Earticle

Uncharacterized genes from the mesophilic bacterium Clostridium difficile and hyperthermophilic bacterium Thermotoga maritima that were previously proposed to be ribose-5-phosphate isomerases (RpiBs) were cloned and expressed in Escherichia coli. The RpiBs were compared to RpiB from the thermophilic bacterium C. thermocellum. The maximal activity of l-talose isomerization was observed at pH 7.5 and 40 °C in C. difficile RpiB, at pH 7.5 and 65 °C in C. thermocellum RpiB, and at pH 8.0 and 70 °C in T. maritima RpiB. C. difficile RpiB exhibited activity only with aldose substrates possessing hydroxyl groups oriented in the right-handed configuration (Fischer projections) at the C2 and C3 positions, such as d-ribose, d-allose, l-talose, llyxose, d-gulose, and l-mannose. In contrast, C. thermocellum and T. maritima RpiBs displayed activity only with aldose substrates possessing hydroxyl groups configured the same direction at the C2, C3, and C4 positions, such as the d- and l-forms of ribose, talose, and allose.

Levulinic acid (LA) is a versatile chemical building block that can be produced from cellulosic biomass and can be converted into valuable chemicals and fuels. In this study, enzymatic reduction of LA was performed for the synthesis of 4-hydroxyvaleric acid (4HV), which can be used as a monomer of bio-polyester and a precursor of bio-fuels. Because no enzyme that has LA as its native or major substrate has been reported, engineering of the substrate specificity of an enzyme is required to yield the enzymatic reduction of LA. 3-Hydroxybutyrate dehydrogenase (3HBDH) from Alcaligenes faecalis was selected as a target enzyme for the engineering of substrate specificity. A rational design approach with molecular docking simulation was performed for the engineering of the substrate specificity of the enzyme. Through the mutational studies of 3HBDH, the substrate specificity of the enzyme was engineered and a double mutant which has catalytic activity toward LA was selected as a best mutant. Approximately 57% conversion of LA was achieved with this mutant in 24 hr, while no conversion was achieved with the wild-type enzyme.

The mpi gene encoding mannose-6-phosphate isomerase (GTMpi) from Geobacillus thermodenitrificans was cloned and expressed in Escherichia coli. GTMpi converted aldose substrates possessing hydroxyl groups oriented in the same direction at the C2 and C3 positions, such as the D- and L-forms of ribose, lyxose, talose, mannose, and allose. D-Lyxose isomerization was optimal at pH 7.0 and 70°C with 1mM Co2+. The half-lives of the enzyme at 60°C, 65°C, and 70°C were 388 h, 73h, and 27 h, respectively. D-Lyxose was converted to D-xylulose by GTMpi with 38% conversion yields after about 3 h, whereas L-ribose was converted to L-ribulose with 29% conversion yields after about 3 h.

Abstract The uncharacterized gene previously proposed as a myosin cross antigen from Macrococcus caseolyticus was cloned and expressed in Escherichia coli. The putative protein was identified as an oleate hydratase by the determination of the substrate specificity and the analysis of the amino acid sequence. The native enzyme was a 69-kDa dimer with a molecular mass of 138 kDa, as measured by gel filtration. The maximal activity of the oleate hydratase for oleate hydration was observed at pH 6.5 and 25ºC in the presence of 0.5 mM flavin adenine dinucleotide. The hydroxylated products formed from unsaturated fatty acids by the enzyme were made as the standards with a high purity (>99%) by thin layer chromatography and solvent extraction and then were identified. Using the products, the accurate specific activity and kinetic parameters for fatty acids as substrates were determined. Hydratase from Macrococcus caseolyticus exhibited hydration activity only for 9(Z)-double bonds of C16 and C18 unsaturated fatty acids and showed the highest specific activity and catalytic efficiency (kcat/Km) for oleic acid among the unsaturated fatty acid substrates. Thus, 10-hydroxystearic acid was produced from oleic acid by the recombinant enzyme and E. coli.

