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 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 Rational design of a highly efficient catalytic system for the production of 3′-phosphoadenosine-5′-phosphosulfate from ATP

2021 ◽  
Author(s):  
Kaifang Liu ◽  
Xiulai Chen ◽  
Yunlu Zhong ◽  
Jia Liu ◽  
Guipeng Hu ◽  
...  
Keyword(s):  
Catalytic System ◽  
Rational Design ◽  
Specific Activity ◽  
Catalytic Efficiency ◽  
Disodium Salt ◽  
In Vivo Testing ◽  
Dynamics Simulations ◽  
Rate Limiting

The compound 3′-phosphoadenosine-5′-phosphosulfate (PAPS) serves as a sulfate group donor in the production of valuable sulfated compounds, such as glycosaminoglycan and oxamniquine. However, elevated costs and low conversion efficiency limit the industrial applicability of PAPS. Here, we designed and constructed an efficient and controllable catalytic system for the conversion of ATP (disodium salt) into PAPS without inhibition from by-products. In vitro and in vivo testing in Escherichia coli identified adenosine-5′-phosphosulfate kinase from Penicillium chrysogenum (PcAPSK) as the rate-limiting enzyme. Based on analysis of the catalytic steps and molecular dynamics simulations, a mechanism-guided “ADP expulsion” strategy was developed to generate an improved PcAPSK variant (L7), with a specific activity of 48.94 U·mg-1 and 73.27-fold higher catalytic efficiency (kcat/Km) that of the wild-type enzyme. The improvement was attained chiefly by reducing the ADP-binding affinity of PcAPSK, as well as by changing the enzyme’s flexibility and lid structure to a more open conformation. By introducing PcAPSK L7 in an in vivo catalytic system, 73.59 mM (37.32 g·L-1) PAPS was produced from 150 mM ATP in 18.5 h using a 3-L bioreactor. The achieved titer is the highest reported to date and corresponds to a 98.13% conversion rate. The proposed strategy will facilitate industrial production of PAPS as well as the engineering of similar enzymes.

scholarly journals Ag2O–MnO2/Graphene Oxide Nanocomposite

2020 ◽  
Author(s):  
Farooq Syed ◽  
Mujeeb Khan ◽  
Mohammed Rafi Shaik ◽  
Mufsir Kuniyil ◽  
M Rafiq Siddiqui ◽  
...  
Keyword(s):  
Catalytic Properties ◽  
Specific Activity ◽  
Catalytic Efficiency ◽  
Cost Effective ◽  
Catalytic Performance ◽  
Industrial Applications ◽  
Recoverable Catalyst ◽  
Aerial Oxidation ◽  
Milling Method ◽  
State Mixing

In this study, we reported the eco-friendly fabrication of Ag2O–MnO2/GRO nanocomposites by the solid-state mixing of separately prepared GRO and Ag2O–MnO2 NPs using ball milling method, a mechanochemical approach. The prepared material was studied for the catalytic effect of GRO in the system for the aerial oxidation of a variety of alcohols. It was found that the (1%)Ag2O–MnO2/(5 wt.%)GRO nanocatalyst demonstrated a high conversion ability (~100%) and excellent selectivity in the presence of O2 as a clean oxidant. The higher catalytic properties of the nanocomposite were attributed to the presence of GRO, which exhibited extraordinary catalytic properties like improved surface area, excellent chemical compatibility, and stability, as well as the introduction of several defects in the obtained nanocomposite that enhance the catalytic performance. The specific activity of 13.3 mmol·g−1·h−1 is obtained for the catalyst i.e. (1%)Ag2O–MnO2/(5 wt.%)GRO, which is reportedly superior to the various other catalysts previously reported in the literature for the same conversion reaction. Our catalytic strategy was highly selective, producing only desired products with no over-oxygenation to carboxylic acids. The merits of our catalytic methodology were: (a) facile process, (b) inexpensive and clean oxidant, (c) no surfactants or nitrogenous bases were required, (d) mild catalytic conditions, (e) cost-effective recoverable catalyst, (f) complete convertibility, (g) full selectivity, (h) rapid process, and (i) applicable to virtually all types of alcohols. So, these highlights made this catalytic strategy to be highly applicable in the industrial applications for manufacturing of carbonyls. To the best of our knowledge, this was the first study of utilizing Ag2O–MnO2/GRO composite as a catalyst for the oxidation of alcohols, highlighting the catalytic efficiency of GRO.

scholarly journals Facet-Dependent Reactivity of Fe2O3/CeO2 Nanocomposites: Effect of Ceria Morphology on CO Oxidation

Catalysts ◽  
2019 ◽  
Vol 9 (4) ◽  
pp. 371 ◽  
Author(s):  
Maria Lykaki ◽  
Sofia Stefa ◽  
Sόnia Carabineiro ◽  
Pavlos Pandis ◽  
Vassilis Stathopoulos ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Co Oxidation ◽  
Photoelectron Spectroscopy ◽  
Rational Design ◽  
Mixed Oxides ◽  
Specific Activity ◽  
Catalytic Performance ◽  
Catalytic Systems ◽  
X Ray ◽  
Full Characterization

