Microorganisms
·
2025
·Vol. 13
·期 7
·1540
·
IF 4.2
Wenwen Peng,Haocheng Xu,Meiting Zhang,Baoyang Xu,Bing Dai,Caimei Yang
With 80% of bacterial infections occurring as biofilms, biofilm-related infections have evolved into a critical public health concern. Probiotics such asBacillus licheniformishave emerged as promising alternatives, offering new avenues for effective treatment. This study aimed to evaluate the activity oflicheniformisagainst the growth, biofilm formation, motility, and quorum sensing (QS) ofSalmonella typhimurium. Several experiments were conducted: The minimum inhibitory concentration (MIC) ofBacillus licheniformisagainstSalmonella typhimuriumwas determined to be 0.5 mg/mL using the broth microdilution method. The inhibition zone of 100 mg/mL ofB. licheniformisagainstSalmonella typhimuriumwas 19.98 ± 1.38 mm; the time-growth curve showed thatB. licheniformiscan effectively inhibit the growth ofSalmonella typhimurium. In biofilm experiments, at the MIC ofB. licheniformis, the inhibition rate of immature biofilm ofSalmonella typhimuriumwas 86.9%, and it significantly reduced the production of biofilm components (EPS, e-DNA, and extracellular proteases) (p< 0.05). The disruption rate of mature biofilm byB. licheniformisat the MIC was 66.89%, and it significantly decreased the levels of biofilm components (EPS and e-DNA) (p< 0.5). Microscopic observation showed that both the MIC and 1/2 MIC ofB. licheniformiscould reduce the number of bacteria in theSalmonella typhimuriumbiofilm, which was not conducive to the formation and maintenance of the biofilm structure. Swimming/Swarming assays and QS experiments confirmed thatB. licheniformisinhibits the motility ofSalmonella typhimuriumand the secretion of AI-1-type quorum sensing molecules and downregulates the AI-2 quorum sensing system by upregulatinglsrgene expression. These findings suggest thatB. licheniformiscould be a potential antimicrobial agent and biofilm inhibitor.
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INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
·
2023
·
IF 8.2
Shiyi Wang,Yiwen Zhao,Shufen Mao,Jiang Zhu,Yangyang Zhan,Dongbo Cai,Xin Ma,Dong Wang,Shouwen Chen
Disulfide bonds in proteins have strongly influence on the folding efficiency by constraining the conformational space. The inefficient disulfide bond formation of proteins is the main limiting factor of enzyme activity and stability. This study aimed to increase the activity of disulfide-bond-containing proteins via promoting disulfide bonds formation in Bacillus licheniformis . Initially, the glutamate decarboxylase GAD from Escherichia coli was selected as the model protein and introduced into the B. licheniformis . Then, the disulfide isomerase and oxidoreductase from different sources were excavated and overexpressed successively to improve the catalytic efficiency of GAD. The final engineered B. licheniformis showed significantly improved GAD specific activity (from 10.4 U/mg to 80.0 U/mg), which also presented perfect adaptability for other disulfide-bond-containing proteins, for instance, UDP-glucosyltransferase from Arabidopsis thaliana . Taken together, our work demonstrated that the activity of GAD in B. licheniformis was regulated by the disulfide bonds formation status and provided a promising platform for the expression of disulfide-bond-containing proteins.
