A combination of bioinformatics analysis and rational design strategies to enhance keratinase thermostability for efficient biodegradation of feathers

Su, Chang; Gong, Jin‐Song; Qin, Anqi X.; Li, Heng; Li, Hui; Qin, Jiufu; Qian, Jianying; Xu, Zhenghong; Shi, Jin‐Song

doi:10.1016/j.scitotenv.2021.151824

Cited by 22 publications

(22 citation statements)

References 42 publications

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“…subtilis shuttle plasmid pMA5 with the strong constitutive promoter P Hpa II . 23 Keratinase gene kerBp under the control of this promoter showed a continuous and steady expression. Herein, considering the host toxicity of keratinase, the processes of cell growth and keratinase production were regulated by adopting the growth phase-specific promoters P aprE and P srfA , which can control the expression of downstream genes by sensing cell density.…”

Section: ■ Results and Discussionmentioning

confidence: 97%

“…coli – B. subtilis shuttle plasmid pMA5 with the strong constitutive promoter P Hpa II . Keratinase gene kerBp under the control of this promoter showed a continuous and steady expression.…”

Section: Resultsmentioning

confidence: 99%

“…subtilis WB600, a protease-deficient strain used for higher heterologous expression, was adopted as the host strain. The keratinase gene kerBp was mined, and the keratinase-producing strain was constructed using the shuttle plasmid pMA5 with the strong constitutive promoter P HpaII as described in our previous studies. − Strains were cultivated in LB medium for 12 h at 37 °C, 220 rpm for seed preparation, inoculated at 3% into 50 mL TB medium (12 g/L tryptone, 24 g/L yeast extract, 9.4 g/L K 2 HPO 4 , 2.2 g/L KH 2 PO 4 , 4 mL/L glycerol) containing 50 mg/L kanamycin, and cultivated at 37 °C, 220 rpm.…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Development of a Growth-Dependent System to Regulate Cell Growth and Keratinase Production in B. subtilis

Gong

et al. 2023

J. Agric. Food Chem.

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Keratinases specifically degrade insoluble keratin waste, thus contributing to environmental protection and sustainable biomass development. However, their industrial application is hindered by inefficient enzyme production and poor biomass generation. In this study, the heterologous expression of keratinase was found to have cytotoxicity and might block host cell growth due to its proteolytic property. To address this problem, an autoregulatory expression system based on quorum sensing was developed to synergistically regulate cell growth and keratinase production in Bacillus subtilis. The growth-dependent promoter P aprE was chosen and shown to be effective in delaying keratinase production while promoting host cell proliferation. Copy number screening and core region mutations further balanced the two states. Carbon supplement optimization indicated that addition of 2% glucose facilitated biomass accumulation during the early stage of fermentation. Cell density increased to 15.6 (OD 600 nm ) from 8 with keratinase activity raised to 4200 U•mL −1 from 1162 U•mL −1 . Keratinase was then utilized in the bioconversion of feather waste to prepare soluble keratins, polypeptides, and amino acids. This study provides a powerful system for efficient production of keratinase and paves the way for keratin waste recycling.

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Section: ■ Results and Discussionmentioning

confidence: 97%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Development of a Growth-Dependent System to Regulate Cell Growth and Keratinase Production in B. subtilis

Gong

et al. 2023

J. Agric. Food Chem.

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“…Their efficiency can be evaluated by measuring the enzymic activity, the concentration of both the released thiol groups and soluble proteins, and the incurred weight loss [ 8 ]. Since the cost of keratinase production is high, several researchers have increased the yield and stability of keratinases by altering the genome of keratinolytic organisms via physical and chemical mutagenesis or recombinant DNA technologies [ 80 , 82 , 90 , 93 , 94 , 95 , 96 ]. Keratinases have become increasingly popular, and many studies have reviewed their discovery, production, classification, structure, hydrolysis function, and industrial application [ 9 , 97 , 98 , 99 , 100 ].…”

Section: Methods For the Valorization Of Livestock Keratinmentioning

confidence: 99%

Valorization of Livestock Keratin Waste: Application in Agricultural Fields

Chen

Gao

et al. 2022

IJERPH

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Livestock keratin waste is a rich source of protein. However, the unique structure of livestock keratin waste makes its valorization a great challenge. This paper reviews the main methods for the valorization of livestock keratin waste, which include chemical, biological, and other novel methods, and summarizes the main agricultural applications of keratin-based material. Livestock keratin waste is mainly used as animal feed and fertilizer. However, it has promising potential for biosorbents and in other fields. In the future, researchers should focus on the biological extraction and carbonization methods of processing and keratin-based biosorbents for the soil remediation of farmland.

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“…Ashraf et al ( 2019 ) designed and obtained 2 eight‐point mutations using the online server, FireProt, which showed increased activity (1.4‐fold) and thermostability. Su et al ( 2022 ) combined bioinformatic analysis and rational design to increase the optimal temperature of keratinase by 20°C. However, the effectiveness of directed evolution depends on high‐throughput screening methods, an important factor limiting the widespread application of directed evolution.…”

Section: Introductionmentioning

confidence: 99%

Improving the catalytic activity of a detergent‐compatible serine protease by rational design

Wang

Qin

Tong

et al. 2023

Microbial Biotechnology

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Serine proteases are among the most important biological additives in various industries such as detergents, leather, animal feed and food. A serine protease gene, Fgapt4, from Fusarium graminearum 2697 was identified, cloned and expressed in Pichia pastoris. The optimal pH and temperature of FgAPT4 were 8.5 and 40°C, respectively. The relative activity was >30% even at 10°C. It had a wide range of pH stability (4.0-12.0) and detergent compatibility. To improve the catalytic activity, a strategy combining molecular docking and evolutionary analysis was adopted. Twelve amino acid residue sites and three loops (A, B and C) were selected as potential hot spots that might play critical roles in the enzyme's functional properties. Twentyeight mutants targeting changes in individual sites or loops were designed, and mutations with good performance were combined. The best mutant was FgAPT4-M3 (Q70N/D142S/A143S/loop C). The specific activity and catalytic efficiency of FgAPT4-M3 increased by 1.6 (1008.5 vs. 385.9 U/mg) and 2.2fold (3565.1 vs. 1106.3/s/mM), respectively. Computational analyses showed that the greater flexibility of the substrate pocket may be responsible for the increased catalytic activity. In addition, its application in detergents indicated that FgAPT4-M3 has great potential in washing.

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A combination of bioinformatics analysis and rational design strategies to enhance keratinase thermostability for efficient biodegradation of feathers

Cited by 22 publications

References 42 publications

Development of a Growth-Dependent System to Regulate Cell Growth and Keratinase Production in B. subtilis

Development of a Growth-Dependent System to Regulate Cell Growth and Keratinase Production in B. subtilis

Valorization of Livestock Keratin Waste: Application in Agricultural Fields

Improving the catalytic activity of a detergent‐compatible serine protease by rational design

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