Enhancement of alkaline protease production in recombinant Bacillus licheniformis by response surface methodology
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ABSTRACT:
SUBMITTER: Zhang Y
PROVIDER: S-EPMC10991860 | biostudies-literature | 2023 Apr
REPOSITORIES: biostudies-literature
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Project description:BACKGROUND:Bacillus licheniformis 2709 is extensively applied as a host for the high-level production of heterologous proteins, but Bacillus cells often possess unfavorable wild-type properties, such as production of viscous materials and foam during fermentation, which seriously influenced the application in industrial fermentation. How to develop it from a soil bacterium to a super-secreting cell factory harboring less undomesticated properties always plays vital role in industrial production. Besides, the optimal expression pattern of the inducible enzymes like alkaline protease has not been optimized by comparing the transcriptional efficiency of different plasmids and genomic integration sites in B. licheniformis. RESULT:Bacillus licheniformis 2709 was genetically modified by disrupting the native lchAC genes related to foaming and the eps cluster encoding the extracellular mucopolysaccharide via a markerless genome-editing method. We further optimized the expression of the alkaline protease gene (aprE) by screening the most efficient expression system among different modular plasmids and genomic loci. The results indicated that genomic expression of aprE was superior to plasmid expression and finally the transcriptional level of aprE greatly increased 1.67-fold through host optimization and chromosomal integration in the vicinity of the origin of replication, while the enzyme activity significantly improved 62.19% compared with the wild-type alkaline protease-producing strain B. licheniformis. CONCLUSION:We successfully engineered an AprE high-yielding strain free of undesirable properties and its fermentation traits could be applied to bulk-production by host genetic modification and expression optimization. In summary, host optimization is an enabling technology for improving enzyme production by eliminating the harmful traits of the host and optimizing expression patterns. We believe that these strategies can be applied to improve heterologous protein expression in other Bacillus species.
| S-EPMC7041084 | biostudies-literature
Project description:BackgroundOur laboratory has constructed a Bacillus licheniformis strain that secretes alkaline protease (AprE) with excellent enzymatic properties. B. licheniformis is generally regarded as safe and has a high industrial exoenzyme secretion capacity, but the host retains some undomesticated characteristic that increase its competitiveness and survival, such as spore-formation, which increases the requirements and difficulties in industrial operations (e.g. sterilization and enzyme activity control). Furthermore, the influence of sporulation on alkaline protease production in B. licheniformis has not been elucidated in detail.ResultA series of asporogenic variants of the parent strain were constructed by individually knocking out the master regulator genes (spo0A, sigF and sigE) involved in sporulation. Most of the variants formed abortively disporic cells characterized by asymmetric septa at the poles and unable to survive incubation at 75 °C for 10 min. Two of them (ΔsigF and ΔsigE) exhibited superior characteristics in protease production, especially improving the expression of the aprE gene. Under the currently used fermentation conditions, the vegetative production phase of ΔsigF can be prolonged to 72 h, and the highest protease production of ΔsigF reached 29,494 ± 1053 U/mL, which was about 19.7% higher than that of the wild-type strain.ConclusionWe first constructed three key sporulation-deficient strain to investigate the effect of sporulation on alkaline protease synthesis. The sigF mutant retained important industrial properties such as facilitating the sterilization process, a prolonged stable phase of enzyme production and slower decreasing trend, which will be superior in energy conservation, simpler operations and target product controlling effect. In summary, the work provides a useful industrial host with preferable characteristics and a novel strategy to enhance the production of protease.
| S-EPMC6657089 | biostudies-literature
Project description:The enormous increase in world population has resulted in generation of million tons of agricultural wastes. Biotechnological process for production of green chemicals, namely, enzymes, provides the best utilization of these otherwise unutilized wastes. The present study elaborates concomitant production of protease and amylase in solid state fermentation (SSF) by a newly isolated Bacillus megaterium B69, using agroindustrial wastes. Two-level statistical model employing Plackett-Burman and response surface methodology was designed for optimization of various physicochemical conditions affecting the production of two enzymes concomitantly. The studies revealed that the new strain concomitantly produced 1242 U/g of protease and 1666.6 U/g of amylase by best utilizing mustard oilseed cake as the substrate at 20% substrate concentration and 45% moisture content after 84 h of incubation. An increase of 2.95- and 2.04-fold from basal media was observed in protease and amylase production, respectively. ANOVA of both the design models showed high accuracy of the polynomial model with significant similarities between the predicted and the observed results. The model stood accurate at the bench level validation, suggesting that the design model could be used for multienzyme production at mass scale.
