scholarly journals Rational design and structure-based engineering of alkaline pectate lyase from Paenibacillus sp. 0602 to improve thermostability

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Zhanping Zhou ◽  
Xiao Wang
Keyword(s):  
Rational Design ◽  
Specific Activity ◽  
Intramolecular Interaction ◽  
Pectate Lyase ◽  
Life Time ◽  
Industrial Applications ◽  
Wild Type ◽  
Discovery Studio ◽  
Paenibacillus Sp ◽  
Alkaline Pectate Lyase

Abstract Background Ramie degumming is often carried out at high temperatures; therefore, thermostable alkaline pectate lyase (PL) is beneficial for ramie degumming for industrial applications. Thermostable PLs are usually obtained by exploring new enzymes or reconstructing existing enzyme by rational design. Here, we improved the thermostability of an alkaline pectate lyase (PelN) from Paenibacillus sp. 0602 with rational design and structure-based engineering. Results From 26 mutants, two mutants of G241A and G241V showed a higher thermostability compared with the wild-type PL. The mutant K93I showed increasing specific activity at 45 °C. Subsequently, we obtained combinational mutations (K93I/G241A) and found that their thermostability and specific activity improved simultaneously. The K93I/G241A mutant showed a half-life time of 15.9 min longer at 60 °C and a melting temperature of 1.6 °C higher than those of the wild PL. The optimum temperature decreased remarkably from 67.5 °C to 60 °C, accompanied by a 57% decrease in Km compared with the Km value of the wild-type strain. Finally, we found that the intramolecular interaction in PelN was the source in the improvements of molecular properties by comparing the model structures. Rational design of PelN was performed by stabilizing the α-helices with high conservation and increasing the stability of the overall structure of the protein. Two engineering strategies were applied by decreasing the mutation energy calculated by Discovery Studio and predicting the free energy in the process of protein folding by the PoPMuSiC algorithm. Conclusions The results demonstrated that the K93I/G241A mutant was more suitable for industrial production than the wild-type enzyme. Furthermore, the two forementioned strategies could be extended to reveal engineering of other kinds of industrial enzymes.

scholarly journals Directed Evolution and Structural Analysis of Alkaline Pectate Lyase from the Alkaliphilic Bacterium Bacillus sp. Strain N16-5 To Improve Its Thermostability for Efficient Ramie Degumming

2015 ◽  
Vol 81 (17) ◽  
pp. 5714-5723 ◽  
Author(s):  
Cheng Zhou ◽  
Jintong Ye ◽  
Yanfen Xue ◽  
Yanhe Ma
Keyword(s):  
Directed Evolution ◽  
Textile Industry ◽  
Specific Activity ◽  
High Specific Activity ◽  
Pectate Lyase ◽  
Fold Increase ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Content Type ◽  
Alkaline Pectate Lyase

ABSTRACTThermostable alkaline pectate lyases have potential applications in the textile industry as an alternative to chemical-based ramie degumming processes. In particular, the alkaline pectate lyase fromBacillussp. strain N16-5 (BspPelA) has potential for enzymatic ramie degumming because of its high specific activity under extremely alkaline conditions without the requirement for additional Ca2+. However, BspPelA displays poor thermostability and is inactive after incubation at 50°C for only 30 min. Here, directed evolution was used to improve the thermostability of BspPelA for efficient and stable degumming. After two rounds of error-prone PCR and screening of >12,000 mutants, 10 mutants with improved thermostability were obtained. Sequence analysis and site-directed mutagenesis revealed that single E124I, T178A, and S271G substitutions were responsible for improving thermostability. Structural and molecular dynamic simulation analysis indicated that the formation of a hydrophobic cluster and new H-bond networks was the key factor contributing to the improvement in thermostability with these three substitutions. The most thermostable combined mutant, EAET, exhibited a 140-fold increase in thet50(time at which the enzyme loses 50% of its initial activity) value at 50°C, accompanied by an 84.3% decrease in activity compared with that of wild-type BspPelA, while the most advantageous combined mutant, EA, exhibited a 24-fold increase in thet50value at 50°C, with a 23.3% increase in activity. Ramie degumming with the EA mutant was more efficient than that with wild-type BspPelA. Collectively, our results suggest that the EA mutant, exhibiting remarkable improvements in thermostability and activity, has the potential for applications in ramie degumming in the textile industry.

