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

Author(s):  
Xiaoli Zhou ◽  
Zhiqiang Xu ◽  
Yueqiu Li ◽  
Jia He ◽  
Honghui Zhu

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.

2019 ◽  
Vol 20 (7) ◽  
pp. 1602 ◽  
Author(s):  
Anna Bashirova ◽  
Subrata Pramanik ◽  
Pavel Volkov ◽  
Aleksandra Rozhkova ◽  
Vitaly Nemashkalov ◽  
...  

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.


2021 ◽  
Vol 12 ◽  
Author(s):  
Jing Si ◽  
Hongfei Ma ◽  
Yongjia Cao ◽  
Baokai Cui ◽  
Yucheng Dai

This study introduces a valuable laccase, designated ThLacc-S, purified from white rot fungus Trametes hirsuta. ThLacc-S is a monomeric protein in nature with a molecular weight of 57.0 kDa and can efficiently metabolize endocrine disrupting chemicals. The enzyme was successfully purified to homogeneity via three consecutive steps consisting of salt precipitation and column chromatography, resulting in a 20.76-fold increase in purity and 46.79% yield, with specific activity of 22.111 U/mg protein. ThLacc-S was deciphered as a novel member of the laccase family and is a rare metalloenzyme that contains cysteine, serine, histidine, and tyrosine residues in its catalytic site, and follows Michaelis-Menten kinetic behavior with a Km and a kcat/Km of 87.466 μM and 1.479 s–1μM–1, respectively. ThLacc-S exerted excellent thermo-alkali stability, since it was markedly active after a 2-h incubation at temperatures ranging from 20 to 70°C and retained more than 50% of its activity after incubation for 72 h in a broad pH range of 5.0–10.0. Enzymatic activities of ThLacc-S were enhanced and preserved when exposed to metallic ions, surfactants, and organic solvents, rendering this novel enzyme of interest as a green catalyst for versatile biotechnological and industrial applications that require these singularities of laccases, particularly biodegradation and bioremediation of environmental pollutants.


Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4059-4059
Author(s):  
Osheiza Abdulmalik ◽  
J. Eric Russell

Abstract 4059 Poster Board III-994 Transgenic approaches to β thalassemia and sickle cell disease require viral vectors that express high levels of therapeutic β-like globin proteins. We recently proposed that the overall expression of these transgenes would likely be improved by structural modifications that prolong the cytoplasmic half-lives of their encoded mRNAs. Relevant experiments from our laboratory have previously linked the constitutively high stability of β-globin mRNA to a region of its 3'UTR that appears to interact with at least two distinct cytoplasmic mRNA-stabilizing factors, and is predicted to form an imperfect stem-loop (SL) structure. Based upon these findings, we conducted enzymatic secondary-structure mapping studies of the β-globin 3'UTR, unequivocally validating the existence of the predicted functional stem-loop element. We subsequently reasoned that the constitutive half-life of β-globin mRNA might be prolonged by the insertion of multiple SL motifs into its 3'UTR, resulting in increased levels of the mRNA–and its encoded β-globin product–in terminally differentiating erythroid cells. To test this hypothesis, we constructed full-length β-globin genes containing either wild-type 3'UTRs, or variant 3'UTRs that had been modified to contain either two or three tandem SL motifs. Each gene was identically linked to a tetracycline-suppressible promoter, permitting pulse-chase mRNA stability analyses to be conducted in vivo in intact cultured cells. Erythroid-phenotype K562 cells were transiently transfected with SL-variant and control wild-type β-globin genes, exposed to tetracycline, and levels of β-globin mRNA determined by qRT-PCR at defined intervals using tet-indifferent β-actin mRNA as internal control. Relative to wild-type β-globin mRNA, SL-duplicate β-globin mRNAs displayed a position-dependent two-fold increase in cytoplasmic half-life; SL-triplicate β-globin mRNAs did not exhibit any additional stability. These experiments confirm the existence of a defined SL structure within the β-globin 3'UTR, and demonstrate that duplication of this motif can substantially increase the stability of β-globin mRNA. We subsequently designed a series of experiments to elucidate post-transcriptional processes involved in mRNA hyperstability. These studies required the construction of HeLa cells that stably express either wild-type β-globin mRNA (11 subclones) or SL-duplicate β-globin mRNAs (10 subclones). Preliminary analyses indicate an approximate 1.5-fold increase in the median steady-state expression of SL-duplicate genes, consistent with a prolongation in the half-life of its encoded mRNA. While formal mRNA stability studies are not yet complete, early data appear to replicate results from experiments conducted in transiently transfected cells. We have also initiated structural studies to link differences in the stability of SL-variant β-globin mRNA to alterations in its poly(A) tail. Using an RNase H-based strategy, we identified a previously unknown poly(A)-site heterogeneity–of undetermined significance–affecting both wild-type and SL-duplicate β-globin mRNAs. Finally, we recently isolated fifty-four K562 subclones expressing SL-duplicate or control β-globin mRNAs; parallel analyses of these cells will permit the cell-specificity of β-globin SL-directed mRNA stabilization to be investigated in detail. Results from each of these studies will be immediately applicable to the design of high-efficiency therapeutic transgenes for β thalassemia and sickle-cell disease. Disclosures: No relevant conflicts of interest to declare.