| S-EPMC4244924 | biostudies-literature
Project description:A new potent halophilic protease producer, Halobacterium sp. strain LBU50301 was isolated from salt-fermented fish samples (budu) and identified by phenotypic analysis, and 16S rDNA gene sequencing. Thereafter, sequential statistical strategy was used to optimize halophilic protease production from Halobacterium sp. strain LBU50301 by shake-flask fermentation. The classical one-factor-at-a-time (OFAT) approach determined gelatin was the best nitrogen source. Based on Plackett-Burman (PB) experimental design; gelatin, MgSO4·7H2O, NaCl and pH significantly influenced the halophilic protease production. Central composite design (CCD) determined the optimum level of medium components. Subsequently, an 8.78-fold increase in corresponding halophilic protease yield (156.22 U/mL) was obtained, compared with that produced in the original medium (17.80 U/mL). Validation experiments proved the adequacy and accuracy of model, and the results showed the predicted value agreed well with the experimental values. An overall 13-fold increase in halophilic protease yield was achieved using a 3 L laboratory fermenter and optimized medium (231.33 U/mL).
| S-EPMC5040864 | biostudies-literature
Project description:A soil bacterium capable of degrading chitin on chitin agar plates was isolated and identified as Bacillus pumilus isolate U5 on the basis of 16S rDNA sequence analysis. In order to optimize culture conditions for chitinase production by this bacterium, a two step approach was employed. First, the effects of several medium components were studied using the Plackett-Burman design. Among various components tested, chitin and yeast extract showed positive effect on enzyme production while MgSO4 and FeSO4 had negative effect. However, the linear model proved to be insufficient for determining the optimum levels for these components due to a highly significant curvature effect. In the second step, Box-Behnken response surface methodology was used to determine the optimum values. It was noticed that a quadratic polynomial equation fitted he experimental data appropriately. The optimum concentrations for chitin, yeast extract, MgSO4 and FeSO4 were found to be 4.76, 0.439, 0.0055 and 0.019 g/L, respectively, with a predicted value of chitinase production of 97.67 U/100 mL. Using this statistically optimized medium, the practical chitinase production reached 96.1 U/100 mL.
| S-EPMC3813072 | biostudies-literature
Project description:BackgroundDifferent types of exogenous protease supplements have a positive impact on animal performance, but their effects on the nutritional value of meat and the gut microbial community of broilers have not been extensively studied. The objective of this investigation was to determine the impact of supplementation with a novel alkaline protease derived from Bacillus licheniformis (at doses of 0, 100, 200, 300, and 400 g/t) on the fatty acid and amino acid profiles, inosine monophosphate (IMP) levels, total volatile basic nitrogen (TVB-N) content found within the breast muscle, as well as the impact on the cecal microbiota and metabolites.ResultsSupplementation with 200-400 g/t of the novel protease resulted in a significant elevation in the concentration of essential amino acids (P < 0.001), flavor amino acids (P < 0.001), and total protein (P = 0.013) within the breast muscle. Results derived from the 16S rRNA sequencing and untargeted metabolomics analysis of the cecal content revealed that the novel protease reshaped the cecal microbial and metabolite profiles. In particular, it led to increased relative abundances of Bacteroides, Lactobacillus, Alistipes, and Eubacterium, while simultaneously causing a reduction in the metabolites of D-lactic acid and malonic acid. Moreover, correlation analyses unveiled significant relationships between distinct microbes and metabolites with the contents of IMP, fatty acids, and amino acids in the broiler's breast muscle.ConclusionIn summary, the novel protease regulated the intestinal microbial community and metabolism, thereby inducing changes in the compositions of fatty acids and amino acids profiles, as well as IMP levels in broiler meat. These alterations significantly contributed to the enhancement of the nutritional value and flavor of the meat.
| S-EPMC10770948 | biostudies-literature
Project description:The production of halophilic thermostable protease by Haloferax lucentensis VKMM 007 was optimized using a statistical approach. In accordance with factorial design, soluble starch, gelatin, KCl and MgSO4 were selected among 27 variables tested. Next, a second-order quadratic model was estimated and optimal medium concentrations were determined based on quadratic regression equation generated by model. These were 5.14 g L(-1) of KCl, 6.57 g L(-1)of MgSO4, 9.05 g L(-1)of gelatin and 5.27 g L(-1)of soluble starch in high salts media supplemented with 0.5% (w/v) of beef extract and peptone, respectively. In these optimal conditions, the obtained protease concentration of 6.80 U mL(-1) was in agreement with the predicted protease concentration and was further improved to 7.02 U mL(-1) by increasing the concentration of NaCl in the medium to 25% (w/v). An overall 4.0-fold increase in protease production was achieved in the optimized medium compared to activity obtained in initial medium.