scholarly journals Single Residue Substitution at N-Terminal Affects Temperature Stability and Activity of L2 Lipase

Molecules ◽  
2020 ◽  
Vol 25 (15) ◽  
pp. 3433
Author(s):  
Noramirah Bukhari ◽  
Adam Thean Chor Leow ◽  
Raja Noor Zaliha Raja Abd Rahman ◽  
Fairolniza Mohd Shariff
Keyword(s):  
Rational Design ◽  
Thermal Denaturation ◽  
Temperature Stability ◽  
Industrial Applications ◽  
Terminal Region ◽  
Wild Type ◽  
Functional Region ◽  
Target Region ◽  
Residue Substitution

Rational design is widely employed in protein engineering to tailor wild-type enzymes for industrial applications. The typical target region for mutation is a functional region like the catalytic site to improve stability and activity. However, few have explored the role of other regions which, in principle, have no evident functionality such as the N-terminal region. In this study, stability prediction software was used to identify the critical point in the non-functional N-terminal region of L2 lipase and the effects of the substitution towards temperature stability and activity were determined. The results showed 3 mutant lipases: A8V, A8P and A8E with 29% better thermostability, 4 h increase in half-life and 6.6 °C higher thermal denaturation point, respectively. A8V showed 1.6-fold enhancement in activity compared to wild-type. To conclude, the improvement in temperature stability upon substitution showed that the N-terminal region plays a role in temperature stability and activity of L2 lipase.

scholarly journals Rational design to change product specificities and thermostability of cyclodextrin glycosyltransferase from Paenibacillus sp.

RSC Advances ◽  
2017 ◽  
Vol 7 (23) ◽  
pp. 13726-13732 ◽  
Author(s):  
Yu Li ◽  
Likun Wei ◽  
Zhangliang Zhu ◽  
Songtao Li ◽  
Jian-Wen Wang ◽  
...  
Keyword(s):  
Rational Design ◽  
Industrial Applications ◽  
Cyclodextrin Glycosyltransferase ◽  
Product Specificity ◽  
Functional Modification ◽  
Paenibacillus Sp

Functional modification of cyclodextrin glycosyltransferase (CGTases) for better product specificity and thermostability is of great importance for industrial applications.

scholarly journals Improvement of the Stability and Activity of an LPMO Through Rational Disulfide Bonds Design

2022 ◽  
Vol 9 ◽  
Author(s):  
Xiaoli Zhou ◽  
Zhiqiang Xu ◽  
Yueqiu Li ◽  
Jia He ◽  
Honghui Zhu
Keyword(s):  
Disulfide Bonds ◽  
Specific Activity ◽  
Fold Increase ◽  
Catalytic Performance ◽  
Industrial Applications ◽  
Enzyme Engineering ◽  
Wild Type ◽  
Polysaccharide Monooxygenases ◽  
Saccharification Efficiency ◽  
The Stability

Lytic polysaccharide monooxygenases (LPMOs) oxidatively break down the glycosidic bonds of crystalline polysaccharides, significantly improving the saccharification efficiency of recalcitrant biomass, and have broad application prospects in industry. To meet the needs of industrial applications, enzyme engineering is needed to improve the catalytic performance of LPMOs such as enzyme activity and stability. In this study, we engineered the chitin-active CjLPMO10A from Cellvibrio japonicus through a rational disulfide bonds design. Compared with the wild-type, the variant M1 (N78C/H116C) exhibited a 3-fold increase in half-life at 60°C, a 3.5°C higher T5015, and a 7°C rise in the apparent Tm. Furthermore, the resistance of M1 to chemical denaturation was significantly improved. Most importantly, the introduction of the disulfide bond improved the thermal and chemical stability of the enzyme without causing damage to catalytic activity, and M1 showed 1.5 times the specific activity of the wild-type. Our study shows that the stability and activity of LPMOs could be improved simultaneously by selecting suitable engineering sites reasonably, thereby improving the industrial adaptability of the enzymes, which is of great significance for applications.

scholarly journals Production and Characterization of Recombinant Wild Type Uricase from Indonesian Coelacanth (L. menadoensis) and Improvement of Its Thermostability by In Silico Rational Design and Disulphide Bridges Engineering

