Enhanced Extracellular Production and Characterization of Sucrose Isomerase in Bacillus subtilis with Optimized Signal Peptides

Abstract

Sucrose isomerase (SIase) catalyzes the hydrolysis and isomerization of sucrose into isomaltulose, which is an important functional sugar widely used in the food industry. However, the lack of safe and efficient expression systems for recombinant SIase has impeded its production and application. In this study, enhanced expression of a SIase from Klebsiella sp. LX3 (referred to as KsLX3-SIase) was achieved in Bacillus subtilis WB800N, by optimizing the signal peptides. First, 13 candidate signal peptides were selected using a semi-rational approach, and their effects on KsLX3-SIase secretion were compared. The signal peptide WapA was most efficient in directing the secretion of KsLX3-SIase into the culture medium, producing a specific activity of 23.0 U/mL, as demonstrated by shake flask culture. Using a fed-batch strategy, the activity of KsLX3-SIase in the culture medium was increased to 125.0 U/mL in a 5-L fermentor. Finally, the expressed KsLX3-SIase was purified and was found to have maximum activity at 45 °C and pH 5.5. Its Km for sucrose was 267.6 ± 18.6 mmol/L, and its kcat/Km was 10.1 ± 0.2 s−1mM−1. These findings demonstrated an efficient expression of SIase in B. subtilis, and this is thought to be the highest level of SIase produced in a food-grade bacteria to date.

Development of an engineered Bacillus subtilis strain for antibiotic‐free sucrose isomerase production

“…In our previous research, using B. subtilis strain WB800N with a strong IPTG‐inducible promoter P grac100 yielded a 23.0 U mL ‐1 SIase activity in the shake flask cultivation. [ 12 ] Therefore, future research should further optimize the fermentation and feeding conditions for the engineered strain TEA4‐PogSI to enhance its fermentation process. Additionally, this study has improved the food‐grade expression system by optimizing the host strain through genetic modifications using CRISPR/Cas9 and enhancing the promoters on the expression vector.…”

“…[11] SIase has been expressed and secreted in B. subtilis WB800N, yielding a 23.0 U mL -1 in the shake flask cultivation and 125.0 U mL -1 in a 5-L fermentor. [12] B. subtilis, a well-known GRAS microorganism, is extensively used for industrial enzyme production due to its robust protein secretion, rapid growth rate, and suitability for scale-up fermentation. [13][14][15] However, the requirement of antibiotics for plasmid maintenance in B. subtilis raises safety concerns.…”

Analyzing Current Trends and Possible Strategies to Improve Sucrose Isomerases’ Thermostability

“…With the exception of the isoform from Enterobacter sp. FMB-1 (T op 50 • C) [50], most SIase exhibit their peak activity between 20 and 40 • C [89,90] (Table 2). Even so, the isoform from Enterobacter sp.…”

Enhanced extracellular production of raw starch-degrading α-amylase in Bacillus subtilis through expression regulatory element modification and fermentation optimization

“…For example, based on high-throughput screening technology, Yao et al randomly screened 173 B. subtilis signal peptides and obtained the optimal signal peptide SP YojL which can increase α-amylase activity by 3.5 times compared with control SP AmyQ’ [ 11 ]. In another study, Guo et al used a semi-rational screening method to obtain the optimal signal peptide SP WapA from 13 B. subtilis signal peptides, which can increase the sucrose isomerase activity by more than 2-fold compared with control SP WapA [ 12 ].…”