Journal of Molecular Catalysis B-enzymatic最新文献

Biocatalytic oxidations can offer clear advantages compared to chemically catalyzed oxidations in terms of chemo, regio and stereoselectivity as well as a reduced environmental impact. One of the most industrially important reactions is the oxidation of alcohols, which can be carried out using alcohol dehydrogenases. However, their effective use requires an effective regeneration of the oxidized nicotinamide cofactor (NAD(P)⁺), which is critical for the economic feasibility of the process. NAD(P)H oxidase is an enzyme class of particular interest for this cofactor regeneration since it enables the use of molecular oxygen as a substrate, generating either water or hydrogen peroxide as a by-product. The use of these enzymes is now gaining an increased interest, and several different enzymes of both types have been applied for proof-of-concept. In this review, we give an overview of the state-of-the-art, and discuss several important issues for future implementation in a production process.

The aim of this study was to investigate the effect of 3 different surface plasma treatments on the immobilization of β-galactosidase on a fibrous PET nonwoven membrane. Two methods 1. Entrapment in a thin calcium alginate coating and 2. Direct sorption, were used to immobilize the enzyme. The three different plasma treatments for surface activation of PET nonwovens were: 1. Air atmospheric DBD plasma, 2. Cold remote plasma-CRP with 100% N₂ and 3.- CRP with a mixture of N₂/O₂ gases. Plasma treatment of the PET fiber surface increased the quantity of immobilized enzyme using the entrapment method, and the degree of alginate film cross-linking highly influenced the enzyme activity. Highest enzyme activity was reached for the PET treated with air atmospheric plasma and cross-linked with 0.25 g/l of CaCl₂. With the direct sorption method, greater amounts of enzyme were immobilized as compared to the entrapment method, but a considerable proportion of enzyme lost their catalytic activity. Only with the CRP N₂/O₂ plasma treatment, up to 90% of sorbed enzyme maintained their activity. Reusability study showed that for the optimized entrapment method, a progressive decrease in activity was observed after each use cycle. With the optimized sorption method using N₂/O₂ CRP plasma, no decrease in enzyme activity was detected, and the immobilized enzyme could be used over more than 15 cycles.

In this study, we developed a whole-cell biocatalysis process for high-level conversion of l-lysine into 5-aminovalerate. To obtain the highly efficient whole-cell biocatalyst, five expression plasmids were constructed to optimize the expression of 5-aminovaleramide amidohydrolase and l-lysine 2-monooxygenase in Escherichia coli. The engineered strain BL-22A-RB-YB harboring plasmid pET22b-davA, pRSFDuet-davB and pACYCDuet-davB was correspondingly obtained. Subsequently, the effects of induction conditions, reaction temperature, metal ion additives, and cell permeability on the whole-cell biocatalyst system were evaluated to improve biocatalytic efficiency. Under optimized reaction conditions, 95.3 g/L 5-aminovalerate was synthesized from 120 g/L l-lysine with a yield of 99.1%, and 103.1 g/L 5-aminovalerate was produced from 150 g/L l-lysine with a molar yield of 85.7%. The 5-aminovalerate production was then further improved using a l-lysine fed-batch strategy, and a hyper 5-aminovalerate production of 240.7 g/L was achieved within 28 h with a yield of 86.8%. The whole-cell biocatalytic system described here demonstrated an environmentally friendly strategy for industrial production of 5-aminovalerate.

p-Hydroxyphenylacetate (HPA) can be derived from the biodegradation of lignin or from man-made compounds. The pathway involved for HPA degradation has been characterized for several species, but little is known on the degradation of HPA in Acinetobacter sp. In this report, the HPA degradation operon in A. baumannii TH was investigated using genome walking and PCR amplification to identify the genes encoded by the operon. The results showed that there are thirteen ORFs that are involved in this process and their arrangement in the operon of A. baumannii TH is different from that in the operons of other previously reported species. ORFs 8-12 show clear variation compared to orthologous genes from other species, particularly at ORF9 which encodes for succinic semialdehyde dehydrogenase (SSADH) that is absent in other species. The ssadh gene was overexpressed and the results confirmed that this enzyme is indeed succinate semialdehyde dehydrogenase. The results suggest that the final metabolites in this pathway are pyruvate and succinate, different from other species which have pyruvate and succinic semialdehyde as final products. Functional studies of the proteins encoded by ORF 8 and 10-12 have confirmed their roles in the HPA degradation pathway as an aldolase, a transporter protein, a hydroxylase and a reductase. Analysis of the sequence similarity network of enzymes encoded by ORFs 8-12 has revealed several interesting features. The designation of enzymes homologous to the oxygenase component of p-hydroxyphenylacetate 3-hydroxylase in the database should be reassigned, as they were mostly incorrectly assigned as acyl-CoA dehydrogenases. An understanding of the enzymatic reactions which convert aromatic compounds into pyruvate and succinate should be highly useful for future metabolic engineering for converting waste-derived aromatic compounds into useful biochemicals.

