Enhancing protease activity of Bacillus subtilis using UV-laser random mutagenesis and high-throughput screening

Lu, Feng; Chao, Jiapin; Zhao, Xiaoxue; Betchem, Garba; Ding, Yanheng; Yang, Xue; Li, Yunliang; Ma, Haile

doi:10.1016/j.procbio.2022.05.018

Cited by 13 publications

(7 citation statements)

References 33 publications

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“…After treating for 150, [10] For chemical mutagenesis, besides time, the affecting factors also include the concentration of mutagen and reaction temperature. Fig.…”

Section: Resultsmentioning

confidence: 99%

“…1 mL spore suspension (1 × 108 mL -1 ) was transferred into a 10 mL sterilized tube and mixed with 1 mL DES ethanol solution. In order to determine the best mutagenic reaction conditions, it adjust different mutagenic time (5,10,15,20,25,30,35, and 40 min), different concentrations (0.5, 1.0, 1.5, 2.0, and 2.5%), and different temperature (40, 45, 50, 55, 60, 65 o C). The mixture was stirred at 200 r• min-1 for reaction.…”

Section: Mutagenesis and Screening By Ultraviolet And Des Combined Me...mentioning

confidence: 99%

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Study on mutagenesis of high protease producing thermophilic bacteria by UV-DES and hydrolysis of excess sludge

Wang,

Ning,

Zhong

et al. 2023

E3S Web Conf.

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Excess sludge a resource-rich organic waste, can be solubilized by thermophilic enzymes to extract proteins and anaerobic digestion of methane production to reduce sludge and use resources. At the same time, it can improve new ideas to relieve the pressure of the energy industry. To solve the problems of low enzyme-producing activity of wild strains and low hydrolysis rate of ES, this study utilized UV-DES compound mutagenesis to screen dominant mutant strains under optimized conditions, which were added to ES hydrolysis reaction system. The results showed that UV-DES mutagenesis could effectively improve the ability of S-TE and promote the hydrolysis of ES. The protease activity of the mutant strain KT16 obtained by screening was 46.7% higher than that of the original strain DC8. KT16 effectively degraded insoluble organic matter, reduced the average particle size of ES, and increased the concentrations of extracellular proteins, polypeptides, and amino acids to 1731.8, 521.3, and 510 mg·L-1, respectively. This study will provide a reference for the resource utilized.

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“…After treating for 150, [10] For chemical mutagenesis, besides time, the affecting factors also include the concentration of mutagen and reaction temperature. Fig.…”

Section: Resultsmentioning

confidence: 99%

Section: Mutagenesis and Screening By Ultraviolet And Des Combined Me...mentioning

confidence: 99%

Study on mutagenesis of high protease producing thermophilic bacteria by UV-DES and hydrolysis of excess sludge

Wang,

Ning,

Zhong

et al. 2023

E3S Web Conf.

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“…They replaced the native promoter of the mycosubtilin operon with P repU , and that increased mycosubtilin up to 15-fold more than B. subtilis ATCC 6633 as a wild type. Hussein and Fahim (44) created a mutant strain, BMG06, that overexpressed plipastatin and fengycin by about 35 and 4 folds, respectively, more than wild-type B. subtilis 168, using constitutive promotor PrepU to replace Ppps (the plipastatin promoter) and Pfen (the fengycin promoter).Different genetic approaches were employed to improve the protease production from microbial sources for different applications; for example, UV-laser random mutagenesis was employed to treat B. subtilis to select high protease producers for increasing the peptide level of fermented soybean meal, which helps to improve its nutritional value (45) . Suberu etal (46) established the heterologous expression of the serine alkaline protease gene from Bacillus subtilis RD7 using the pET15b vector in E. coli BL21 cells, and the puri ed recombinant protease enzyme was employed to eliminate egg yolk stains at 40 °C and pH 10.…”

Section: Discussionmentioning

confidence: 99%

Effect of neutral protease overproduction in Bacillus subtilis 168 via site-directed mutation against Meloidogyne incognita infecting eggplant under greenhouse conditions

