scholarly journals Computational design of highly stable and soluble alcohol dehydrogenase for NADPH regeneration

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Jinling Xu ◽  
Haisheng Zhou ◽  
Haoran Yu ◽  
Tong Deng ◽  
Ziyuan Wang ◽  
...  
Keyword(s):  
Alcohol Dehydrogenase ◽  
Specific Activity ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
High Specific Activity ◽  
Computational Design ◽  
Clostridium Beijerinckii ◽  
Protein Repair ◽  
Research Attention ◽  
Nadph Regeneration ◽  
Highly Active

AbstractNicotinamide adenine dinucleotide phosphate (NADPH), as a well-known cofactor, is widely used in the most of enzymatic redox reactions, playing an important role in industrial catalysis. However, the absence of a comparable method for efficient NADP+ to NADPH cofactor regeneration radically impairs efficient green chemical synthesis. Alcohol dehydrogenase (ADH) enzymes, allowing the in situ regeneration of the redox cofactor NADPH with high specific activity and easy by-product separation process, are provided with great industrial application potential and research attention. Accordingly, herein a NADP+-specific ADH from Clostridium beijerinckii was selected to be engineered for cofactor recycle, using an automated algorithm named Protein Repair One-stop Shop (PROSS). The mutant CbADH-6M (S24P/G182A/G196A/H222D/S250E/S254R) exhibited a favorable soluble and highly active expression with an activity of 46.3 U/mL, which was 16 times higher than the wild type (2.9 U/mL), and a more stable protein conformation with an enhanced thermal stability: Δ $${T}_{1/2}^{60\mathrm{min}}$$ T 1 / 2 60 min =  + 3.6 °C (temperature of 50% inactivation after incubation for 60 min). Furthermore, the activity of CbADH-6M was up-graded to 2401.8 U/mL by high cell density fermentation strategy using recombinant Escherichia coli, demonstrating its industrial potential. Finally, the superb efficiency for NADPH regeneration of the mutant enzyme was testified in the synthesis of some fine chiral aromatic alcohols coupling with another ADH from Lactobacillus kefir (LkADH).

scholarly journals Computational Design of Stable, Soluble and Highly Active Alcohol Dehydrogenase for NADPH Regeneration

2020 ◽  
Author(s):  
jinling Xu ◽  
Haisheng Zhou ◽  
Tong Deng ◽  
Haoran Yu ◽  
Ziyuan Wang ◽  
...  
Keyword(s):  
Alcohol Dehydrogenase ◽  
Specific Activity ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
High Specific Activity ◽  
Clostridium Beijerinckii ◽  
Protein Repair ◽  
Research Attention ◽  
Nadph Regeneration ◽  
Highly Active ◽  
Fermentation Strategy

Abstract Nicotinamide adenine dinucleotide phosphate (NADPH), as a well-known cofactor, is widely used in the most of enzymatic redox reactions, playing an important role in industrial catalysis. However, the absence of a comparable method for efficient NADP + to NADPH cofactor regeneration radically impairs efficient green chemical synthesis. Alcohol dehydrogenase (ADH) enzymes, allowing the in situ regeneration of the redox cofactor NADPH with high specific activity and easy by-product separation process, are provided with great industrial application potential and research attention. Accordingly, herein a NADP + specific ADH from Clostridium beijerinckii was selected to be engineered for cofactor recycle, using an automated algorithm named Protein Repair One-stop Shop (PROSS). The mutant CbADH-6M exhibited a favorable soluble and highly active expression with an activity of 46.3 U/mL, which was 16 times higher than the wild type (2.9 U/mL), and a more stable protein conformation with an enhanced thermal stability: Δ = +3.6°C (temperature of 50% inactivation after incubation for 60 min). Furthermore, the activity of CbADH-6M was up-graded to 2401.8 U/mL by high cell density fermentation strategy, demonstrating its industrial potential. Finally, the superb efficiency for NADPH regeneration of the mutant enzyme was testified in the synthesis of some fine chiral aromatic alcohols coupling with anther ADH from Lactobacillus kefir (LkADH). Nicotinamide adenine dinucleotide phosphate (NADPH), as a well-known cofactor, is widely used in the most of enzymatic redox reactions, playing an important role in industrial catalysis. However, the absence of a comparable method for efficient NADP + to NADPH cofactor regeneration radically impairs efficient green chemical synthesis. Alcohol dehydrogenase (ADH) enzymes, allowing the in situ regeneration of the redox cofactor NADPH with high specific activity and easy by-product separation process, are provided with great industrial application potential and research attention. Accordingly, herein a NADP + specific ADH from Clostridium beijerinckii was selected to be engineered for cofactor recycle, using an automated algorithm named Protein Repair One-stop Shop (PROSS). The mutant CbADH-6M exhibited a favorable soluble and highly active expression with an activity of 46.3 U/mL, which was 16 times higher than the wild type (2.9 U/mL), and a more stable protein conformation with an enhanced thermal stability: ΔT60 min 1/2 = +3.6°C (temperature of 50% inactivation after incubation for 60 min). Furthermore, the activity of CbADH-6M was up-graded to 2401.8 U/mL by high cell density fermentation strategy, demonstrating its industrial potential. Finally, the superb efficiency for NADPH regeneration of the mutant enzyme was testified in the synthesis of some fine chiral aromatic alcohols coupling with anther ADH from Lactobacillus kefir (LkADH).