Author(s):  
Farooq Syed ◽  
Mujeeb Khan ◽  
Mohammed Rafi Shaik ◽  
Mufsir Kuniyil ◽  
M Rafiq Siddiqui ◽  
...  

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.


2007 ◽  
Vol 73 (22) ◽  
pp. 7291-7299 ◽  
Author(s):  
Mirella Di Lorenzo ◽  
Aurelio Hidalgo ◽  
Rafael Molina ◽  
Juan A. Hermoso ◽  
Domenico Pirozzi ◽  
...  

ABSTRACT A prolipase from Rhizopus oryzae (proROL) was engineered in order to increase its stability toward lipid oxidation products such as aldehydes with the aim of improving its performance in oleochemical industries. Out of 22 amino acid residues (15 Lys and 7 His) prone to react with aldehydes, 6 Lys and all His residues (except for the catalytic histidine) were chosen and subjected to saturation mutagenesis. In order to quickly and reliably identify stability mutants within the resulting libraries, active variants were prescreened by an activity staining method on agar plates. Active mutants were expressed in Escherichia coli Origami in a 96-well microtiterplate format, and a stability test using octanal as a model deactivating agent was performed. The most stable histidine mutant (H201S) conferred a stability increase of 60%, which was further enhanced to 100% by combination with a lysine mutant (H201S/K168I). This increase in stability was also confirmed for other aldehydes. Interestingly, the mutations did not affect specific activity, as this was still similar to the wild-type enzyme.


Blood ◽  
2008 ◽  
Vol 112 (7) ◽  
pp. 2761-2769 ◽  
Author(s):  
Hironao Wakabayashi ◽  
Fatbardha Varfaj ◽  
Jennifer DeAngelis ◽  
Philip J. Fay

AbstractFactor VIII consists of a heavy chain (A1A2B domains) and light chain (A3C1C2 domains), whereas the contiguous A1A2 domains are separate subunits in the cofactor, factor VIIIa. The intrinsic instability of the cofactor results from weak affinity interactions of the A2 subunit within factor VIIIa. The charged residues Glu272, Asp519, Glu665, and Glu1984 appear buried at the interface of the A2 domain with either the A1 or A3 domain, and thus may impact protein stability. To determine the effects of these residues on procofactor/cofactor stability, these residues were individually replaced with either Ala or Val, and stable BHK cell lines expressing the B-domainless proteins were prepared. Specific activity and thrombin generation parameters for 7 of the 8 variants were more than 80% the wild-type value. Factor VIII activity at 52°C to 60°C and the decay of factor VIIIa activity after thrombin activation were monitored. Six of the 7 variants showing wild-type-like activity demonstrated enhanced stability, with the Glu1984Val variant showing a 2-fold increase in thermostability and an approximately 4- to 8-fold increase in stability of factor VIIIa. These results indicate that replacement of buried charged residues is an effective alternative to covalent modification in increasing factor VIII (VIIIa) stability.


