Screening signal peptidase based on split-GFP assembly technology to promote the secretion of alkaline protease AprE in Bacillus amyloliquefaciens.

Improving the ability of bacteria to secrete protein is essential for large-scale production of food enzymes. However, due to the lack of effective tracking technology for target proteins, the optimization of the secretory system is facing many problems. In this study, we utilized the split-GFP system to achieve self-assembly into mature GFP in Bacillus amyloliquefaciens and successfully tracked the alkaline protease AprE. The split-GFP system was employed to assess the signal peptidases, a crucial component in the secretory system, and signal peptidase sipA was identified as playing a role in the secretion of AprE. Deletion of sipA resulted in a higher accumulation of the precursor protein of AprE compared to other signal peptidase deletion strains. To explore the mechanism of signal peptidase on signal peptide, molecular docking and calculation of free energy were performed. The action strength of the signal peptidase is determined by its binding affinity with the tripeptides at the C-terminal of the signal peptide. The functions of signal peptides YdbK and NucB rely on sipA, and overexpression of sipA by integrating it into genome of B. amyloliquefaciens increased the activity of extracellular AprE by 19.9 %. These findings provide insights into enhancing the secretion efficiency of chassis strains.

Sequence composition and location of CRE motifs affect the binding ability of CcpA protein.

Bacillus catabolite control protein (CcpA) mediates carbon catabolite repression (CCR) by binding with catabolite response elements (CREs) of genes or operons. Although numerous CREs had been predicted and identified, the influence of the changes in sequence and structure of CREs on recognition and binding for CcpA has yet to be unclear. This study aimed at revealing how CcpA could bind such diverse sites and focused on the analysis of multiple mutants of the CRE motif derived from the α-amylase promoter. Molecular docking and free energy calculation insights into the binding ability between the CRE sequences composition and CcpA protein. Disruption of conserved nucleotides in the CRE motifs, as well as altering the symmetric structure of the CRE sequences and the relative position of the displaced CRE motifs near the transcription start site contribute to some extent to weakening the strength of CcpA - dependent regulation. These main factors contribute to the understanding of the subtle changes in CRE motifs leading to differential regulatory effects of CcpA. Finally, an engineered promoter with a high level of transcription was obtained, and elevated extracellular enzyme activity was achieved in the expression system of Bacillus amyloliquefaciens, including alkaline protease, keratinase, aminopeptidase and acid-stable alpha amylase. The study also provides a reference for the application of other promoters with CRE motifts.

Hofmeister anion effects synergize with microbial transglutaminase to enhance the techno-functional properties of pea protein.

Pea protein are emerging as the most potential alternative for meat products, but its application was hindered by their weaker gelling properties. Here, the feasibility of combining the Hofmeister anion (CO32-, Citrate3-, and SO42-) effect with microbial transglutaminase (MTG) cross-linking strategy to improve the techno-functional properties of pea protein was studied. Hofmeister anions or/and MTG treatment of pea protein caused a clear shift in far-UV CD spectra towards ß-turn and random coil structures. Furthermore, Hofmeister anion and MTG-induced crosslinking caused a reduction of surface hydrophobicity in contrast with anions-treated. Compared to CO32- and SO42-, Citrate3- treatment can better improve the efficiency of MTG-crosslinking, as demonstrated by a reduction in free amino group contents and an increase in mean diameter size. Using MTG in combination with Hofmeister anions showed significantly improved foam property and gel hardness as well as decrease gelation temperature of pea protein, specifically Citrate3- treatment. Thus, this research provides a novel and effective method to improve the effect of MTG-cross-linked pea protein, which will play an essential role in future food production.

[Effect of hemX gene deletion on heme synthesis in Bacillus amyloliquefaciens].

Heme, which exists widely in living organisms, is a porphyrin compound with a variety of physiological functions. Bacillus amyloliquefaciens is an important industrial strain with the characteristics of easy cultivation and strong ability for expression and secretion of proteins. In order to screen the optimal starting strain for heme synthesis, the laboratory preserved strains were screened with and without addition of 5-aminolevulinic acid (ALA). There was no significant difference in the heme production of strains BA, BAΔ6 and BAΔ6ΔsigF. However, upon addition of ALA, the heme titer and specific heme production of strain BAΔ6ΔsigF were the highest, reaching 200.77 µmol/L and 615.70 µmol/(L·g DCW), respectively. Subsequently, the hemX gene (encoding the cytochrome assembly protein HemX) of strain BAΔ6ΔsigF was knocked out to explore its role in heme synthesis. It was found that the fermentation broth of the knockout strain turned red, while the growth was not significantly affected. The highest ALA concentration in flask fermentation reached 82.13 mg/L at 12 h, which was slightly higher than that of the control 75.11 mg/L. When ALA was not added, the heme titer and specific heme production were 1.99 times and 1.45 times that of the control, respectively. After adding ALA, the heme titer and specific heme production were 2.08 times and 1.72 times higher than that of the control, respectively. Real-time quantitative fluorescent PCR showed that the expressions of hemA, hemL, hemB, hemC, hemD, and hemQ genes at transcription level were up-regulated. We demonstrated that deletion of hemX gene can improve the production of heme, which may facilitate future development of heme-producing strain.

