Heterologous expression and characterisation of a keratinase produced by Chryseobacterium carnipullorum

Mwanza, Elebert Pauline; Westhuizen, Wouter André van der; Boucher, Charlotte E.; Charimba, George; Hugo, Celia J.

doi:10.1016/j.pep.2021.105926

Cited by 7 publications

(4 citation statements)

References 44 publications

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“…The relative abundance of Zoogloea in AOA 90 was the highest among three bioreactors at the 90th day of operation. Chryseobacterium with the highest richness in AOA 90 would also induce EPS degradation due to its good proteolytic ability [ 52 ]; therefore, resulting in the cell lysis in AOA 90 . These findings confirmed that the slow-growing and hydrolytic microorganisms played a critical role on the biomass reduction in AOA 90 and AOA 80 [ 53 , 54 ].…”

Section: Resultsmentioning

confidence: 99%

In-Situ Sludge Reduction in Membrane-Controlled Anoxic-Oxic-Anoxic Bioreactor: Performance and Mechanism

Maqbool

Kang

et al. 2022

Membranes

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Conventional and advanced biological wastewater treatment systems generate excess sludge, which causes socio-economic and environmental issues. This study investigated the performance of membrane-controlled anoxic-oxic-anoxic (AOA) bioreactors for in-situ sludge reduction compared to the conventional anoxic-oxic-oxic membrane bioreactor (MBRcontrol). The membrane units in the AOA bioreactors were operated as anoxic reactors at lower sludge recirculation rates to achieve hydrolysis of extracellular polymeric substances (EPS) and extensive endogenous respiration. Compared to MBRcontrol, the AOA bioreactors operated with 90%, and 80% recirculation rates reduced the sludge growth up to 19% and 30%, respectively. Protein-like components were enriched in AOA bioreactors while fulvic-like components were dominant in MBRcontrol. The growth of Dechloromonas and Zoogloea genra was promoted in AOA bioreactors and thus sludge reduction was facilitated. Metagenomics analysis uncovered that AOA bioreactors exhibited higher proportions of key genes encoding enzymes involved in the glycolysis and denitrification processes, which contributed to the utilization of carbon sources and nitrogen consumption and thus sludge reduction.

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Section: Resultsmentioning

confidence: 99%

In-Situ Sludge Reduction in Membrane-Controlled Anoxic-Oxic-Anoxic Bioreactor: Performance and Mechanism

Maqbool

Kang

et al. 2022

Membranes

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“…Keratinases have received increasing attention from researchers due to their multiple promising applications, and more keratinase-producing microorganisms have been identi ed [31]. In recent years, several keratinase genes have been cloned, and the heterologous expression of some of these genes has been successfully achieved [32,33]. For example, some keratinases encoded by the kerA gene extracted from bacteria of the genus Bacillus (most commonly Bacillus licheniformis) have been cloned and expressed [34][35][36][37].…”

Section: Discussionmentioning

confidence: 99%

“…are 6.5 and 50°C, respectively [48]. The optimum temperature and pH of recombinant keratinases derived from Chryseobacterium carnipullorum are 50°C and 8.5, respectively [49]. A few keratases can maintain high enzymatic activity at very alkaline and higher temperatures; for example, the recombinant keratinase AprE539 derived from Bacillus tequilensis has an optimal pH of 11.0 and an optimal temperature of 65-70°C [50].…”

Section: Discussionmentioning

confidence: 99%

Isolation and identification of feather-degrading bacteria and cloning and expression of the keratinase gene