Streptococcus pneumoniae was cloned and expressed in E.coli Er2566. The recombinant RpiB was purified by HisTrap HP chromatography. The enzyme exhibited maximal activity at pH 7.5 and 35°C, and its activity was independent of metal ions. The molecular mass of native RpiB was estimated to be 98.2 kDa as a tetramer using Sephacryl S-300 gel filtration. The enzyme showed specificity for aldose substrates possessing hydroxyl groups oriented in the same direction at the C2 and C3 positions such as L-lysose,L-talose, D-ribose, D-allose, Dgulose, D-xylose, and L-mannose. Especially, the RpiB exhibited high enzyme activity for the L-form aldoses such as L-lyxose and L-talose among various aldoses. The catalytic efficiency (kcat/Km) of RpiB for L-lyxose and L-talose were 3.86 and 1.2 mM-1s-1, respectively. Recently, L-form sugars have been increased the interest as a potential functional material in the food and pharmaceutical industry. Thus, it is suggested strongly that of RpiB from Streptococcus pneumonia is applied usefully for the production of L-form sugars.

The rpiB gene, encoding ribose-5-phosphate isomerase (RpiB) from Clostridium thermocellum, was cloned and expressed in Escherichia coli. Substrate specificity of the expressed enzyme was investigated with hexose and pentose. The RpiB enzyme had narrow specificity for L-talose, D-ribose, L-ribose, D-allose, L-allose or D-talose which converted to the respective ketose. Interestingly, structures of those substrates showed the same OH direction of carbon C2, C3 and C4. Also the enzyme had a inverse reaction such as D-ribulose, D-psicose, L-tagatose or L-ribulose. Among substrate, L-talose as aldose show the highest Km and Kcat/Km values of 35.3 mM and 467.2 μM-1/s-1, respectively.

We purified recombinant galactose 6-phosphate isomerase (LacAB) from Lactococcus lactis using HiTrap Q HP and Phenyl-Sepharose columns. The purified LacAB had a final specific activity of 1.79 units/mg to produce D-allose. The molecular mass of native galactose 6-phosphate isomerase was estimated at 135.5 kDa using Sephacryl S-300 gel filtration, and the enzyme exists as a hetero-octamer of LacA and LacB subunits. The activity of galactose 6-phosphate isomerase was maximal at pH 7.0 and 30°C, and enzyme activity was independent of metal ions. When 100 g/L of D-psicose was used as the substrate, 25 g/L of D-allose and 13 g/L of D-altrose were simultaneously produced at pH 7.0 and 30°C after 12 h of incubation. The enzyme had broad specificity for various aldoses and ketoses. The interconversion of sugars with the same configuration except at the C2 position was driven by using a large amount of enzyme in extended reactions. The interconversion occurred via two isomerization reactions, i.e., the interconversion of D-allose↔D-psicose↔Daltrose, and D-allose to D-psicose reaction was faster than D-altrose to D-psicose reaction.

Cytochrome P450s act on the inactive carbon-hydrogen bonds of alkanes, fatty acids, terpenes, and steroids [1]. And some exhibits high regio and stereoselective monooxygenation activity. The CYP102A5 is a 120 kDa (1065 aa) self-sufficient monooxygenase which belongs to Class III, consisting of an FMN/FAD-containing reductase domain and a heme domain. The gene encoding CYP102A5, a novel P450 monooxygenase from Bacillus cereus, was cloned and expressed in Escherichia coliBL21 using pET expression system. The CYP102A5 of wild type catalyses the oxidation of alkanes, alkanoic acids and unsaturated fatty acids. Furthermore, double mutant F90V T441G L442G exhibited its activity towards daidzein of biphenolic compound. Furthermore, this triple mutant catalyzed regioselective hydroxylation at the 6, 8 and 3' position of daidzein.

Benzoate-CoA ligase (BCL) from Burkholderia xenovorans LB400 has narrow substrate specificity toward benzoate. Compared to the activity with benzoate, specific activity of BCL is much less than that with m-/p-hyroxybenzoate. In order to find BCL mutants which have higher activity, site-directed mutagenesis within substrate binding pocket based on rational design were carried out. The comparative structural analysis revealed that active site of 2,3-dihydroxybenzoate AMP ligase(DhbE) and 4-chlorobenzoate CoA ligase(4-CBAL) with that of BCL has shown absolutely different amino acids. Especially, substratebinding pocket of BCL showed relatively narrow circumstanse than that of BCL from Bulkholderia xenovorans due to the surrounding residues(i. e, HisXXX) located at the bottom of the active sites. Their structural analysis allows us to generate thirteen mutants and in-vitro analysis of specific activities using indirect assay monitoring NADH consumption were examined for understanding their structure-activity relationship. In conclusion, BCL mutants screened by above mentioned structural understanding and mutagenesis showed higher activity against benzoate as we are targeting.