Enhancing the functional characteristics of soy protein isolate via cross-linking catalyzed by Bacillus subtilis transglutaminase.

BACKGROUND: Although Streptomyces mobaraense transglutaminase (MTG) has been extensively applied to enhance the functional characteristics of soy protein isolate (SPI) through cross-linking, various transglutaminases (TGs) in nature may provide more choice in the food industry. Previous research reported that TG derived from Bacillus subtilis (BTG) exhibited better pH stability and thermostability than MTG. RESULTS: An attempt was made to study the influence of BTG induced cross-linking on the properties of SPI. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results indicated that almost all protein constituents (α', α, ß, AS, and BS) in SPI could be cross-linked with BTG treatment. The BTG treatment also resulted in a significant increase (*P < 0.05) in SPI mean particle size. Emulsifying activity and stability were improved from 0.11535 m2  g-1 and 48.3% for native SPI to 0.13252 m2  g-1 and 83.9% for SPI treated with BTG at 6 h. Similarly, the modified SPI showed better foam activity (1.32 mL) and stability (87.6%) than the original SPI (0.93 mL and 56.8%). The water-holding capacity of SPI gel was found to increase with time, with a value of 95.43% at 6 h. Furthermore, SPI gel's texture profiles were greatly improved by adding BTG (*P < 0.05). CONCLUSION: The results of the present study indicated that BTG could be a promising cross-linking agent for improving the functional characteristics of SPI. As a substitute for MTG, BTG could thus potentially be used for food structure engineering to enhance the functional characteristics of multiple proteins to advance the development of food chemistry. © 2020 Society of Chemical Industry.

Tunable physical and mechanical properties of gelatin hydrogel after transglutaminase crosslinking on two gelatin types.

The crosslinking and related gel properties of 3 wt% gelatin (type-A and type-B) catalyzed by microbial transglutaminase (MTG, dose of 0-20 U/g gelatin) have been investigated. A MTG-depended increase in the molecular weight and mean diameter of both gelatins was observed, where type-A presented a higher crosslinking efficiency than type-B due to more acyl donors of the former. As MTG concentration increased, the surface hydrophobicity and thermal stability of type-A gelatin increased. Textural profile analysis (TPA) of type-A gelatin hydrogel showed a decrease in hardness and slight increase in springiness, while type-B gelatin gel was not affected generally. Rheological measurements confirmed the melting point of type-A gelatin hydrogel continually increased until the disappearance of gel thermo-reversibility at higher MTG levels (≥12 U/g gelatin), while type-B gelatin hydrogel always showed a sol-gel transition, suggesting that the gel performance was depended on the dominance of whether physical crosslinking or chemical crosslinking. Scanning electron microscope (SEM) results showed that the network structure of the type-A gelatin became more irregular as MTG increasing which indicated that introducing additional covalent cross-links within or between gelatin chains had a profound influence on gel's network structure, closely associated with the gel properties mentioned above. In summary, the superiority of type-A in MTG-crosslinking efficiency than type-B, can be used to modulate the physical and mechanical properties of gelatin hydrogel, governing by the combing of weak physical crosslinking and strong covalent crosslinking, which will be suitable for numerous industrial applications.

An innovative biotransformation to produce resveratrol by Bacillus safensis.

Resveratrol is considered as a potential food supplement, cosmetic ingredient and nutraceutical. In this study, resveratrol was produced by biotransformation successfully. In detail, a ß-glucosidase producing strain was isolated and identified as Bacillus safensis, and it could convert polydatin to resveratrol efficiently and rapidly. Further research showed that the conversion rate to resveratrol reached 93.1% in 8 h at 37 °C. The production of resveratrol was confirmed by HPLC, LC-MS and 1H-NMR to identify its structure and it was verified to possess antibacterial properties especially against Escherchia coli. To illustrate the resveratrol transformation mechanism, several glucosidases from B. safensis CGMCC 13129 were expressed and analyzed. The results showed that BGL4 and BGL5 had higher transformation activity compared with other tested glucosidases. This research provides a novel approach to produce resveratrol, and would promote the application of resveratrol in health-promoting pharmaceutical and food products.