A novel di-D-furanose 1,2′:2,3′-dianhydride hydrolase (DFA-IIIase) from Duffyella gerundensis A4
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The prebiotic fructan inulin can provide energy to organisms via several pathways. One pathway is that inulin fructotransferase (IFTase) firstly converted inulin to III-type difructose anhydride (DFA-III). Then DFA-III is hydrolyzed to inulobiose via difructose anhydride hydrolase (DFA-IIIase). However, only five DFA-IIIases have been reported to date and all of them are from Arthrobacter genus. Whether other microbes except Arthrobacter genus can utilize DFA-III through DFA-IIIase are unknown. In this work, a DFA-IIIase from Duffyella gerundensis A4 (D. gerundensis A4), abbreviated as DgDFA-IIIase, was cloned, expressed, purified, identified, and characterized. It was approximately 50 kDa assayed by SDS-PAGE and showed the highest catalytic activity for DFA-III at pH 6.0 and 35 °C with specific activity of 56 U mg-1. The enzyme was metal-independent and had a high pH stability but low thermostability, which kept only 28% of residual activity after incubation under 50 °C for 3 h. Moreover, Km and kcat/Km for DFA-III was 122 mM and 0.39 mM-1 s-1, respectively. The constructed model of DgDFA-IIIase showed that it has identical residues around substrate at active site with AcDFA-IIIase whose crystal structure has been revealed, indicating that DgDFA-IIIase probably adopts the same catalytic mechanism with the reported AcDFA-IIIase. The work finds that DFA-III can be catalyzed by DFA-IIIase from microorganism of non-Arthrobacter genus, which also extends the enzymatic sources of DFA-IIIase.Keywords:
Thermostability
Hydrolase
Enzyme Kinetics
Thermostability
Enzyme Kinetics
Site-directed mutagenesis
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Thermostability
Hydrolase
Thermal Stability
Protein Engineering
Site-directed mutagenesis
Parathion methyl
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The encoding region of mpd gene for methyl parathion hydrolase was subcloned by PCR. The recombinant plasmid pET29a-mpd was constructed and the methyl parathion hydrolase was expressed in E. coli BL21 (DE3) as a fusion protein tagged with (His) 6 at C-terminus. The fusion protein was purified to homogeneity by Ni-affinity chromatography under undenaturing condition, the protein could degrade methyl parathion effectively. A convenient and reliable method to assay the activity of methyl parathion hydrolase was established. The effects of environmental factors on the activity of methyl parathion hydrolase and its enzymatic kinetics were analyzed. The optimum pH value for enzymatic hydrolysis of methyl parathion was 8 6～8 8, the optimum temperature was 15℃. Mn 2+, Zn 2+, Cu 2+ increased the activity of methyl parathion hydrolase by 15%～20%. Ca 2+, Mg 2+ and Ni 2+ decreased enzyme activity slightly. 1mmol/L EDTA·Na 2+ almost had no effect on the enzyme activity, but 10mmol/L EDTA·Na 2+ inhibited enzyme activity strongly. The K m and kcat against methyl parathion at 25℃ was (68 6 ± 5 1)μmol/L and (45 ± 6)S -1 respectively. The optimum pH value for hydrolysis of parathion by this hydrolase was also 8 6～8 8, the K m and kcat against parathion at 25℃ was (59 5 ± 6 0)μmol/L and (8 ± 1) S -1 respectively. The values of kcat/K m of methyl parathion hydrolase to two substrates showed that the enzyme hydrolyzes methyl parathion more efficiently.
Hydrolase
Parathion methyl
Enzyme Kinetics
Parathion
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Inulin hydrolysis catalyzed with strongly acidic cation resins as solid acids was investigated by suing desalted inulin solutions and high fructose syrups with high quality were obtained after being vacuum concentrated.The optimal conditions for inulin hydrolysis were:D061 strongly acidic cation resin as solid acid;the ratio of solid acid and inulin solution(20 %)1:3;hydrolysis temperature 75 ℃;hydrolysis time 120 min.Percentage of hydrolysed Inulin with this method was 92.1 %.Desalting from inulin solution was one of the key technologies for successful inulin hydrolysis with solid acid.
Solid acid
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Objective To find the optimum condition of the hydrolysis process of oligosaccharides from Inulin.Methods Inulin is hydrolyzed via acid and enzymatic means respectively.Single factor experiments were set to get the best method.Results As for acid hydrolysis,the best method is undertaken under the condition of 80 ℃(pH=2.0) for 30 min.With regard to the enzymatic hydrolysis approach,the best one is as follows:substrate concentration ratio is 1∶1,reacting at 60 ℃ with 0.4 g enzyme for 18 h.Conclusion The acid hydrolysis,with higher convert rate and simpler working condition,is better than the enzymatic one.
Enzymatic Hydrolysis
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Thermostability
Enzyme Kinetics
Bacteroides thetaiotaomicron
Catalytic efficiency
Protein Engineering
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Solid acid
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As a member of the α/β-hydrolase superfamily,Rhodococcus sp.R04 derived BphD C-C hydrolase(2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate,HOPDA hydrolase) catalyses the hydrolytic C-C cleavage of the meta-ring fission products from the biphenyl catabolic pathway.We constructed six mutants of C-C hydrolase to investigate its catalytic mechanism.Substitutions of Ser-110,Asp-237 and His-265 in HOPDA hydrolase with Ala caused to 17~19-fold reduction in kcat,and reduced the kcat/Km values by 104~103 folds in comparison to the wild-type.The Ala substitution of Trp-85 and Trp-219 led to 3.6-and 3.3-fold reduction in kcat/Km,respectively.Mutating of Trp-266 to Ala resulted in a 104-fold reduction of kcat/Km.From the circular dichroism(CD)spectra,S110A,D237A,H265A and W266A were predominately showed in α-helix conformation as the wild-type.The chemical modification results showed that the modifiers did not influence the activities of S110A,D237A,H265A and Trp266 mutants,except 0.5 mmol/L NBS reduced the activity of W85A by 90% and W219A by70%.Our data indicated that Ser110,Asp237 and His265 were necessary for the catalytic reaction of HOPDA hydrolase,and Trp266 was suggested to play a key role in the enzymatic reaction.
Enzyme Kinetics
Hydrolase
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Triazophos is a broad-spectrum and highly effective insecticide, and the residues of triazophos have been frequently detected in the environment. A triazophos-degrading bacterium, Burkholderia sp. SZL-1, was isolated from a long-term triazophos-polluted soil. Strain SZL-1 could hydrolyze triazophos to 1-phenyl-3-hydroxy-1,2,4-triazole, which was further utilized as the carbon sources for growth. The triazophos hydrolase gene trhA, cloned from strain SZL-1, was expressed and homogenously purified using Ni-nitrilotriacetic acid affinity chromatography. TrhA is 55 kDa and displays maximum activity at 25°C, pH 8.0. This enzyme still has nearly 60% activity at the range of 15°C–50°C for 30 min. TrhA was mutated by sequential error prone PCR and screened for improved activity for triazophos degradation. One purified variant protein (Val89-Gly89) named TrhA-M1 showed up to 3-fold improvement in specific activity against triazophos, and the specificity constants of Kcat and Kcat/Km for TrhA-M1 were improved up to 2.3- and 8.28-fold, respectively, compared to the wild-type enzyme. The results in this paper provided potential material for the contaminated soil remediation and hydrolase genetic structure research.
Cloning (programming)
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