scholarly journals Application Potential of Cyanide Hydratase from Exidia glandulosa: Free Cyanide Removal from Simulated Industrial Effluents

Catalysts ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 1410
Author(s):  
Anastasia Sedova ◽  
Lenka Rucká ◽  
Pavla Bojarová ◽  
Michaela Glozlová ◽  
Petr Novotný ◽  
...  
Keyword(s):  
Specific Activity ◽  
Copper Ions ◽  
High Specific Activity ◽  
Industrial Effluents ◽  
Free Cyanide ◽  
Highly Active ◽  
Physico Chemical ◽  
Application Potential ◽  
Ph Range ◽  
Few Data

Industries such as mining, cokemaking, (petro)chemical and electroplating produce effluents that contain free cyanide (fCN = HCN + CN−). Currently, fCN is mainly removed by (physico)chemical methods or by biotreatment with activated sludge. Cyanide hydratases (CynHs) (EC 4.2.1.66), which convert fCN to the much less toxic formamide, have been considered for a mild approach to wastewater decyanation. However, few data are available to evaluate the application potential of CynHs. In this study, we used a new CynH from Exidia glandulosa (protein KZV92691.1 designated NitEg by us), which was overproduced in Escherichia coli. The purified NitEg was highly active for fCN with 784 U/mg protein, kcat 927/s and kcat/KM 42/s/mM. It exhibited optimal activities at pH approximately 6–9 and 40–45 °C. It was quite stable in this pH range, and retained approximately 40% activity at 37 °C after 1 day. Silver and copper ions (1 mM) decreased its activity by 30–40%. The removal of 98–100% fCN was achieved for 0.6–100 mM fCN. Moreover, thiocyanate, sulfide, ammonia or phenol added in amounts typical of industrial effluents did not significantly reduce the fCN conversion, while electroplating effluents may need to be diluted due to high fCN and metal content. The ease of preparation of NitEg, its high specific activity, robustness and long shelf life make it a promising biocatalyst for the detoxification of fCN.

scholarly journals Biosynthesis of microsomal nicotinamide–adenine dinucleotide phosphate–cytochrome c reductase by membrane-bound and free polysomes from rat liver

1969 ◽  
Vol 112 (2) ◽  
pp. 139-147 ◽  
Author(s):  
G. Ragnotti ◽  
G. R. Lawford ◽  
P. N. Campbell
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Specific Activity ◽  
Reductase Activity ◽  
Gel Filtration ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
High Specific Activity ◽  
Rat Liver Cells ◽  
Microsome Fraction ◽  
Membrane Bound