Ceria has been widely studied either as catalyst itself or support of various active phases in many catalytic reactions, due to its unique redox and surface properties in conjunction to its lower cost, compared to noble metal-based catalytic systems. The rational design of catalytic materials, through appropriate tailoring of the particles’ shape and size, in order to acquire highly efficient nanocatalysts, is of major significance. Iron is considered to be one of the cheapest transition metals while its interaction with ceria support and their shape-dependent catalytic activity has not been fully investigated. In this work, we report on ceria nanostructures morphological effects (cubes, polyhedra, rods) on the textural, structural, surface, redox properties and, consequently, on the CO oxidation performance of the iron-ceria mixed oxides (Fe2O3/CeO2). A full characterization study involving N2 adsorption at –196 °C, X-ray diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscopy-energy dispersive X-ray spectroscopy (SEM-EDS), temperature programmed reduction (TPR), and X-ray photoelectron spectroscopy (XPS) was performed. The results clearly revealed the key role of support morphology on the physicochemical properties and the catalytic behavior of the iron-ceria binary system, with the rod-shaped sample exhibiting the highest catalytic performance, both in terms of conversion and specific activity, due to its improved reducibility and oxygen mobility, along with its abundance in Fe2+ species.

scholarly journals Characterizing Activity and Thermostability of GH5 Cellulase Chimeras from Mesophilic and Thermophilic Parents

2018 ◽  
Author(s):  
Fei Zheng ◽  
Josh V. Vermaas ◽  
Jie Zheng ◽  
Yuan Wang ◽  
Tao Tu ◽  
...  
Keyword(s):  
Thermal Properties ◽  
Rational Design ◽  
Enzyme Stability ◽  
Catalytic Efficiency ◽  
Industrial Applications ◽  
Essential Property ◽  
Tim Barrel ◽  
Dynamics Simulations ◽  
Temperature Optima ◽  
Hybrid Enzymes

ABSTRACTCellulases from glycoside hydrolase (GH) family 5 are key enzymes in the degradation of diverse polysaccharide substrates and are used in industrial enzyme cocktails to break down biomass. The GH5 family shares a canonical (βα)8-barrel structure, where each (βα) module is essential for the enzyme stability and activity. Despite their shared topology, the thermostability of GH5 enzymes can vary significantly, and highly thermostable variants are often sought for industrial applications. Based on a previously characterized thermophilic GH5 cellulase from Talaromyces emersonii (TeEgl5A, with an optimal temperature of 90°C), we created ten hybrid enzymes with the mesophilic cellulase from Prosthecium opalus (PoCel5) to determine which elements are responsible for enhanced thermostability. Five of the expressed hybrid enzymes exhibit enzyme activity. Two of these hybrids exhibited pronounced increases in the temperature optima (10 and 20°C), T50 (15 and 19°C), Tm (16.5 and 22.9°C), and extended half life, t1/2 (~240- and 650-fold at 55°C) relative to the mesophilic parent enzyme, and demonstrated improved catalytic efficiency on selected substrates. The successful hybridization strategies were validated experimentally in another GH5 cellulase from Aspergillus nidulans (AnCel5), which demonstrated a similar increase in thermostability. Based on molecular dynamics simulations (MD) of both PoCel5 and TeEgl5A parent enzymes as well as their hybrids, we hypothesize that improved hydrophobic packing of the interface between α2 and α3 is the primary mechanism by which the hybrid enzymes increase their thermostability relative to the mesophilic parent PoCel5.IMPORTANCEThermal stability is an essential property of enzymes in many industrial biotechnological applications, as high temperatures improve bioreactor throughput. Many protein engineering approaches, such as rational design and directed evolution, have been employed to improve the thermal properties of mesophilic enzymes. Structure-based recombination has also been used to fuse TIM-barrel fragments and even fragments from unrelated folds, to generate new structures. However, there are not many research on GH5 cellulases. In this study, two GH5 cellulases, which showed TIM-barrel structure, PoCel5 and TeEgl5A with different thermal properties were hybridized to study the roles of different (βα) motifs. This work illustrates the role that structure guided recombination can play in helping to identify sequence function relationships within GH5 enzymes by supplementing natural diversity with synthetic diversity.

scholarly journals Understanding the Enhanced Catalytic Performance of Ultrafine Transition Metal Nanoparticles–Graphene Composites

2015 ◽  
Vol 03 (01n02) ◽  
pp. 1540002 ◽  
Author(s):  
Xin Liu ◽  
Changgong Meng ◽  
Yu Han
Keyword(s):  
Transition Metal ◽  
Metal Nanoparticles ◽  
Rational Design ◽  
Energy Requirement ◽  
Catalytic Performance ◽  
Industrial Applications ◽  
Interfacial Structure ◽  
Transition Metal Nanoparticles ◽  
Energy And Environment ◽  
Environment Control

