Probing protein stability and proteolytic resistance by loop scanning: A comprehensive mutational analysis

Ahmad, Shoeb; Kumar, Virender; Ramanand, K. Bhanu; Rao, Nalam Madhusudhana

doi:10.1002/pro.2029

Cited by 74 publications

(50 citation statements)

References 53 publications

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“…Ordered structures (α‐helix and β‐strand) have been reported to be helpful for stabilizing an enzyme structure suffering from thermal denaturation . Concurrently, loop regions in the spatial structure of proteins are apt to unfold and destabilize because of their great flexibility under the externally detrimental conditions . Therefore, the more stable spatial structure of AP could be due to its higher contents of α‐helix and β‐strand and smaller content of loop regions than those in NP I.…”

Section: Resultsmentioning

confidence: 99%

Separation, biochemical characterization and salt‐tolerant mechanisms of alkaline protease from Aspergillus oryzae

et al. 2019

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BACKGROUND The salt tolerance of proteases secreted by Aspergillus oryzae 3.042 closely relates to the utilization of raw materials and the quality of soy sauce. However, little is known about the salt‐tolerant proteases and their salt‐tolerant mechanisms. RESULTS In this study, we isolated and identified a salt‐tolerant alkaline protease (AP, approximately 29 kDa) produced by A. oryzae 3.042. It was considered as a metal‐ion‐independent serine protease. The optimum and stable pH values were both pH 9.0 and the optimum temperature was 40 °C. Over 20% relative activity of AP remained in the presence of 3.0 mol L−1 NaCl after 7 days, but its Km and Vmax were only mildly influenced by the presence of 3.0 mol L−1 NaCl, indicating its outstanding salt tolerance. Furthermore, AP was more stable than non‐salt‐tolerant protease at high salinity. The salt‐tolerant mechanisms of AP could be due to more salt bridges, higher proportion of ordered secondary structures and stronger hydrophobic amino acid residues in the interior. CONCLUSIONS The above results are vital for maintaining, activating and/or modulating the activity of AP in high‐salt environments. They would also provide theoretical guidance for the modification of AP and the engineering of A. oryzae 3.042 so as to secrete more AP. © 2018 Society of Chemical Industry

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Section: Resultsmentioning

confidence: 99%

Separation, biochemical characterization and salt‐tolerant mechanisms of alkaline protease from Aspergillus oryzae

et al. 2019

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“…S23). Therefore, by monitoring the first-order exponential decay of the full-length CBM using quantitative MALDI-TOF MS, the CBM glycoform half-life to thermolysin degradation was calculated (33)(34)(35). As shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Each sample was analyzed by quantitative MALDI-TOF MS (described below) to calculate the change in CBM concentration with time. The digestion rate was determined by monitoring and fitting data to the first-order exponential decay of the full-length CBM glycoform over time (33)(34)(35).…”

Section: Methodsmentioning

confidence: 99%

Specificity of O -glycosylation in enhancing the stability and cellulose binding affinity of Family 1 carbohydrate-binding modules

Chen

Drake

Resch

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

154

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The majority of biological turnover of lignocellulosic biomass in nature is conducted by fungi, which commonly use Family 1 carbohydrate-binding modules (CBMs) for targeting enzymes to cellulose. Family 1 CBMs are glycosylated, but the effects of glycosylation on CBM function remain unknown. Here, the effects of O-mannosylation are examined on the Family 1 CBM from the Trichoderma reesei Family 7 cellobiohydrolase at three glycosylation sites. To enable this work, a procedure to synthesize glycosylated Family 1 CBMs was developed. Subsequently, a library of 20 CBMs was synthesized with mono-, di-, or trisaccharides at each site for comparison of binding affinity, proteolytic stability, and thermostability. The results show that, although CBM mannosylation does not induce major conformational changes, it can increase the thermolysin cleavage resistance up to 50-fold depending on the number of mannose units on the CBM and the attachment site. O-Mannosylation also increases the thermostability of CBM glycoforms up to 16°C, and a mannose disaccharide at Ser3 seems to have the largest themostabilizing effect. Interestingly, the glycoforms with small glycans at each site displayed higher binding affinities for crystalline cellulose, and the glycoform with a single mannose at each of three positions conferred the highest affinity enhancement of 7.4-fold. Overall, by combining chemical glycoprotein synthesis and functional studies, we show that specific glycosylation events confer multiple beneficial properties on Family 1 CBMs. chemical synthesis | cellulase | biofuels | protein engineering

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“…Site saturation mutagenesis (SSM) approach has been used successfully in the recent past to understand the structure-function relationship and also to improve stability of proteins under a variety of in vitro evolution context. [27][28][29][30] Thus, a thorough analysis by SSM would seek optimal substitution for residue susceptible to deamidation to improve the thermotolerance of the protein.…”

Section: -21mentioning

confidence: 99%

Engineering deamidation‐susceptible asparagines leads to improved stability to thermal cycling in a lipase

2014

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At high temperatures, protein stability is influenced by chemical alterations; most important among them is deamidation of asparagines. Deamidation kinetics of asparagines depends on the local sequence, solvent, pH, temperature, and the tertiary structure. Suitable replacement of deamidated asparagines could be a viable strategy to improve deamidation-mediated loss in protein properties, specifically protein thermostability. In this study, we have used nano RP-HPLC coupled ESI MS/MS approach to identify residues susceptible to deamidation in a lipase (6B) on heat treatment. Out of 15 asparagines and six glutamines in 6B, only five asparagines were susceptible to deamidation at temperatures higher than 75 C. These five positions were subjected to site saturation mutagenesis followed by activity screen to identify the most suitable substitutions. Only three of the five asparagines were found to be tolerant to substitutions. Best substitutions at these positions were combined into a mutant. The resultant lipase (mutC) has near identical secondary structure and improved thermal tolerance as compared to its parent. The triple mutant has shown almost two-fold higher residual activity compared to 6B after four cycles at 90 C. MutC has retained more than 50% activity even after incubation at 100 C. Engineering asparagines susceptible to deamidation would be a potential strategy to improve proteins to withstand very high temperatures.

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Probing protein stability and proteolytic resistance by loop scanning: A comprehensive mutational analysis

Cited by 74 publications

References 53 publications

Separation, biochemical characterization and salt‐tolerant mechanisms of alkaline protease from Aspergillus oryzae

Separation, biochemical characterization and salt‐tolerant mechanisms of alkaline protease from Aspergillus oryzae

Specificity of O -glycosylation in enhancing the stability and cellulose binding affinity of Family 1 carbohydrate-binding modules

Engineering deamidation‐susceptible asparagines leads to improved stability to thermal cycling in a lipase

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