1. NADPH–ferricytochrome c oxidoreductase (EC 1.6.2.3) was purified from the endoplasmic reticulum of rat liver cells. The methods, which involved digestion of membrane with Steapsin, a crude pancreatic extract containing diastase and trypsin, gel filtration and preparative electrophoresis on polyacrylamide, provided an enzyme with a high specific activity in good yield. 2. The incorporation of 14C-labelled amino acids into the purified reductase by the incubation of various subcellular fractions was studied. The microsome fraction, bound polysomes, free polysomes and detergent-treated polysomes effected the synthesis of the enzyme. 3. The reductase that had been synthesized by the polysomes was tightly bound to preparations of smooth-surfaced endoplasmic reticulum that were added to the incubation medium. 4. Reductase activity could be detected on both free and detergent-treated polysomes. Evidence is presented to show that this activity was due, at least in part, to the presence on the ribosomes of nascent enzyme. The association of enzyme with detergent-treated polysomes did not appear to be due to contamination of the ribosomes with either membrane or cell sap but it is possible for such ribosomes to adsorb some enzyme. 5. The amount of reductase activity associated with the detergent-treated polysomes was increased when the rats from which the polysomes were derived had been previously injected with phenobarbitone. 6. The results are discussed with respect to their relevance for the question of the existence of two functionally different groups of polysomes in the liver and for current ideas on the biogenesis of membranes.

Effects of Heparin Oligosaccharides with High Affinity for Antithrombin III in Experimental Venous Thrombosis

1982 ◽  
Vol 47 (03) ◽  
pp. 244-248 ◽  
Author(s):  
D P Thomas ◽  
Rosemary E Merton ◽  
T W Barrowcliffe ◽  
L Thunberg ◽  
U Lindahl
Keyword(s):  
Antithrombin Iii ◽  
Specific Activity ◽  
High Specific Activity ◽  
Factor Xa ◽  
Blood Levels ◽  
Thrombin Time ◽  
High Affinity ◽  
Very High

SummaryThe in vitro and in vivo characteristics of two oligosaccharide heparin fragments have been compared to those of unfractionated mucosal heparin. A decasaccharide fragment had essentially no activity by APTT or calcium thrombin time assays in vitro, but possessed very high specific activity by anti-Factor Xa assays. When injected into rabbits at doses of up to 80 ¼g/kg, this fragment was relatively ineffective in impairing stasis thrombosis despite producing high blood levels by anti-Xa assays. A 16-18 monosaccharide fragment had even higher specific activity (almost 2000 iu/mg) by chromogenic substrate anti-Xa assay, with minimal activity by APTT. When injected in vivo, this fragment gave low blood levels by APTT, very high anti-Xa levels, and was more effective in preventing thrombosis than the decasaccharide fragment. However, in comparison with unfractionated heparin, the 16-18 monosaccharide fragment was only partially effective in preventing thrombosis, despite producing much higher blood levels by anti-Xa assays.It is concluded that the high-affinity binding of a heparin fragment to antithrombin III does not by itself impair venous thrombogenesis, and that the anti-Factor Xa activity of heparin is only a partial expression of its therapeutic potential.

Autoprothrombin C Activity from Prothrombin with Snake Venom or Trypsin

1962 ◽  
Vol 08 (03) ◽  
pp. 425-433 ◽  
Author(s):  
Ewa Marciniak ◽  
Edmond R Cole ◽  
Walter H Seegers
Keyword(s):  
Snake Venom ◽  
Thrombolytic Agent ◽  
Sodium Citrate ◽  
Specific Activity ◽  
High Specific Activity ◽  
Citrate Solution ◽  
Clotting Factors ◽  
Activation Products ◽  
Russell’S Viper ◽  
Autoprothrombin C

SummarySuitable conditions were found for the generation of autoprothrombin C from purified prothrombin with the use of Russell’s viper venom or trypsin. DEAE chromatographed prothrombin is structurally altered and has never been found to yield autoprothrombin C and also did not yield it when Russell’s viper venom or trypsin were used. Autoprothrombin C is derived from prothrombin with tissue extract thromboplastin, but not in large amounts with the intrinsic clotting factors. With the latter thrombin and autoprothrombin III are the chief activation products. Autoprothrombin III concentrates were prepared from serum and upon activation with 25% sodium citrate solution or with Russell’s viper venom large amounts of autoprothrombin C were obtained, and this was of high specific activity. Theoretically trypsin is not a thrombolytic agent, but on the contrary should lead to intravascular clotting.