Molecular modeling and site-directed mutagenesis were used to probe the molecular determinants of substrate specificity for aliphatic and aromatic nitriles of nitrilase from Rhodococcus rhodochrous tg1-A6. Ten putative expected substrate-binding residues within 4.5 Å of active site were replaced with respective Ala and then only the substitution of position 142 resulted in the high activity at aromatic substrates but no activity at aliphatic substrates. When Tyr-142 was further mutated to glycine, valine, leucine, isoleucine, serine, asparagine, arginine, aspartic acid and phenylalanine respectively, interestingly uncharged and smaller amino acid substituted mutants exhibited higher catalytic efficiency with aromatic substrates than the wild-type enzyme exhibited. Whereas smaller amino acid substituted mutants had no activity on aliphatic substrates. These results Tyr-142 position modulated substrate specificity of aromatic and aliphatic substrates.

There are three types of alginate lyases: the first type is specific for α-L-guluronate (G) block guluronate lyase, the second type for β-Dmannuronate (M) block mannuronate lyase, and the third type is bifunctional for G and M blocks. These enzymes depolymerize alginate through a β-elimination reaction. Their structure/function relationships are expected to provide information valuable to future industrial alginate processing and drug design for Pseudomonas aeruginosa alginate biofilm-dependent infection. Alginate lyase AlyVI is specific for polyguluronate and has a molecular mass of 34 KDa. Site-directed mutagenesis was used to examine their roles in binding alginate. Several mutants were prepared, expressed in Escherichia coli, and tested by TBI method using supernatant of culture. Mutants D226K, D226A, and D226G had higher activity than the wild type. Especially, the activities of D226A and D226K were increased nearly 4- and 5-fold, respectively. A relatively conserved tryptophan residue corresponding to tryptophan 165 of alginate lyase AlyVI is proposed to be important for the functioning of the enzyme. Tryptophan 165 was individually changed to an alanine, aspartic acid, arginine, and glycine. All of these mutants were completely inactive. These results indicates that W165 is critical part for enzyme activit

The nitrilase from Rhodococcus rhodochrous ATCC 33278 hydrolyzes both aliphatic and aromatic nitriles. When the enzyme containing Tyr at the position of 142 was substituted into aromatic amino acid (Phe), the mutant retained activity for both class of substrates. However, the mutants containing non-polar aliphatic amino acid (Ala, Val, or Leu) at the position of 142 had activity only for aromatic substrates such as benzonitrile, m-tolunitrile, and 2-cyanopyridine, not for aliphatic substrates. The results suggest that the enzyme reaction is likely to involve conjugated phi-electron system of aromatic ring as electron acceptor. The mutants containing charged amino acid such as Asp, Glu, Arg, or Asn at the position 142 had no activity for all nitriles due to disruption of hydrophobic interaction with substrate. Thus, the amino acid at the position 142 is a molecular determinant of substrate specificity in the nitrilase.

Carbohydrate microarray might have powerful potential for assaying substrate specificity or activity of carbohydrate-processing enzymes such as glycosyltransferase and glycosidase. However, carbohydrate microarray system has been rarely utilized because complex chemical reactions are demanded for microarray preparation. Recently, we have been developed a simple and cost-effective method for fabrication of carbohydrate microarray which uses the reaction of amine-conjugated carbohydrates with glass surface. In the present work, we employed the developed carbohydrate microarray system to rapidly determine substrate specificity or activity of glycosyltransferase. Pasteurella multocidaoriginated sialyltransferase was used as a target glycosyltransferase because it is a wonderful target to prove the potential of carbohydrate microarray. Collectively, the functional carbohydrate microarray would be a powerful analytical tool to screen substrate specificity or other properties of carbohydrate-processing enzymes.