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INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
·
2022
·Vol. 23
·期 17
·9663
·
IF 6.208
Yawen Zhou,Linshu Jiao,Juan Shen,Huibing Chi,Zhaoxin Lu,Huawei Liu,Fengxia Lu,Ping Zhu
Low catalytic activity is a key factor limiting the widespread application of type II L-asparaginase (ASNase) in the food and pharmaceutical industries. In this study, smart libraries were constructed by semi-rational design to improve the catalytic activity of type II ASNase fromBacillus licheniformis. Mutants with greatly enhanced catalytic efficiency were screened by saturation mutations and combinatorial mutations. A quintuple mutant ILRAC was ultimately obtained with specific activity of 841.62 IU/mg andkcat/Kmof 537.15 min−1·mM−1, which were 4.24-fold and 6.32-fold more than those of wild-type ASNase. The highest specific activity andkcat/Kmwere firstly reported in type II ASNase fromBacillus licheniformis. Additionally, enhanced pH stability and superior thermostability were both achieved in mutant ILRAC. Meanwhile, structural alignment and molecular dynamic simulation demonstrated that high structure stability and strong substrate binding were beneficial for the improved thermal stability and enzymatic activity of mutant ILRAC. This is the first time that enzymatic activity of type II ASNase fromBacillus licheniformishas been enhanced by the semi-rational approach, and results provide new insights into enzymatic modification of L-asparaginase for industrial applications.Keywords:L-asparaginase;protein engineering;saturation mutagenesis;catalytic efficiency;molecular dynamics simulation
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Minerals
·
2019
·Vol. 9
·期 7
·392
·
IF 2.25
Yangjian Cheng,Zhibin Ke,Xiaojing Bian,Jianhua Zhang,Zhen Huang,Yuancai Lv,Minghua Liu
The recovery of precious metals is a project with both economic and environmental significance. In this paper, how to use bacterial mineralization to selectively recover gold from multi-ionic aqueous systems is presented. TheBacillus licheniformisFZUL-63, isolated from a landscape lake in Fuzhou University, was shown to selectively mineralize and precipitate gold from coexisting ions in aqueous solution. The removal of Au(III) almost happened in the first hour. Scanning electron microscope with X-ray energy dispersive spectroscopy (SEM/EDS-mapping) results and fourier transform infrared spectroscopy (FTIR) data show that the amino, carboxyl, and phosphate groups on the surface of the bacteria are related to the adsorption of gold ions. X-ray photoelectron spectroscopy (XPS) results implied that Au(III) ions were reduced to those that were monovalent, and the Au(I) was then adsorbed on the bacterial surface at the beginning stage (in the first hour). X-ray diffraction (XRD) results showed that the gold biomineralization began about 10 h after the interaction between Au(III) ions and bacteria. Au(III) mineralization has rarely been influenced by other co-existing metal ions. Transmission electron microscope (TEM) analysis shows that the gold nanoparticles have a polyhedral structure with a particle size of ~20 nm. TheBacillus licheniformisFZUL-63 could selectively mineralize and recover 478 mg/g (dry biomass) gold from aqua regia-based metal wastewater through four cycles. This could be of great potential in practical applications.Keywords:selective biomineralization;recovery of Au(III);AuNP;Bacillus licheniformisFZUL-63;aqua regia-based metal wastewater
研究领域:
微生物学
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环境科学
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材料科学
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FOOD CHEMISTRY
·
2020
·
IF 6.306
Yingqi Ruan,Yan Xu,Wenchi Zhang,Rongzhen Zhang
Maltogenic amylase suppressed starch retrogradation in baked products. Here, a maltogenic amylase-producing strain of bacteria was screened and identified as Bacillus licheniformis R-53. Its coding gene was cloned and over-expressed in Bacillus subtilis WB600. Recombinant maltogenic amylase BLMA exhibited activity of 3235 U/mg under optimal conditions (60 °C and pH 6.5), with a good thermostability and pH stability. Mixolab experiment showed that a concentration of 60 ppm BLMA significantly improved the operating characteristics of dough. Baking test indicated the recombinant BLMA reduced bread hardness by 2.12 times compared with the control. Compared with maltogenic amylase from Novozymes (Novamyl 3D BG) and Angel Yeast Co. Ltd. (MAM100), BLMA has better effect on improving the bread volume, and almost the same effect on reducing hardness, improving elasticity and maintaining sensory as Novamyl 3D BG. Adding BLMA improved bread quality, increased bread volume and decreased hardness during storage, thus extending its shelf life.
研究领域:
食品科学
·
微生物学
·
酶学
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FOOD MICROBIOLOGY
·
2024
·
IF 4.5
Yang Liu,Wenwen Ge,Yuxin Sun,Hongchao Dai,Luyao Fan,Lei Yuan,Zhenquan Yang,Xinan Jiao
Bacillus cereus and Bacillus licheniformis are widely presented in dairy products. They can form thick biofilms on surfaces of dairy processing equipment, which may pose serious safety issues and spoilage of final dairy products. However, how ecological interactions between B. cereus and B. licheniformis affect the functions and stability of mixed-species biofilm remains uncovered. In this work, the altered profiles of a dual-species biofilm by dairy-derived B. cereus 121 and B. licheniformis 919 were investigated by RNA-sequencing analysis in combined with phenotype validation (bacterial growth, biofilm-forming capacity, biofilm EPS production, and biofilm structures). The results confirmed that the presence of B. cereus 121 reduced the growth of B. licheniformis 919 planktonic cells, and decreased the biofilm cell numbers of B. licheniformis 919 in the dual-species biofilm when compared to that in its single-species biofilm. The bacterial interaction also reduced the amount of proteins and carbohydrates in the biofilm matrix, and decreased the coverage, average thickness, and total biomass of biofilms. In addition, results from RNA-sequencing analysis showed that the bacterial interaction caused a total of 128 ( B. licheniformis 919) and 216 ( B. cereus 121) differentially expressed genes (DEGs) during the co-culture of planktonic cells. Functional annotation revealed that the DEGs of B. licheniformis 919 were mainly involved in 10 downregulated pathways including citrate cycle, pyruvate metabolism, nonribosomal peptide structures, glycolysis/gluconeogenesis, quorum sensing, alanine, aspartate and glutamate metabolism, oxidative phosphorylation, beta-Lactam resistance, arginine and proline metabolism, and beta-Alanine metabolism when co-cultured with B. cereus 121. On the other hand, the DEGs from B. cereus 121 were significantly enriched for two downregulated pathways (cysteine and methionine metabolism, and inositol phosphate metabolism) and four upregulated pathways (nitrogen metabolism, glyoxylate and dicarboxylate metabolism, glycine, serine and threonine metabolism, and propanoate metabolism). Results of this study facilitate updated knowledge of how bacterial interaction during the biofilm formation shapes the features of the mixed-species biofilm.