| S-EPMC3769814 | biostudies-literature
Project description:BackgroundPolyhydroxybutyrate (PHB) is a biopolymer formed by some microbes in response to excess carbon sources or essential nutrient depletion. PHBs are entirely biodegradable into CO2 and H2O under aerobic and anaerobic conditions. It has several applications in various fields such as medicine, pharmacy, agriculture, and food packaging due to its biocompatibility and nontoxicity nature.ResultIn the present study, PHB-producing bacterium was isolated from the Dirout channel at Assiut Governorate. This isolate was characterized phenotypically and genetically as Bacillus cereus SH-02 (OM992297). According to one-way ANOVA test, the maximum PHB content was observed after 72 h of incubation at 35 °C using glucose and peptone as carbon and nitrogen source. Response surface methodology (RSM) was used to study the interactive effects of glucose concentration, peptone concentration, and pH on PHB production. This result proved that all variables have a significant effect on PHB production either independently or in the interaction with each other. The optimized medium conditions with the constraint to maximize PHB content and concentration were 22.315 g/L glucose, and 15.625 g/L peptone at pH 7.048. The maximum PHB content and concentration were 3100.799 mg/L and 28.799% which was close to the actual value (3051 mg/l and 28.7%). The polymer was identified as PHB using FTIR, NMR, and mass spectrometry. FT-IR analysis showed a strong band at 1724 cm- 1 which attributed to the ester group's carbonyl while NMR analysis has different peaks at 169.15, 67.6, 40.77, and 19.75 ppm that were corresponding to carbonyl, methine, methylene, and methyl resonance. Mass spectroscopy exhibited molecular weight for methyl 3- hydroxybutyric acid.ConclusionPHB-producing strain was identified as Bacillus cereus SH-02 (OM992297). Under optimum conditions from RSM analysis, the maximum PHB content and concentration of this strain can reach (3100.799 mg/L and 28.799%); respectively. FTIR, NMR, and Mass spectrometry were used to confirm the polymer as PHB. Our results demonstrated that optimization using RSM is one of the strategies used for reducing the production cost. RSM can determine the optimal factors to produce the polymer in a better way and in a larger quantity without consuming time.
| S-EPMC9306189 | biostudies-literature
Project description:BackgroundCholesterol oxidase has numerous biomedical and industrial applications. In the current study, a new bacterial strain was isolated from sewage and was selected for its high potency for cholesterol degradation (%) and production of high cholesterol oxidase activity (U/OD600).ResultsBased on the sequence of 16S rRNA gene, the bacterium was identified as Bacillus subtilis. The fermentation conditions affecting cholesterol degradation (%) and the activity of cholesterol oxidase (U/OD600) of B. subtilis were optimized through fractional factorial design (FFD) and response surface methodology (RSM). According to this sequential optimization approach, 80.152% cholesterol degradation was achieved by setting the concentrations of cholesterol, inoculum size, and magnesium sulphate at 0.05 g/l, 6%, and 0.05 g/l, respectively. Moreover, 85.461 U of cholesterol oxidase/OD600 were attained by adjusting the fermentation conditions at initial pH, 6; volume of the fermentation medium, 15 ml/flask; and concentration of cholesterol, 0.05 g/l. The optimization process improved cholesterol degradation (%) and the activity of cholesterol oxidase (U/OD600) by 139% and 154%, respectively. No cholesterol was detected in the spectroscopic analysis of the optimized fermented medium via gas chromatography-mass spectroscopy (GC-MS).ConclusionThe current study provides principal information for the development of efficient production of cholesterol oxidase by B. subtilis that could be used in various applications.
| S-EPMC10673797 | biostudies-literature
Project description:The alkaline protease from Bacillus licheniformis strain 2709 (AprE 2709) is widely used in Chinese industries but faces stability challenges under high-temperature conditions. This study employed molecular modeling and mutagenesis to identify Asn residues at positions 61, 160, and 211 as key sites affecting the stability of AprE 2709. By leveraging the additive and cooperative effects of mutations, the mutant enzyme AprE 2709 (N61G/N160G/N211G) was engineered, exhibiting enhanced thermostability and catalytic activity. The mutant demonstrated a 2.89-fold increase in half-life at 60 °C and a 1.56-fold improvement in catalytic efficiency compared to the wild-type enzyme. Structural analysis revealed that the improved thermostability was due to altered electrostatic interactions and strengthened hydrophobic contacts. Targeting Asn residues prone to deamidation presents a promising strategy for improving protein heat tolerance. These findings not only enhance our understanding of enzyme stability but also lay a foundation for future research aimed at optimizing alkaline proteases for diverse industrial applications, particularly in high-temperature processes.
| S-EPMC11901772 | biostudies-literature