α-l-Rhamnosidases (α-RHAs) are a group of glycosyl hydrolases of biotechnological potential in industrial processes, which catalyze the hydrolysis of α-l-rhamnose terminal residues from several natural compounds. A novel α–RHA activity was identified in the crude extract of Novosphingobium sp. PP1Y, a marine bacterium able to grow on a wide range of aromatic polycyclic compounds. In this work, this α-RHA activity was isolated from the native microorganism and the corresponding orf was identified in the completely sequenced and annotated genome of strain PP1Y. The coding gene was expressed in Escherichia coli, strain BL21(DE3), and the recombinant protein, rRHA-P, was purified and characterized as an inverting monomeric glycosidase of ca. 120 kDa belonging to the GH106 family. A biochemical characterization of this enzyme using pNPR as substrate was performed, which showed that rRHA-P had a moderate tolerance to organic solvents, a significant thermal stability up to 45 °C and a catalytic efficiency, at pH 6.9, significantly higher than other bacterial α-RHAs described in literature. Moreover, rRHA-P was able to hydrolyze natural glycosylated flavonoids (naringin, rutin, neohesperidin dihydrochalcone) containing α-l-rhamnose bound to β-d-glucose with either α-1,2 or α-1,6 glycosidic linkages. Data presented in this manuscript strongly support the potential use of RHA-P as a biocatalyst for diverse biotechnological applications.

Both optical antipodes of the cis-isomers of osmundalactone, a hydroxypyranone natural product and core structure of the angiopterlactones, have been synthesized from acetylfuran in only three steps through a redox cascade utilizing oxidoreductases and transition metal catalysis in a concerted fashion. The key step in this fully catalytic strategy is the enzyme-mediated Achmatowicz reaction via selective furan oxygenation to furnish the pyran core structure.

Mannitol-2-dehydrogenase (MtDH) (E.C. 1.1.1.67) gene was cloned from Thermotoga neapolitana DSM 4359 and expressed in Escherichia coli BL21. The purified enzyme showed a predicted clear band of 36 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), native molecular mas was 135 kDa. K_m and V_max values for reduction of D-fructose to D-mannitol were 20 mM and 200 U mg-1 respectively. k_cat for reduction direction was 180 s⁻¹ and k_cat/K_m were 9 mM⁻¹ s⁻¹. The enzyme showed optimal pH at 6.5 and the optimum temperature was 90 °C with 100% relative activity. The purified enzyme was quite stable at 75 °C and had half of initial activity after 1 h of incubation at 90 °C. (TnMtDH) showed no activity with xylitol, inositol, sorbitol, rahmanose, mannose and xylose, and with NADPH and NADP⁺ as co factors. The presence of some divalent metals in the reaction enhanced the enzyme activity. The enzyme might be utilizing to produce mannitol without other sugar conformation under high temperature.

L-arginase from Rummeliibacillus pycnus SK31.001 is newly discovered. A 906 bp complete open reading frame, which encodes a 301 amino acid protein, was identified using degenerate PCR and inverse PCR techniques. The arginase was found to have a conserved active site with 6 amino acid residues binding to 2 manganese ions: D123, H125, D228, D230, H100 and D127. Bioinformatics analysis revealed that R. pycnus arginase is a hexamer with a subunit molecular mass of 33 kDa and whole molecular mass of 195 kDa. R. pycnus arginase is thermostable with an optimal temperature of 80 °C and maintains 85% of its initial activity after 24 h of incubation at 40 or 50 °C. An arginase activity assay showed that R. pycnus arginase has an optimum pH of 9.5 and a preference for Mn²⁺. Using arginine as the substrate, the Michaelis-Menten constant (K_m) and catalytic efficiency (k_cat/K_m) were measured to be 0.212 mM and 2970 mM⁻¹s⁻¹, respectively. The biosynthesis yield of L-ornithine by the purified enzyme was 144.4 g/L, and the molar yield was 95.2%.

Aspergillus flavipes l-methionine γ-lyase (AfMGL) has been recognized as a powerful broad range anticancer agent. However, catalytic instability and antigenicity are the main challenges of its applications in vivo. Thus, the objective of this study was to investigate the influence of conjugation with dextran on AfMGL biochemical properties and functionality. The activity of dextran AfMGL conjugates was 50% of the free MGL that consistent with the extent of occupied surface reactive amino groups. The accessibility of reactive ε-amino groups on dextran AfMGL surface was reduced by 70% normalizing to free enzyme. The thermal stability of dextran AfMGL was increased by two folds over the free enzyme. The dextran AfMGL had a higher resistance to proteinase K, retaining about 70% of its initial activity comparing to 20% to the native enzyme after 30 min of proteolysis at 37 °C. The in vivo half-life time of dextran modified AfMGL in New Zealand rabbits was increased by 2.3 folds comparing to free enzyme. The in vitro anticancer activity of the free and modified AfMGL was evaluated against five tumor cell lines (MCF-7, HEPG-2, HCT, PC3, HEP-2). The activity of AfMGL towards the tested tumor cells was significantly increased upon dextran conjugation suggesting the dramatic increasing of MGL hydrophilicity and catalytic efficiency. Dextran solution was used as negative control.