Coprinus cinereus peroxidase (CIP) is a kind of fungi peroxidase which catalyzes alkyl phenol compounds. It can catalyze the polymerization of ortho-, meta-, and para- phenol compounds. The phenol compounds are phenol, cresol, ethyl phenol, isopropyl phenol, and tert-butyl phenol. All meta- and para- phenol compounds are catalyzed by CIP but o-isopropyl phenol and o-tert-butyl phenol are not catalyzed by CIP. Horseradish peroxidase (HRP) is a kind of plant peroxidase which also catalyzes alkyl phenol compounds. The substrate specificity spectrum of HRP is narrower than CIP : HRP cannot catalyze o-isopropyl phenol, o-tert-butyl phenol, and m-tert-butyl phenol. Also, the yield of m-isopropyl phenol is very low. This was confirmed from the docking result that the yield is higher, and docking distance is closer. In this presentation, docking energy and distance between Fe atom in heme group and oxygen atom in phenol compounds were calculated. And the relationships between Mn (number-average molecular weight), yield, docking energy and distance were investigated by QSAR methods. It was found that there is a linear relationship among docking energy, docking distance and the yield.

ω-Transaminase has a great potential in pharmaceutical industry due to its ability to synthesize chiral amines and amino acids that are used as intermediate for chiral drugs. However size constraints in the active site pocket of ω-transaminase limits production of various enantiopure amino acids. To overcome this stringency of substrate specificity, protein engineering was applied. From the docking model, a single point mutations were introduced to the active site residue to expand the small pocket. The resulting mutants showed enhanced activity toward keto acids, amino acids, and amines carrying bulky substituents. This work was supported by the Advanced Biomass R&D Center (ABC- 2010-0029737) and the Basic Science Research Program (2010-0024448) through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology.

ω-Aminotransferase (ωAT) is an interesting biocatalyst for the preparation of chiral amines which are widely used as building blocks for pharmaceuticals, agrochemicals and fine chemicals. With the assumption that substrate and sequence spaces are in the process of co-evolution, we explored sequences space to screen the enzymes showing activity to new target compounds. 527 bacterial genome sequences were analyzed by the profiles of subgroups in aminotransferase group II including ornithine aminotransferase (orAT), acetylornithine aminotransferase (aoAT), ω-aminotransferase (ωAT), γ-aminobutyrate aminotransferase (GABAAT) and 7,8-diaminopelargonate aminotransferase (DAPAAT). We selected the sequences having a Z-score of 0~1.8 to guarantee the ωAT reaction and to avoid the typical ωAT sequences. Among the selected sequences, we filtered out the sequences having very low Z-scores for the rest of four subgroups in aminotransferase group II to consider the diversity. For the selected sequences, we performed protein-ligand docking simulations to predict the docking pose of amino acceptor. Throughout all the analysis procedures, several candidate aminotransferase sequences for the asymmetric synthesis of chiral amines were obtained. An efficient procedure for virtual screening of novel enzymes was demonstrated.

Although barley α-amylase isozyme 1 (AMY1) and 2 (AMY2) share up to 80% of amino acid sequence identity, their enzymatic properties differ remarkably. In this study, the 42nd alanine residue of AMY2 was replaced with another random amino acid via saturation mutagenesis. Eight out of 370 recombinant E. coli cells showing enhanced starch-hydrolyzing activity were characterized as possessing the same proline residue instead of alanine. Even though the specific activity of AMY2-A42P is reduced to 81% of wild-type, its expression level and purification yield were enhanced by approximately 2 and 4 times that of AMY2, respectively. Characterization of its enzymatic properties confirmed that AMY2-A42P is similar to that of wild-type. However, its specificity to starch substrates is likely to be intermediate between AMY1 and AMY2.

Polyphenol oxidase (PPO) was separated from Magnolia (Magnolia kobus) flower by acetone precipitation and CM-Sepharose and Phenyl-Sepharose chromatographies. Molecular weight of the purified PPO from Magnolia flower was assumed to be just over 20 kDa on the sodiumdodecylsulfate-polyacrylamide gel electrophoresis and around 40 kDa under non-boiling without ${\beta}$-mercaptoethanol. Magnolia flower PPO showed the highest enzyme activity with chlorogenic acid as a substrate.