2019 ◽  
Vol 20 (6) ◽  
pp. 1269 ◽  
Author(s):  
Sakda Yainoy ◽  
Thanawat Phuadraksa ◽  
Sineewanlaya Wichit ◽  
Maprang Sompoppokakul ◽  
Napat Songtawee ◽  
...  
Keyword(s):  
Rational Design ◽  
Specific Activity ◽  
Homology Modelling ◽  
Biochemical Properties ◽  
Wild Type ◽  
E Coli ◽  
Sequence Identity ◽  
High Expression Level ◽  
The Ideal

The ideal therapeutic uricase (UOX) is expected to have the following properties; high expression level, high activity, high thermostability, high solubility and low immunogenicity. The latter property is believed to depend largely on sequence identity to the deduced human UOX (dH-UOX). Herein, we explored L. menadoensis uricase (LM-UOX) and found that it has 65% sequence identity to dH-UOX, 68% to the therapeutic chimeric porcine-baboon UOX (PBC) and 70% to the resurrected ancient mammal UOX. To study its biochemical properties, recombinant LM-UOX was produced in E. coli and purified to more than 95% homogeneity. The enzyme had specific activity up to 10.45 unit/mg, which was about 2-fold higher than that of the PBC. One-litre culture yielded purified protein up to 132 mg. Based on homology modelling, we successfully engineered I27C/N289C mutant, which was proven to contain inter-subunit disulphide bridges. The mutant had similar specific activity and production yield to that of wild type (WT) but its thermostability was dramatically improved. Up on storage at −20 °C and 4 °C, the mutant retained ~100% activity for at least 60 days. By keeping at 37 °C, the mutant retained ~100% activity for 15 days, which was 120-fold longer than that of the wild type. Thus, the I27C/N289C mutant has potential to be developed for treatment of hyperuricemia.

scholarly journals Rational Design of Alginate Lyase from Microbulbifer sp. Q7 to Improve Thermal Stability

Marine Drugs ◽  
2019 ◽  
Vol 17 (6) ◽  
pp. 378 ◽  
Author(s):  
Min Yang ◽  
Su-Xiao Yang ◽  
Zhe-Min Liu ◽  
Nan-Nan Li ◽  
Li Li ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Disulfide Bonds ◽  
Rational Design ◽  
Spatial Configuration ◽  
Specific Activity ◽  
Alginate Lyase ◽  
Life Time ◽  
Enzyme Specific Activity ◽  
Elimination Mechanism ◽  
Thermal Stability Of

Alginate lyase degrades alginate by the β-elimination mechanism to produce oligosaccharides with special bioactivities. The low thermal stability of alginate lyase limits its industrial application. In this study, introducing the disulfide bonds while using the rational design methodology enhanced the thermal stability of alginate lyase cAlyM from Microbulbifer sp. Q7. Enzyme catalytic sites, secondary structure, spatial configuration, and molecular dynamic simulation were comprehensively analyzed. When compared with cAlyM, the mutants D102C-A300C and G103C-T113C showed an increase by 2.25 and 1.16 h, respectively, in half-life time at 45 °C, in addition to increases by 1.7 °C and 0.4 °C in the melting temperature, respectively. The enzyme-specific activity and kcat/Km values of D102C-A300C were 1.8- and 1.5-times higher than those of cAlyM, respectively. The rational design strategy that was used in this study provides a valuable method for improving the thermal stability of the alginate lyase.

scholarly journals Disulfide Bond Engineering of an Endoglucanase from Penicillium verruculosum to Improve Its Thermostability

2019 ◽  
Vol 20 (7) ◽  
pp. 1602 ◽  
Author(s):  
Anna Bashirova ◽  
Subrata Pramanik ◽  
Pavel Volkov ◽  
Aleksandra Rozhkova ◽  
Vitaly Nemashkalov ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Rational Design ◽  
Specific Activity ◽  
Catalytic Performance ◽  
Industrial Applications ◽  
Penicillium Verruculosum ◽  
Bulk Chemicals ◽  
Commercial Applications ◽  
The Stability ◽  
Dynamics Simulations