研究领域:
微生物学
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食品科学
·
分子生物学
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JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
·
2022
·
IF 5.895
Huibing Chi,Yilian Wang,Bingjie Xia,Yawen Zhou,Zhaoxin Lu,Fengxia Lu,Ping Zhu
l-Asparaginase has gained much attention for effectively treating acute lymphoblastic leukemia (ALL) and mitigating carcinogenic acrylamide in fried foods. Due to high-dose dependence for clinical treatment and low mitigation efficiency for thermal food processes caused by poor thermal stability, a method to achieve thermostable l-asparaginase has become a critical bottleneck. In this study, a rational design including free energy combined with structural and conservative analyses was applied to engineer the thermostability of l-asparaginase from Bacillus licheniformis (BlAsnase). Two enhanced thermostability mutants D172W and E207A were screened out by site-directed saturation mutagenesis. The double mutant D172W/E207A exhibited highly remarkable thermostability with a 65.8-fold longer half-life at 55 °C and 5 °C higher optimum reaction temperature and melting temperature (Tm) than those of wild-type BlAsnase. Further, secondary structure, sequence, molecular dynamics (MD), and 3D-structure analysis revealed that the excellent thermostability of the mutant D172W/E207A was on account of increased hydrophobicity and decreased flexibility, highly rigid structure, hydrophobic interactions, and favorable electrostatic potential. As the first report of rationally designing l-asparaginase with improved thermostability from B. licheniformis, this study offers a facile and efficient process to improve the thermostability of l-asparaginase for industrial applications.
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ACS Sustainable Chemistry & Engineering
·
2022
·
IF 9.224
Ze-Wang Guo,Zi-Fu Ni,Min-Hua Zong,Wen-Yong Lou
Consolidated bioprocessing (CBP) has emerged as a promising strategy for green synthesis of 2,3-butanediol (BDO) from Jerusalem artichoke tubers (JAT). Here, modular metabolic engineering of Bacillus licheniformis MW3 was implemented, in which the overall BDO synthesis process was divided into four modules (inulin hydrolysis, sporulation elimination, carbon flux redistribution, and enantiopure meso-BDO synthesis). First, endo-inulinase was introduced to boost inulin utilization. Second, sporulation was blocked by spoIIE knockout, thus reducing the leakage risk of the genetically engineered strain from the fermentation plant. Third, more carbon flux was shunted into BDO production via exchanging the alsS start codon, blocking acetoin catabolism, and competing pathways. Finally, enantiopure meso-BDO was synthesized by gdh knockout. Encouragingly, the final engineered strain, MWO-8, produced meso-BDO with 99.3% purity at a titer, productivity, and yield of 82.51 g/L, 1.38 g/L/h, and 0.439 g/g, respectively. To our knowledge, this is the highest titer of BDO from JAT by CBP so far. Collectively, this work provides a highly promising alternative approach for sustainable and cost-effective production of BDO.
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FOOD BIOTECHNOLOGY
·
2023
·
IF 1.8
Junxin Zhao,Maomao Ma,Zheling Zeng,Dongman Wan,Xianghui Yan,Jiaheng Xia,Ping Yu,Deming Gong
This study aimed to investigate the effect of mutating the consensus pentapeptide on the enzymatic properties and structural characteristics of Bacillus licheniformis lipase. Site-directed mutagenesis was used to construct mutants and then expressed in Escherichia coli BL21. After purification and enzyme activity analysis, only A75C and A75G retained activity. Compared to the wild-type lipase and A75G, the A75C showed higher thermostability at 40°C and better alkali resistance in pH 9.0–10.0 after incubation for 1 h. The kinetic parameters, structural characteristics and surface hydrophobicity of the lipases were determined. The results indicated that the secondary structure was hardly changed, while mutants exhibited higher activity, thermostability and hydrophobicity. Finally, the reasons for the increased enzyme activity were conducted through molecular dynamics simulation. The strategy of mutating the first residue of the lipase consensus pentapeptide may provide new insights into the enzyme engineering for industrial applications.
