Overproduction of a thermo-stable halo-alkaline protease on agro-waste-based optimized medium through alternate combinatorial random mutagenesis of Stenotrophomonas acidaminiphila

Asitok, Atim; Ekpenyong, Maurice; Takon, Iquo; Antai, S. P.; Ogarekpe, Nkpa; Antigha, Richard; Edet, Philomena; Ben, Ubong C.; Akpan, Anthony E.; Antai, Agnes; Essien, Joseph P.

doi:10.1016/j.btre.2022.e00746

Cited by 13 publications

(5 citation statements)

References 40 publications

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“…The tolerance and stability exhibited by B950 and to a lesser extent by B961 are rarely reported among the B. licheniformis representatives, although these species otherwise had good output as enzyme producers (Díaz-Cornejo et al, 2023). However, a previous study reported by Asitok et al, (2022) observed that the ability of a bacterial strain to withstand multiple extreme conditions, such as high pH, temperature, and salinity is crucial; as the enzyme yield can be reasonably optimized.…”

Section: Discussionmentioning

confidence: 99%

Quantification of thermo-halotolerant alkaline protease activity derived from Bacillus licheniformis strains isolated from extreme environments in Morocco

Chouati,

Ayadi,

Ajdig

et al. 2023

Novel Research in Microbiology Journal

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Proteases; especially alkaline proteases, constitute the most used group of enzymes in industry. The wide scope of their application requires varying properties; most commonly stability throughout the conditional changes that would occur during the various industrial processes. This study aimed to identify the potentially interesting bacterial strains obtained from Moroccan extreme environment, and evaluate their proteolytic activity under the various growing conditions. In this study, the impact of temperature and salinity on the alkaline protease activity (pH 11) of 33 Bacillus strains deposited in the Moroccan Coordinated Culture Collection, which originated from the various extreme environments from Morocco, was studied. Bacterial identification to the species level was performed using 16S rRNA gene sequencing and MultiLocus Sequence Typing (MLST), a technique that is much more precise and can identify the bacteria to the strain level. Strain B950 showed a relatively stable protease activity at the tested extreme conditions (i.e., 130.8 U/ ml at 60 °C and 10 % NaCl). On the other hand, strain B961 displayed a better overall activity at either high temperature (140.8 U/ ml) or salinity (137 U/ ml); however, it was not as stable as when grown under both extreme conditions. Regarding identity of the bacterial strains, they were all representatives of Bacillus licheniformis; although B950 and B961 strains belonged to Sequence Types 3 and 5, respectively.

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Section: Discussionmentioning

confidence: 99%

Quantification of thermo-halotolerant alkaline protease activity derived from Bacillus licheniformis strains isolated from extreme environments in Morocco

Chouati,

Ayadi,

Ajdig

et al. 2023

Novel Research in Microbiology Journal

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“…Sterilization was conducted in situ and inoculation with 3% (v.v −1 ) spore suspension. The bioreactor was operated as described in Asitok et al ( 2022a ) and determinations of total protein (Bradford 1976 ), biomass concentration (Rodrigues et al 2006 ), l -ASNase activity (Imada et al 1973 ) and residual carbohydrate (Miller 1959 ) were made at 6 h interval for 96 h. Briefly, l -ASNase assay protocol by Imada et al ( 1973 ) involved dissolution of 0.04 M of l -asparagine (0.5 mL) in 0.5 M (0.5 mL) Tris–HCl buffer (pH 7.2) and adding the mixture to 0.5 mL of cell-free fermentation broth of fusant mold and making up total reaction volume to 2 mL with sterile distilled water. The preparation was incubated in a water bath at 37 ºC for 30 min and reaction stopped by adding 0.5 mL of 1.5 M trichloroacetic acid.…”

Section: Methodsmentioning

confidence: 99%

“…Analysis was as described for batch and fed-batch modes. Performances of models were evaluated by adjusted goodness-of-fit, r 2 , root mean squared error (rmse) and mean absolute error (mse) using the equations detailed in Asitok et al ( 2022a ).…”

Section: Methodsmentioning

confidence: 99%

“…A common huddle with fermentative production of L-ASNases and other microbially-derived value-added products is low yield because microorganisms maintain tight control over biosynthesis of metabolites, especially extracellularly-secreted ones (Sadh et al 2018 ; Pinu et al 2018 ). A combined use of hyper-producing strains obtained naturally or through mutagenesis, protoplast fusion and genetic engineering, especially with CRISPR/Cas9 system (Zou et al 2019 ; Zhang et al 2021 ; Hu et al 2021 ), and design of experiments by response surface methodology and artificial neural network, has significantly improved yield (Farjaminezhad and Garoosi 2021 ; Asitok et al 2022a ; Kusuma et al 2022 ).…”

