Extracellular expression of agarase rAgaM1 in Bacillus subtilis and its ability for neoagaro‐oligosaccharide production

Abstract

An agarase gene (agaM1) was cloned, expressed and characterized by using Escherichia coli as host strain, revealing the outstanding properties of recombinant AgaM1 (rAgaM1) in agarose degradation and neoagaro‐oligosaccharides (NAs) production in our previous work. In current study, agaM1 was extracellularly expressed in Bacillus subtilis, and we aim to assess the ability of the supernatant of recombinant B. subtilis fermentation broth containing rAgaM1 to degrade agarose without protein purification, which would save the cost of purification and avoid the activity loss during purification. The pH and temperature optima for the supernatant were 7.0 and 50 °C, respectively. The supernatant containing rAgaM1 has outstanding stability against 40 °C and 50 °C. Besides, we detailedly studied the possible influence factors of rAgaM1 expression in the supernatant, including pH, temperature, isopropyl β‐D‐thiogalactoside (IPTG) concentration, initial optical density at a wavelength of 600 nm (OD600), and induction time, and the optimum conditions for rAgaM1 expression by B. subtilis were confirmed. Moreover, the supernatant was able to produce NAs by using the Gracilaria lemaneiformis, whose cells were broken by autoclaving, as substrate, and a total of 1.41 µmol ml−1 of NA, including neoagarotetraose and neoagarohexaose, was produced after degradation for 48 h. This ability could save the cost of substrates in NA production, although the method requires a further study. Our results reveal that the NAs with great potential in food and pharmaceutical industries could be inexpensive to make by the supernatant containing rAgaM1 of B. subtilis fermentation broth in the foreseeable future.

Exploring and Engineering Novel Strong Promoters for High-Level Protein Expression in Bacillus subtilis DB104 through Transcriptome Analysis

“…A plasmid stability test was performed, following the methodology outlined by Li et al [51], to determine the durability of the recombinant plasmid throughout protein production. Briefly, a single colony of the recombinant B. subtilis DB104 strain was inoculated into LB broth containing 10 µg/mL of kanamycin.…”