Pullulanase with high temperature and low pH optima improved starch saccharification efficiency

Abstract

Pullulanase, a starch debranching enzyme, is required for the preparation of high glucose/maltose syrup from starch. In order to expand its narrow reaction conditions and improve its application value, Bacillus naganoensis pullulanase (PulA) was mutated by site-directed mutagenesis and the biochemical characteristics of the mutants were studied. The mutant PulA-N3 with mutations at asparagine 467, 492 and 709 residues was obtained. It displayed the activity maximum at 60 °C and pH 4.5 and exceeded 90% activities between 45 and 60 °C and from pH 4.0 to pH 5.5, which was improved greatly compared with wild-type PulA. Its thermostability and acidic pH stability were also remarkably improved. Its catalytic rate (kcat/Vmax) was 2.76 times that of PulA. In the preparation of high glucose syrup, the DX (glucose content, %) values of glucose mediated by PulA-N3 and glucoamylase reached 96.08%, which were 0.82% higher than that of PulA. In conclusion, a new pullulanase mutant PulA-N3 was successfully developed, which has high debranching activity in a wide range of temperature and pH, thereby paving the way for highly efficient starch saccharification.

Secretory expression of amylosucrase in Bacillus licheniformis through twin-arginine translocation pathway

“…Of which, the Sec pathway is ubiquitous and essential to directly export a majority number of proteins under their unfolded forms through a well-organized cargo across the cell membrane and cell wall in a process of either co-translational or post-translational (Tsirigotaki et al., 2017 ). The Sec pathway can overexpress and efficiently prepare many kinds of industrially important enzymes (Niu et al., 2009 , 2022 ; Shen et al., 2022 ). Therefore, we tried to examine the possibility of the Sec pathway in B. licheniformis for secretory expression of NpAS.…”

Pullulanase: unleashing the power of enzyme with a promising future in the food industry

“…Pullulanase is used in the corn starch processing industry to obtain mild sweetness-based High fructose maltose syrup (HFMS) (Hii et al, 2012). They were also observed to tolerate a wider temperature and pH ranges, for instance, PulA-N3 (Niu et al, 2022) further enhancing the overall efficiency of the process.…”

Domain analysis and site-directed mutagenesis of a thermophilic pullulanase from Thermotoga maritima MSB8

“…Site-directed mutagenesis was a structureguided consensus about the quaternary structure with sequences, it was considered a reliable way to explore enzyme properties, protein structure, and protein binding patterns (Li et al 2019). In recent years, scientists made some achievements in the research of site-directed mutagenesis of pullulanase (Wang et al 2020;Niu et al 2022; Wang et al 2018). Li obtained four mutants Y477A, Y175C, L215C and R473E, which had different degrees improvement in thermal stability (Li et al 2015).…”