Section: Introductionmentioning

confidence: 99%

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Production, characterization and techno-economic evaluation of Aspergillus fusant l-asparaginase

et al. 2023

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Protoplast fusion is one of the most reliable methods of introducing desirable traits into industrially-promising fungal strains. It harnesses the entire genomic repertoire of fusing microorganisms by routing the natural barrier and genetic incompatibility between them. In the present study, the axenic culture of a thermo-halotolerant strain of Aspergillus candidus (Asp-C) produced an anti-leukemic l-asparaginase (l-ASNase) while a xylan-degrading strain of Aspergillus sydowii (Asp-S) produced the acrylamide-reduction type. Protoplast fusion of the wild strains generated Fusant-06 with improved anti-leukemic and acrylamide reduction potentials. Submerged fed-batch fermentation was preferred to batch and continuous modes on the basis of impressive techno-economics. Fusant-06 l-ASNase was purified by PEG/Na+ citrate aqueous two-phase system (ATPS) to 146.21-fold and global sensitivity analysis report revealed polymer molecular weight and citrate concentration as major determinants of yield and purification factor, respectively. The enzyme was characterized by molecular weight, amino acid profile, activity and stability to chemical agents. Michaelis–Menten kinetics, evaluated under optimum conditions gave Km, Vmax, Kcat, and Kcat/Km as 6.67 × 10–5 M, 1666.67 µmolmin−1 mg−1 protein, 3.88 × 104 min−1 and 5.81 × 108 M−1.min−1 respectively. In-vitro cytotoxicity of HL-60 cell lines by Fusant-06 l-ASNase improved significantly from their respective wild strains. Stability of Fusant-06 l-ASNase over a wide range of pH, temperature and NaCl concentration, coupled with its micromolar Km value, confers commercial and therapeutic value on the product. Free-radical scavenging and acrylamide reduction activities were intermediate and the conferred thermo-halo-stability could be exploited for sustainable clinical and food industry applications.

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“…Microbial proteases have wide ranging applications in several fields, including baking, brewing, detergents, leather making, pharmaceuticals, meat tenderizing, cosmetics, medical diagnosis and so on ( Christensen et al, 2022 ; Reddy et al, 2022 ; Akram et al, 2023 ; Mubeen et al, 2023 ). In addition, with the rapid development of new fields, applications of microbial proteases are expanding to new areas, such feed industries ( Bernardeau et al, 2022 ; Cupi et al, 2022 ), hydrolysis applications to prepare active peptides ( Christensen et al, 2022 ), and environmental protection applications, such as waste treatment and reuse ( Ariaeenejad et al, 2022 ; Asitok et al, 2022 ; Zhai et al, 2022 ). These applications illustrate the diversity and importance of proteases.…”

Section: Application Of Microbial Proteasesmentioning

confidence: 99%

Microbial proteases and their applications

Song,

Zhang,

Wang

et al. 2023

Front. Microbiol.

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Proteases (proteinases or peptidases) are a class of hydrolases that cleave peptide chains in proteins. Endopeptidases are a type of protease that hydrolyze the internal peptide bonds of proteins, forming shorter peptides; exopeptidases hydrolyze the terminal peptide bonds from the C-terminal or N-terminal, forming free amino acids. Microbial proteases are a popular instrument in many industrial applications. In this review, the classification, detection, identification, and sources of microbial proteases are systematically introduced, as well as their applications in food, detergents, waste treatment, and biotechnology processes in the industry fields. In addition, recent studies on techniques used to express heterologous microbial proteases are summarized to describe the process of studying proteases. Finally, future developmental trends for microbial proteases are discussed.

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Overproduction of a thermo-stable halo-alkaline protease on agro-waste-based optimized medium through alternate combinatorial random mutagenesis of Stenotrophomonas acidaminiphila

Cited by 13 publications

References 40 publications

Quantification of thermo-halotolerant alkaline protease activity derived from Bacillus licheniformis strains isolated from extreme environments in Morocco

Quantification of thermo-halotolerant alkaline protease activity derived from Bacillus licheniformis strains isolated from extreme environments in Morocco

Production, characterization and techno-economic evaluation of Aspergillus fusant l-asparaginase

Microbial proteases and their applications

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