Soliman

El-Sayed

Nour

et al. 2023

Preprint

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Root-knot nematodes (RKN), Meloidogyne incognita, caused significant problems for many important crops. Measuring control with low environmental impact has been required since EU legislation revised pesticide laws for crops. Bacteria-based control methods reduce pollutants and stabilise ecological changes, which makes them promising for controlling plant pathogens. In this study, the derivative of Bacillus subtilis168, termed Bs118, was generated by replacing the native promoter of the extracellular neutral metalloprotease-encoding gene (nprE) with a constitutive promoter of the repU gene responsible for replication of the Staphylococcus aureus plasmid pUB110. As a result, protease production increased to twice that of the wild type. Results revealed that the overproduction of neutral metalloprotease conferred Bs118 high nematocidal activity by inducing 98% mortality in the M. incognita J2 in vitro study. Bs118 stated its priority in affecting root-knot nematode reproduction under greenhouse conditions. The soil drench treatment was more promising than root dipping in controlling M. incognita compared with the untreated control treatment. The same trend happened in the eggplant growth parameters, where Bs118 improved plant health more than Bs168. In conclusion, site-directed mutation via homologous recombination to replace the native promoter with another constitutive one is a promising approach to constructing modified strains with higher protease production that can be employed as an efficient biocontrol agent against root-knot nematodes in addition to the positive impacts on plant growth.

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“…Feng et al screened for strains with high protease production using the UV mutagenesis of Bacillus subtilis [25]. Wang et al combined Co-60 γ irradiation with LiCl mutagenesis to obtain strains with high Nosiheptide production [26], and our study combined UV mutagenesis and LiCl mutagenesis to screen for strains with high diosgenin production.…”

Section: Screening Of High Yield Diosgenin Strains By Compound Mutage...mentioning

confidence: 98%

“…Wright (DZW) to diosgenin [10]. Zhao et al (2022) developed and synthesized a new solid acid based on TiO 2 as an effective solid acid catalyst by sulfonating the prepared TiO 2 , which can be used to extract diosgenin from saponin [11]. Although the methods mentioned above solve the problem of the severe environmental harm caused by the large-scale application of sulfuric acid, they have several disadvantages.…”

Section: Introductionmentioning

confidence: 99%

A Method for Improving Microbial Conversion of Diosgenin and Separation and Identification of the Product

Mou

Tian

et al. 2023

Fermentation

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Diosgenin, a hydrolysis product from Dioscorea plants, can be used as a precursor of steroid drugs (e.g., progesterone, testosterone, and glucocorticoid). However, traditional acid hydrolysis production wastes water and causes severe environmental pollution. The extraction of diosgenin through microbial transformation is the most green and environmentally friendly method at present. In order to improve the efficiency of the extraction of diosgenin through microbial transformation, we proposed a new method of strain mutagenesis. After mutagenesis, the response surface methodology was used to optimize the solid-state fermentation medium, thereby improving the diosgenin yield. We found that the optimal formulation was 5.5% sucrose, 0.6% NH4H2PO4, and 26.6% wheat bran. The final extraction rate of diosgenin reached 0.439% (the value of diosgenin per g. of starting plant dry material). Compared with 0.338% before optimization, it had increased 1.29 times. Furthermore, two other compounds were isolated from the fermentation products. These were identified as diosgenone (C27H41O3) and yuccagenone (C27H42O3). Traditional diosgenone is obtained through the oxidation of diosgenin with oxalic acid, but the method in this study is directly obtained from Dioscorea rhizome powder. The price of Dioscorea rhizome powder is much lower than diosgenin, thus greatly reducing the cost of obtaining diosgenone. This method provides a basis for subsequent research on other pharmacological compounds.

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Enhancing protease activity of Bacillus subtilis using UV-laser random mutagenesis and high-throughput screening

Cited by 13 publications

References 33 publications

Study on mutagenesis of high protease producing thermophilic bacteria by UV-DES and hydrolysis of excess sludge

Study on mutagenesis of high protease producing thermophilic bacteria by UV-DES and hydrolysis of excess sludge

Effect of neutral protease overproduction in Bacillus subtilis 168 via site-directed mutation against Meloidogyne incognita infecting eggplant under greenhouse conditions

A Method for Improving Microbial Conversion of Diosgenin and Separation and Identification of the Product

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