Biodegradation and adsorption of benzo[a]pyrene by fungi-bacterial coculture.

In this work, the relationship and kinetics of biodegradation and bio-adsorption of benzo[a]pyrene (BaP) by Bacillus and Ascomycota were explored, and the metabolites of BaP under mixed microbial coculture were analyzed and characterized. The results show that BaP was removed through both biosorption and biodegradation. Under mixed microbial coculture, biosorption played a significant role in the early stage and biodegradation was predominant in the later stage. During the removal of BaP, the fungi exhibited remarkable adsorption capabilities for BaP with an adsorption efficiency (AE) of 38.14 %, while bacteria had a best degradation for BaP with a degradation efficiency (DE) of 56.13 %. Under the mixed microbial culture, the removal efficiency (RE) of BaP by the synergistic action of fungi and bacteria reached up to 76.12 % within 15 days. Kinetics analysis illustrated that the degradation and adsorption process of BaP were well fit to the first-order and the pseudo-second-order kinetic models, respectively. The research on the relationship between degradation and adsorption during microbial removal of BaP, as well as the synergistic effects of fungi and bacteria, will provide a theoretical guidance for two or even synthetic microbial communities.

Computation-aided engineering of starch-debranching pullulanase from Bacillus thermoleovorans for enhanced thermostability.

Pullulanases are widely used in food, medicine, and other industries because they specifically hydrolyze α-1,6-glycosidic linkages in starch and oligosaccharides. In addition, high-temperature thermostable pullulanase has multiple advantages, including decreasing saccharification solution viscosity accompanied with enhanced mass transfer and reducing microbial contamination in starch hydrolysis. However, thermophilic pullulanase availability remains limited. Additionally, most do not meet starch-manufacturing requirements due to weak thermostability. Here, we developed a computation-aided strategy to engineer the thermophilic pullulanase from Bacillus thermoleovorans. First, three computational design predictors (FoldX, I-Mutant 3.0, and dDFIRE) were combined to predict stability changes introduced by mutations. After excluding conserved and catalytic sites, 17 mutants were identified. After further experimental verification, we confirmed six positive mutants. Among them, the G692M mutant had the highest thermostability improvement, with 3.8 °C increased Tm and 2.1-fold longer half-life than the wild type at 70 °C. We then characterized the mechanism underlying increased thermostability, such as rigidity enhancement, closer conformation, and strengthened motion correlation using root mean square fluctuation (RMSF), principal component analysis (PCA), dynamic cross-correlation map (DCCM), and free energy landscape (FEL) analysis. KEY POINTS: • A computation-aided strategy was developed to engineer pullulanase thermostability. • Seventeen mutants were identified by combining three computational design predictors. • The G692M mutant was obtained with increased Tmand half-life at 70 °C.

Enhancement of the production of Bacillus naganoensis pullulanase in recombinant Bacillus subtilis by integrative expression.

Pullulanase is widely used in the starch processing industry as a debranching enzyme. However, extracellular production of pullulanase from recombinant Bacillus subtilis is limited and the loss of plasmids during fermentation of B. subtilis recombinants seriously affects the expression of the foreign protein, especially in large-scale production. In this study, a universal integrated plasmid was conducted harboring the pul cassette that included the pul gene encoding Bacillus naganoensis pullulanase (PUL), a constitutive promoter, PHpaII, and an extracellular signaling peptide, LipA. This cassette was inserted into the genomes of B. subtilis WB800 and B. subtilis WB600 by double homologous recombination. The pullulanase activity of up to 30.32 U/ml and 18.83 U/ml was achieved for B. subtilis WB800-PHpaII-pul and B. subtilis WB600-PHpaII-pul, respectively, under primary conditions. To further enhance the yield of PUL, the effects of four important factors (inoculum size, incubation temperature, shaking speed, and initial pH) on the expression of PUL in shake flask fermentation were evaluated by "one-factor-at-a-time" technique for B. subtilis WB800-PHpaII-pul. Consequently, the extracellular production of PUL was significantly enhanced, resulting in an activity of 60.85 U/ml.