Polymers ◽  
2021 ◽  
Vol 13 (22) ◽  
pp. 3884
Author(s):  
Andrew Rennison ◽  
Jakob R. Winther ◽  
Cristiano Varrone

Polyethylene terephthalate (PET) is the most widely used polyester plastic, with applications in the textile and packaging industry. Currently, re-moulding is the main path for PET recycling, but this eventually leads to an unsustainable loss of quality; thus, other means of recycling are required. Enzymatic hydrolysis offers the possibility of monomer formation under mild conditions and opens up alternative and infinite recycling paths. Here, IsPETase, derived from the bacterium Ideonella sakaiensis, is considered to be the most active enzyme for PET degradation under mild conditions, and although several studies have demonstrated improvements to both the stability and activity of this enzyme, stability at even moderate temperatures is still an issue. In the present study, we have used sequence and structure-based bioinformatic tools to identify mutations to increase the thermal stability of the enzyme so as to increase PET degradation activity during extended hydrolysis reactions. We found that amino acid substitution S136E showed significant increases to activity and stability. S136E is a previously unreported variant that led to a 3.3-fold increase in activity relative to wild type.


2016 ◽  
Vol 83 (2) ◽  
Author(s):  
Junxian Zheng ◽  
Taowei Yang ◽  
Junping Zhou ◽  
Meijuan Xu ◽  
Xian Zhang ◽  
...  

ABSTRACT NAD+-dependent formate dehydrogenase (FDH; EC 1.2.1.2) is an industrial enzyme widely used for NADH regeneration. However, enzyme inactivation caused by the oxidation of cysteine residues is a flaw of native FDH. In this study, we relieved the oxidation of the free cysteine of FDH from Candida boidinii (CboFDH) through the construction of disulfide bonds between A10 and C23 as well as I239 and C262. Variants A10C, I239C, and A10C/I239C were obtained by the site-directed mutagenesis and their properties were studied. Results showed that there were no significant changes in the optimum temperature and pH between variants and wild-type CboFDH. However, the stabilities of all variant enzymes were improved. Specifically, the CboFDH variant A10C (A10C fdh ) showed a significant increase in copper ion resistance and acid resistance, a 6.7-fold increase in half-life at 60°C, and a 1.4-fold increase in catalytic efficiency compared with the wild type. Asymmetric synthesis of l-tert-leucine indicated that the process time was reduced by 40% with variant A10C fdh , which benefited from the increase in catalytic efficiency. Circular dichroism analysis and molecular dynamics simulation indicated that variants that contained disulfide bonds lowered the overall root mean square deviation (RMSD) and consequently increased the protein rigidity without affecting the secondary structure of enzyme. This work is expected to provide a viable strategy to avoid the microbial enzyme inactivation caused by the oxidation of the free cysteine residues and improving their performances. IMPORTANCE FDH is widely used for NADH regeneration in dehydrogenase-based synthesis of optically active compounds to decrease the cost of production. This study highlighted a viable strategy that was used to eliminate the oxidation of free cysteine residues of FDH from Candida boidinii by the introduction of disulfide bonds. Using this strategy, we obtained a variant FDH with improved activity and stability. The improvement of activity and stability of FDH is expected to reduce its price and then further to decrease the cost of its application.


2003 ◽  
Vol 23 (20) ◽  
pp. 7377-7390 ◽  
Author(s):  
Maria Luisa Gándara ◽  
Pilar López ◽  
Raquel Hernando ◽  
José G. Castaño ◽  
Susana Alemany

ABSTRACT Cot, initially identified as an oncogene in a truncated form, is a mitogen-activated protein kinase kinase kinase implicated in cellular activation and proliferation. Here, we show that this truncation of Cot results in a 10-fold increase in its overall kinase activity through two different mechanisms. Truncated Cot protein exhibits a lower turnover rate (half-life, 95 min) than wild-type Cot (half-life, 35 min). The degradation of wild-type and truncated Cot can be specifically inhibited by proteasome inhibitors in situ. The 20S proteasome also degrades wild-type Cot more efficiently than the truncated protein. Furthermore, the amino acid 435 to 457 region within the wild-type Cot COOH-terminal domain confers instability when transferred to the yellow fluorescent protein and targets this fusion protein to degradation via the proteasome. On the other hand, the kinase specific activity of wild-type Cot is 3.8-fold lower than that of truncated Cot, and it appears that the last 43 amino acids of the wild-type Cot COOH-terminal domain are those responsible for this inhibition of kinase activity. In conclusion, these data demonstrate that the oncogenic activity of truncated Cot is the result of its prolonged half-life and its higher kinase specific activity with respect to wild-type Cot.


Membranes ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 137
Author(s):  
Emma Piacentini ◽  
Rosalinda Mazzei ◽  
Lidietta Giorno

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.


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