Highly Active Modified Variants of Recombinant Phospholipase А2 from Streptomyces violaceoruber for Effective Biosynthesis in Yeasts

2019 ◽  
pp. 30-41 ◽  
Author(s):  
E.P. Sannikova ◽  
A.V. Malysheva ◽  
F.A. Klebanov ◽  
D.G. Kozlov
Keyword(s):  
New Technologies ◽  
Specific Activity ◽  
Point Mutations ◽  
Egg Yolk ◽  
Enzyme Preparations ◽  
Highly Active ◽  
High Calcium ◽  
Pla2 Enzyme ◽  
Streptomyces Violaceoruber ◽  
The Cost

The capacity of yeast to produce the highly active variants of PLA2 has been confirmed. The high-active variants were based on the original enzyme from the strain А-2688 of Streptomyces violaceoruber. To reduce the enzyme toxicity and to increase its expression, various approaches were tested including point mutations, construction of artificial N- and/or C-end pro-regions, hybridization with other proteins and engineering or inactivation of glycosylation sites. As a main result, the modified PLA2 enzymes were obtained which have the same secretion level as their low-active predecessors, but specific activity of which was at least tenfold higher. As the main feature, the selected mutants were characterized by a lower affinity for Ca2+ that probably accounts for their low toxicity (and high expression capacity) at the stage of biosynthesis and their ability to activate under special conditions, e.g. during the egg yolk fermentation. The data obtained can provide a basis for the cost reduction of highly active PLA2 enzyme preparations in industries where the application of high calcium concentrations is allowed. recombinant phospholipase А2, Streptomyces violaceoruber, yeasts, secretion, producer strain The work was initiated by the Innovation Center Biriuch - New Technologies, Ltd., and was supported within the framework of the State Assignment no. 595-00004-18 PR.

scholarly journals Cascading Old Yellow Enzyme, Alcohol Dehydrogenase and Glucose Dehydrogenase for Selective Reduction of (E/Z)-Citral to (S)-Citronellol

Catalysts ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 931
Author(s):  
Yunpeng Jia ◽  
Qizhou Wang ◽  
Jingjing Qiao ◽  
Binbin Feng ◽  
Xueting Zhou ◽  
...  
Keyword(s):  
Alcohol Dehydrogenase ◽  
Glucose Dehydrogenase ◽  
Selective Reduction ◽  
Coli Strain ◽  
Severe Reaction ◽  
High Catalytic Activity ◽  
Old Yellow Enzyme ◽  
One Pot ◽  
Nadph Regeneration ◽  
By Products

Citronellol is a kind of unsaturated alcohol with rose-like smell and its (S)-enantiomer serves as an important intermediate for organic synthesis of (-)-cis-rose oxide. Chemical methods are commonly used for the synthesis of citronellol and its (S)-enantiomer, which suffers from severe reaction conditions and poor selectivity. Here, the first one-pot double reduction of (E/Z)-citral to (S)-citronellol was achieved in a multi-enzymatic cascade system: N-ethylmaleimide reductase from Providencia stuartii (NemR-PS) was selected to catalyze the selective reduction of (E/Z)-citral to (S)-citronellal, alcohol dehydrogenase from Yokenella sp. WZY002 (YsADH) performed the further reduction of (S)-citronellal to (S)-citronellol, meanwhile a variant of glucose dehydrogenase from Bacillus megaterium (BmGDHM6), together with glucose, drove efficient NADPH regeneration. The Escherichia coli strain co-expressing NemR-PS, YsADH, and BmGDHM6 was successfully constructed and used as the whole-cell catalyst. Various factors were investigated for achieving high conversion and reducing the accumulation of the intermediate (S)-citronellal and by-products. 0.4 mM NADP+ was essential for maintaining high catalytic activity, while the feeding of the cells expressing BmGDHM6 effectively eliminated the intermediate and by-products and shortened the reaction time. Under optimized conditions, the bio-transformation of 400 mM citral caused nearly complete conversion (>99.5%) to enantio-pure (S)-citronellol within 36 h, demonstrating promise for industrial application.

In-Canal Assay of High Specific Activity 60Co at the Advanced Test Reactor

2021 ◽  
pp. 1-7
Author(s):  
Michael A. Reichenberger ◽  
Jagoda M. Urban-Klaehn ◽  
Jason V. Brookman ◽  
Joshua L. Peterson-Droogh ◽  
Jorge Navarro ◽  
...  
Keyword(s):  
Specific Activity ◽  
High Specific Activity ◽  
Test Reactor
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