Engineering of serine protease for improved thermostability and catalytic activity using rational design

Ashraf, Naeem Mahmood; Krishnagopal, Akshaya; Hussain, Aadil; Kastner, David W.; Sayed, Ahmed M.; Mok, Yu Keung; Swaminathan, Kunchithapadam; Zeeshan, Nadia

doi:10.1016/j.ijbiomac.2018.12.218

Cited by 58 publications

(44 citation statements)

References 69 publications

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“…This protein engineering strategy changes the architecture of an enzyme, modulates the functional properties and catalytic mechanism (Yang et al, 2017). The protein residues at different locations, including noncatalytic residues, catalytic residues, the side chain residues, and residues in the catalytic triad vicinity, have been selectively engineered to obtain the desired biocatalyst (Takagi et al, 1988;Ashraf et al, 2019). Unlike SDM, DE involves nonspecific/random mutagenesis to generate a library of variable genes, which are further screened for the selection of the best performing variants.…”

Section: Keratinase Catalytic Efficiency Enhancement -A Protein Enginmentioning

confidence: 99%

Microbial Keratinase: Next Generation Green Catalyst and Prospective Applications

et al. 2020

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The search for novel renewable products over synthetics hallmarked this decade and those of the recent past. Most economies that are prospecting on biodiversity for improved bio-economy favor renewable resources over synthetics for the potential opportunity they hold. However, this field is still nascent as the bulk of the available resources are non-renewable based. Microbial metabolites, emphasis on secondary metabolites, are viable alternatives; nonetheless, vast microbial resources remain under-exploited; thus, the need for a continuum in the search for new products or bio-modifying existing products for novel functions through an efficient approach. Environmental distress syndrome has been identified as a factor that influences the emergence of genetic diversity in prokaryotes. Still, the process of how the change comes about is poorly understood. The emergence of new traits may present a high prospect for the industrially viable organism. Microbial enzymes have prominence in the bio-economic space, and proteases account for about sixty percent of all enzyme market. Microbial keratinases are versatile proteases which are continuously gaining momentum in biotechnology owing to their effective bio-conversion of recalcitrant keratin-rich wastes and sustainable implementation of cleaner production. Keratinase-assisted biodegradation of keratinous materials has revitalized the prospects for the utilization of cost-effective agro-industrial wastes, as readily available substrates, for the production of high-value products including amino acids and bioactive peptides. This review presented an overview of keratin structural complexity, the potential mechanism of keratin biodegradation, and the environmental impact of keratinous wastes. Equally, it discussed microbial keratinase; vis-à-vis sources, production, and functional properties with considerable emphasis on the ecological implication of microbial producers and catalytic tendency improvement strategies. Keratinase applications and prospective high-end use, including animal hide processing, detergent formulation, cosmetics, livestock feed, and organic fertilizer production, were also articulated.

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Section: Keratinase Catalytic Efficiency Enhancement -A Protein Enginmentioning

confidence: 99%

Microbial Keratinase: Next Generation Green Catalyst and Prospective Applications

et al. 2020

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“…Rational protein engineering has already contributed to generating mutations with enhanced properties 5,[39][40][41] . Frequently, this genetic approach can also simultaneously elucidate the structure-function relationship of a protein [42][43][44] , thus providing better guidance for future design proposals. Niu et al found that substituting three residues (L99, L162 and E230) improved pepsin resistance and that in addition, some of them increased their catalytic efficiency 1.3-2.4-fold and improved their stability at 60 °C and pH 1-2 39 .…”

Section: Discussionmentioning

confidence: 99%

“…Camilloni et al showed that the propensity for aggregation of a protein can be modulated by mutating specific residues that change the average protection of its aggregation-prone surface residues without affecting its structure and stability 42 . Ashraf et al 43 and Han et al 40,44 demonstrated that modifying non-catalytic residues can promote favourable catalytic behaviour and that substituting these residues can provide prospective candidates for industry applications.…”