Ma,

Feng,

Chen

et al. 2024

Preprint

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In this study, the strain CY-A, which has high feather degradation efficiency, was screened from the soil of a chicken pen. Bacillus tequilensis was identified by biological methods. The keratinase gene (bta) of Bacillus tequilensis CY-A was cloned by genetic engineering. The gene is 1110 bp in length, contains an open reading frame of 1089 bp and encodes 369 amino acids. The obtained gene sequence and amino acid sequence have been registered to GenBank under the database accession number OR733336.1. The physicochemical properties, secondary structure and tertiary structure of the protein were analysed by bioinformatics tools, and the relative molecular mass was found to be 37.953 kDa. The tertiary structure of keratin was 98.30% similar to that of Bacillus subtilis arpE. Finally, the keratinase gene bta was ligated to the expression vector pET28a (+) to construct the expression plasmid pET28a(+)-bta, which was subsequently transformed into E. coli BL21 (DE3) to generate engineered recombinant bacteria. Recombinant keratinase Bta was purified using Ni2+ column affinity chromatography with a molecular weight of approximately 37.953 kDa. Further studies on its recombinant enzymatic properties revealed that the enzyme activity of Bta was 283.93 U/mL, the optimum pH was 8, and the optimum reaction temperature was 50°C. Ca2+ has a very significant role in promoting Bta. EDTA and SDS can significantly inhibit the enzyme activity of Bta, which indicates that the enzyme activity requires metal ions. At a concentration of 10 mmol. L-1, PMSF almost completely inhibited the enzyme activity of Bta, indicating that Bta is a typical serine protease. The identification of the keratinase gene provides a theoretical basis for further improving keratinase activity via genetic engineering.

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“…Keratinases have received increasing attention from researchers with reports of their multiple promising applications, and more keratinase-producing microorganisms have been identified [29]. In recent years, several keratinase genes have been cloned and the heterologous expression of some of these genes has been successfully achieved [30,31]. For example, some keratinases encoded by the kerA gene extracted from bacteria of the genus Bacillus (most commonly, Bacillus licheniformis) have been cloned and expressed [32][33][34][35].…”

Section: Discussionmentioning

confidence: 99%

Isolation and Identification of Feather-Degrading Bacteria and Cloning of Their Keratinase Gene

Ma,

Feng,

Chen

et al. 2024

Preprint

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There are a large number of cross-linked chemical bonds such as tight disulfide bonds, hydrogen bonds,and salt bonds in keratin, which makes it difficult to break the structural stability of the protein using conventional methods. The degradation of feather waste using keratinases produced by microorganisms has the advantage of being efficient and environmentally friendly. In this study, 23 strains capable of producing transparent circles were obtained via preliminary screening with skim milk as the sole carbon and nitrogen source, and 9 strains capable of degrading feathers were obtained via rescreening with feathers as the sole carbon and nitrogen source. Finally, through the determination of enzyme activity, the strain CY-A with a high ability to degrade feathers was obtained. The strain was identified as Bacillus tequilensis via morphological observation, physiological and biochemical identification, and 16 S rRNA molecular biology. The keratinase gene (bta) of Bacillus tequila was cloned, and its full-length gene sequence was 1110 bp, containing an open reading frame of 1089, encoding 369 amino acids (GeneBank accession number OR733336). The physicochemical properties, secondary structure, and tertiary structure of the recombinant protein were analyzed using bioinformatics tools such as ProtParm, SOPMA, MEGA, and SWISS-MODELS. It was found that the relative molecular mass was 37.953 kD and that the isoelectric point was 8.48. The protein was a hydrophilic protein. The keratinase bta gene was ligated to the expression vector pET28a(+) using T4 ligase, and the recombination expression plasmid pET28-bta was constructed and transformed into E. coli BL21(DE3) to obtain recombinant engineered bacteria. The expressed keratinase was purified using a Ni2+ affinity chromatography column to obtain recombinant bta keratinase bta with a molecular weight of 37.953 kD. The identification and characterization of this keratinase gene provides some theoretical basis for the further enhancement of keratinase activity by means of genetic engineering.

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Heterologous expression and characterisation of a keratinase produced by Chryseobacterium carnipullorum

Cited by 7 publications

References 44 publications

In-Situ Sludge Reduction in Membrane-Controlled Anoxic-Oxic-Anoxic Bioreactor: Performance and Mechanism

In-Situ Sludge Reduction in Membrane-Controlled Anoxic-Oxic-Anoxic Bioreactor: Performance and Mechanism

Isolation and identification of feather-degrading bacteria and cloning and expression of the keratinase gene

Isolation and Identification of Feather-Degrading Bacteria and Cloning of Their Keratinase Gene

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