity remained after 5 h of treatment. Truncation significantly improved the overall pH tolerance range of Pul-HJΔ782, and its stability in the pH range 4–8 was over 80% relative activity from an average of 60%. The sequence and structural model of Pul-HJΔ782 was analyzed, and its instability index was reduced significantly. Furthermore, the hydrolysates of the truncated and wild-type pullulanase were analyzed, and the enzymatic digestion efficiency of the truncated Pul-HJΔ782 was higher.

Isolation, characterization, and cloning of thermostable pullulanase from Geobacillus stearothermophilus ADM-11

“…The pullulanase gene was reported in mesophilic bacteria such as Bacillus pseudofirmus 703 ( Lu et al, 2018 ) and Bacillus strain KSM-1876 ( Hatada et al, 2001 ), thermophilic bacteria such as Thermotoga maritime MSB8 ( Zhao et al, 2023 ), hyperthermophiles (such as Rhodothermus marinus ( Gomes et al, 2003 ), and Thermococcus siculi HJ21 ( Wu et al, 2023 ). Extremophilic microorganisms, which thrive under severe conditions, can be classified into 7 families; acidophiles, alkaliphilies, halophiles, metalopiles, psychrophlies, and thermophiles ( Taha et al, 2020 ).…”