Section: Discussionmentioning

confidence: 99%

Characterization of Affitin proteolytic digestion in biorelevant media and improvement of their stabilities via protein engineering

Loussouarn

Béhar

Pecorari

et al. 2020

Sci Rep

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Affitins are a novel class of small 7 kDa artificial proteins which can be used as antibody substitutes in therapeutic, diagnostic and biotechnological applications. One challenge for this type of protein agent is their behaviour in the context of oral administration. The digestive system is central, and biorelevant media have fast emerged as relevant and reliable tools for evaluating the bioavailability of drugs. This study describes, for the first time, the stability of Affitins under simulated gastric and intestinal digestion conditions. Affitins appear to be degraded into stable fragments in in vitro gastric medium. We identified cleavage sites generated by pepsin that were silenced by site-directed mutagenesis. This protein engineering allowed us to enhance Affitin properties. We showed that a mutant M1 containing a double mutation of amino acid residues 6 and 7 in H4 and C3 Affitins acquired a resistance against proteolytic digestion. In addition, these mutations were beneficial for target affinity, as well as for production yield. Finally, we found that the mutated residues kept or increased the important pH and temperature stabilities of Affitins. These improvements are particularly sought after in the development of engineered binding proteins for research tools, preclinical studies and clinical applications.

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“…The binding affinities of the compounds were demonstrated by PatchDock server with ACE values ( Table 11). The lower ACE value is considered to be associated with better ligand affinity with the enzyme 30 . Compound 3 showed lower ACE values (À440.29 kJ/mol) as compared to the standard drug Sulindac (À325.99 kJ/mol).…”

Section: Anti-inflammatory Activitymentioning

confidence: 99%

Novel sulindac derivatives: synthesis, characterisation, evaluation of antioxidant, analgesic, anti-inflammatory, ulcerogenic and COX-2 inhibition activity

Bhat

Al-Omar

Alsaif

et al. 2020

Journal of Enzyme Inhibition and Medicinal Chemistry

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A new series of N 0-(substituted phenyl)-2-(1-(4-(methylsulfinyl) benzylidene)À5-fluoro-2-methyl-1H-inden-3yl) acetohydrazide derivatives (1-25) were prepared in good yields in an efficient manner. All the compounds were fully characterised by the elemental analysis and spectral data. Synthesised compounds were evaluated for antioxidant activity by DPPH method. Compounds 7 (R ¼ 3-methoxyphenyl), 3 (R ¼ 4dimethylaminophenyl) and 23 (R ¼ 2,4,5-trimethoxy phenyl) substitutions were found to be having highly potent antioxidant activity. Compound 3, with para dimethylaminophenyl substitution was found to be having highest antioxidant activity. It was further evaluated in vivo for various analgesic, anti-inflammatory, ulcerogenic and COX-2 inhibitory activity in different animal models. Lead compound 3 was found to be significant anti-inflammatory and analgesic agent. It was also evaluated for ulcerogenic activity and demonstrated significant ulcerogenic reduction activity in ethanol and indomethacin model. The LD 50 of compound 3 was found to be 131 mg/kg. The animals treated with compound 3 prior to cisplatin treatment resulted in a significant reduction in COX-2 protein expression when compared to cisplatin-treated group. Sulindac derivative with para dimethylaminophenyl substitution was found to be the most potent antioxidant, anti-inflammatory and analgesic agent as well as with significant gastric sparing activity as compared to standard drug sulindac. Compound 3 significantly downregulated liver tissue COX-2 gene expression.

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Engineering of serine protease for improved thermostability and catalytic activity using rational design

Cited by 58 publications

References 69 publications

Microbial Keratinase: Next Generation Green Catalyst and Prospective Applications

Microbial Keratinase: Next Generation Green Catalyst and Prospective Applications

Characterization of Affitin proteolytic digestion in biorelevant media and improvement of their stabilities via protein engineering

Novel sulindac derivatives: synthesis, characterisation, evaluation of antioxidant, analgesic, anti-inflammatory, ulcerogenic and COX-2 inhibition activity

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