研究领域:
生物化学
·
酶学
·
蛋白质工程
·
分子生物学
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PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY
·
2025
·
IF 1.9
Junxin Zhao,Yiman Wu,Yuhan Yao,Tonglei Zhou,Yiming Xu,Jingyi Gui,Didi Guo,Xinshuo Yang,Deguo Wang,Xianghui Yan,Ping Yu,Zheling Zeng,Fugang Xiao
This study investigates the effects of atmospheric room temperature plasma (ARTP) mutagenesis on the morphological and genetic modifications of the lipase-producing strain Bacillus licheniformis NCU CS-5. While traditional strain improvement methods often suffer from low mutation rates and uneven mutation distribution, ARTP mutagenesis offers a powerful and efficient alternative. The significance of this study lies in its potential to enhance lipase production, a key enzyme in various industrial applications, through the generation of high-performance mutant strains. The gap in existing literature pertains to the limited understanding of ARTP-induced mutations in B. licheniformis and their impact on enzyme production. The novelty of this study is to explore the morphological and genetic changes induced by ARTP and to identify key genetic determinants contributing to these changes. Key findings include significant morphological alterations, higher intracellular Ca2+ levels, and numerous single-nucleotide variants (SNVs) and insertions/deletions (InDels) in the mutant strain. These findings imply that ARTP mutagenesis can effectively enhance lipase production, paving the way for optimized industrial microbial strains.
研究领域:
微生物学
·
基因工程
·
工业生物技术
·
分子遗传学
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NUCLEIC ACIDS RESEARCH
·
2023
·Vol. 51
·期 21
·11952-11966
·
IF 14.9
He Hehe,Li Youran,Ma Xufan,Xu Sha,Zhang Liang,Ding Zhongyang,Shi Guiyang
Synthetic regulation of metabolic fluxes has emerged as a common strategy to improve the performance of microbial cell factories. The present regulatory toolboxes predominantly rely on the control and manipulation of carbon pathways. Nitrogen is an essential nutrient that plays a vital role in growth and metabolism. However, the availability of broadly applicable tools based on nitrogen pathways for metabolic regulation remains limited. In this work, we present a novel regulatory system that harnesses signals associated with nitrogen metabolism to redirect excess carbon flux in Bacillus licheniformis. By engineering the native transcription factor GlnR and incorporating a sorbitol-responsive element, we achieved a remarkable 99% inhibition of the expression of the green fluorescent protein reporter gene. Leveraging this system, we identified the optimal redirection point for the overflow carbon flux, resulting in a substantial 79.5% reduction in acetoin accumulation and a 2.6-fold increase in acetate production. This work highlight the significance of nitrogen metabolism in synthetic biology and its valuable contribution to metabolic engineering. Furthermore, our work paves the way for multidimensional metabolic regulation in future synthetic biology endeavors.
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BIOTECHNOLOGY AND APPLIED BIOCHEMISTRY
·
2019
·Vol. 67
·期 2
·249-256
·
IF 1.559
Bingfeng Li,Bo Fan,Jingping Fan,Siyuan Chang,Xin Pan,Yinan Wang,Yaming Wu,Junsong Song,Xuejun He
Raspberry ketone is a primary aroma component of the red raspberry. The glycosylation of this compound is a potential approach used to improve its pharmaceutical properties. In this work, raspberry ketone glycosides are produced in bacteria for the first time. Bacillus licheniformis PI15, an organic solvent-tolerant glycosyltransferase-producing strain, was isolated from chemically polluted soil. The cloning and heterologous expression of a glycosyltransferase, which was designated PI-GT1, in Escherichia coli BL21 resulted in the expression of an active and soluble protein that accounted for 15% of the total cell protein content. Purified PI-GT1 was highly active and stable over a broad pH range (6.0–10.0) and showed excellent pH stability. PI-GT1 maintained almost 60% of its maximal activity after 3 H of incubation at 20–40 °C and demonstrated optimal activity at 30 °C. Additionally, PI-GT1 displayed high stability and activity in the presence of hydrophilic solvents with log P ≤ −0.2 and retained more than 80% of its activity after 3 H of treatment. Supplementation with 10% DMSO markedly improved the glycosylation of raspberry ketone, resulting in a value 26 times higher than that in aqueous solution. The organic solvent-tolerant PI-GT1 may have potential uses in industrial chemical and pharmaceutical synthesis applications.
研究领域:
生物技术
·
生物化学
·
药物化学
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