Endoglucanases (EGLs) are important components of multienzyme cocktails used in the production of a wide variety of fine and bulk chemicals from lignocellulosic feedstocks. However, a low thermostability and the loss of catalytic performance of EGLs at industrially required temperatures limit their commercial applications. A structure-based disulfide bond (DSB) engineering was carried out in order to improve the thermostability of EGLII from Penicillium verruculosum. Based on in silico prediction, two improved enzyme variants, S127C-A165C (DSB2) and Y171C-L201C (DSB3), were obtained. Both engineered enzymes displayed a 15–21% increase in specific activity against carboxymethylcellulose and β-glucan compared to the wild-type EGLII (EGLII-wt). After incubation at 70 °C for 2 h, they retained 52–58% of their activity, while EGLII-wt retained only 38% of its activity. At 80 °C, the enzyme-engineered forms retained 15–22% of their activity after 2 h, whereas EGLII-wt was completely inactivated after the same incubation time. Molecular dynamics simulations revealed that the introduced DSB rigidified a global structure of DSB2 and DSB3 variants, thus enhancing their thermostability. In conclusion, this work provides an insight into DSB protein engineering as a potential rational design strategy that might be applicable for improving the stability of other enzymes for industrial applications.

scholarly journals Rational engineering of low temperature activity in thermoalkalophilic Geobacillus thermocatenulatus lipase

2021 ◽  
Author(s):  
Weigao Wang ◽  
Siva Dasetty ◽  
Sapna Sarupria ◽  
Mark Blenner
Keyword(s):  
Active Sites ◽  
Specific Activity ◽  
Industrial Applications ◽  
Significant Activity ◽  
Wild Type ◽  
Thermostable Enzymes ◽  
Adaptation Mechanism ◽  
Thermophilic Enzymes ◽  
Cold Activity ◽  
Geobacillus Thermocatenulatus

While thermophilic enzymes have thermostability desired for broad industrial applications, they can lose activity at ambient temperatures far from their optimal. Engineering cold activity into thermophilic enzymes has the potential to broaden the range of temperatures resulting in significant activity (i.e., decreasing the temperature dependence of kcat). Even though it has been widely suggested that cold temperature enzyme activity results from active flexibility that is at odds with the rigidity necessary for thermostable enzymes; however, directed evolution experiments have shown us these properties are not mutually exclusive. In this study, rational protein engineering was used to introduce flexibility inducing mutations around the active sites of Geobacillus thermocatenulatus lipase (GTL). Two mutants were found to have enhanced specific activity compared to wild-type at temperatures between 283 K to 363 K with p-nitrophenol butyrate but not with larger substrates. Kinetics assay revealed both mutations resulted in psychrophilic traits, such as lower activation enthalpy and more negative entropy values compared to wild type in all substrates. Furthermore, the mutants had significantly improved thermostability compared to wild type enzyme, which proves that it is feasible to improve the cold activity without trade-off. Our study provides insight into the enzyme cold adaptation mechanism and design principles for engineering cold activity into thermostable enzymes.

Simultaneous optimization of activity and stability of xylose reductase from D. nepalensis NCYC 3413 using statistical experimental design

2020 ◽  
Vol 27 ◽  
Author(s):  
Shwethashree Malla ◽  
Sathyanarayana N. Gummadi
Keyword(s):  
Half Life ◽  
Specific Activity ◽  
Enzyme Stability ◽  
Reductase Activity ◽  
Xylose Reductase ◽  
Life Time ◽  
Economic Feasibility ◽  
Simultaneous Optimization ◽  
Physical Parameters ◽  
Statistical Experimental Design

Background: Physical parameters like pH and temperature play a major role in the design of an industrial enzymatic process. Enzyme stability and activity are greatly influenced by these parameters; hence optimization and control of these parameters becomes a key point in determining the economic feasibility of the process. Objective: This study was taken up with the objective to optimize physical parameters for maximum stability and activity of xylose reductase from D. nepalensis NCYC 3413 through separate and simultaneous optimization studies and comparison thereof. Method: Effects of pH and temperature on the activity and stability of xylose reductase from Debaryomyces nepalensis NCYC 3413 were investigated by enzyme assays and independent variables were optimised using surface response methodology. Enzyme activity and stability were optimised separately and concurrently to decipher the appropriate conditions. Results: Optimized conditions of pH and temperature for xylose reductase activity were determined to be 7.1 and 27 ℃ respectively, with predicted responses of specific activity (72.3 U/mg) and half-life time (566 min). The experimental values (specific activity 50.2 U/mg, half-life time 818 min) were on par with predicted values indicating the significance of the model. Conclusion: Simultaneous optimization of xylose reductase activity and stability using statistical methods is effective as compared to optimisation of the parameters separately.

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