Catalysis, as the key to minimize the energy requirement and environmental impact of today's chemical industry, plays a vital role in many fields directly related to our daily life and economy, including energy generation, environment control, manufacture of chemicals, medicine synthesis, etc. Rational design and fabrication of highly efficient catalysts have become the ultimate goal of today's catalysis research. For the purpose of handling and product separation, heterogeneous catalysts are highly preferred for industrial applications and a large part of which are the composites of transition metal nanoparticles (TMNPs). With the fast development of nanoscience and nanotechnology and assisted with theoretical investigations, basic understanding on tailoring the electronic structure of these nanocomposites has been gained, mainly by precise control of the composition, morphology, interfacial structure and electronic states. With the rise of graphene, chemical routes to prepare graphene were developed and various graphene-based composites were fabricated. Transition metal nanoparticles-reduced graphene oxide (TMNPs–rGO) composites have attracted considerable attention, because of their intriguing catalytic performance which have been extensively explored for energy- and environment-related applications to date. This review summarizes our recent experimental and theoretical efforts on understanding the superior catalytic performance of subnanosized TMNPs–rGO composites.

scholarly journals Comparison between Lipase Performance Distributed at the O/W Interface by Membrane Emulsification and by Mechanical Stirring

Membranes ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 137
Author(s):  
Emma Piacentini ◽  
Rosalinda Mazzei ◽  
Lidietta Giorno
Keyword(s):  
Methyl Ester ◽  
Kinetic Resolution ◽  
Specific Activity ◽  
Candida Rugosa ◽  
Catalytic Performance ◽  
Membrane Emulsification ◽  
Mechanical Stirring ◽  
Emulsion Droplets ◽  
The Stability ◽  
Naproxen Methyl Ester

Multiphase bioreactors using interfacial biocatalysts are unique tools in life sciences such as pharmaceutical and biotechnology. In such systems, the formation of microdroplets promotes the mass transfer of reagents between two different phases, and the reaction occurs at the liquid–liquid interface. Membrane emulsification is a technique with unique properties in terms of precise manufacturing of emulsion droplets in mild operative conditions suitable to preserve the stability of bioactive labile components. In the present work, membrane emulsification technology was used for the production of a microstructured emulsion bioreactor using lipase as a catalyst and as a surfactant at the same time. An emulsion bioreaction system was also prepared by the stirring method. The kinetic resolution of (S,R)-naproxen methyl ester catalyzed by the lipase from Candida rugosa to obtain (S)-naproxen acid was used as a model reaction. The catalytic performance of the enzyme in the emulsion systems formulated with the two methods was evaluated in a stirred tank reactor and compared. Lipase showed maximum enantioselectivity (100%) and conversion in the hydrolysis of (S)-naproxen methyl ester when the membrane emulsification technique was used for biocatalytic microdroplets production. Moreover, the controlled formulation of uniform and stable droplets permitted the evaluation of lipase amount distributed at the interface and therefore the evaluation of enzyme specific activity as well as the estimation of the hydrodynamic radius of the enzyme at the oil/water (o/w) interface in its maximum enantioselectivity.

scholarly journals Improvement of XYL10C_∆N catalytic performance through loop engineering for lignocellulosic biomass utilization in feed and fuel industries

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Shuai You ◽  
Ziqian Zha ◽  
Jing Li ◽  
Wenxin Zhang ◽  
Zhiyuan Bai ◽  
...  
Keyword(s):  
Low Temperature ◽  
Lignocellulosic Biomass ◽  
Rational Design ◽  
Specific Activity ◽  
Combined Treatment ◽  
Catalytic Efficiency ◽  
Underlying Mechanism ◽  
Catalytic Performance ◽  
Reducing Sugar ◽  
Thermostable Enzymes

Abstract Background Xylanase, an important accessory enzyme that acts in synergy with cellulase, is widely used to degrade lignocellulosic biomass. Thermostable enzymes with good catalytic activity at lower temperatures have great potential for future applications in the feed and fuel industries, which have distinct demands; however, the potential of the enzymes is yet to be researched. Results In this study, a structure-based semi-rational design strategy was applied to enhance the low-temperature catalytic performance of Bispora sp. MEY-1 XYL10C_∆N wild-type (WT). Screening and comparisons were performed for the WT and mutant strains. Compared to the WT, the mutant M53S/F54L/N207G exhibited higher specific activity (2.9-fold; 2090 vs. 710 U/mg) and catalytic efficiency (2.8-fold; 1530 vs. 550 mL/s mg) at 40 °C, and also showed higher thermostability (the melting temperature and temperature of 50% activity loss after 30 min treatment increased by 7.7 °C and 3.5 °C, respectively). Compared with the cellulase-only treatment, combined treatment with M53S/F54L/N207G and cellulase increased the reducing sugar contents from corn stalk, wheat bran, and corn cob by 1.6-, 1.2-, and 1.4-folds, with 1.9, 1.2, and 1.6 as the highest degrees of synergy, respectively. Conclusions This study provides useful insights into the underlying mechanism and methods of xylanase modification for industrial utilization. We identified loop2 as a key functional area affecting the low-temperature catalytic efficiency of GH10 xylanase. The thermostable mutant M53S/F54L/N207G was selected for the highest low-temperature catalytic efficiency and reducing sugar yield in synergy with cellulase in the degradation of different types of lignocellulosic biomass. Graphic Abstract

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.

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