Development of a Novel Biocatalyst for the Resolution of rac‐Pantolactone

A novel L ‐pantolactone hydrolase, Lph, from Agrobacterium tumefaciens Lu681 was characterized, which stereospecifically hydrolyses L ‐pantolactone to L ‐pantoic acid yielding D ‐pantolactone with > 95% enantiomeric excess. The enzyme was found to be a 30 kDa‐Zn²⁺‐hydrolase with a K_m for L ‐pantolactone of 7 mM and a V_max of 30 U/mg. The corresponding lph gene was identified as an 807 bp ORF and cloned into E. coli. It was overexpressed under control of P_tac and P_rha yielding enzyme activities of up to 600 U/g dry weight. Resolution of d,l ‐pantolactone in repeated batches with isolated Lph and enzyme recovery by membrane filtration gave D ‐pantolactone with 50% yield and 90–95% ee over 6 days. Covalent immobilization to EupergitC led to a stable biocatalyst easy to handle in a repeated batch production of D ‐pantolactone. Further improvements in the activity of Lph were achieved by directed evolution of the enzyme. Activities of mutants F62S, K197D and F100L were increased 2.3, 1.7, and 1.5 fold, respectively.     

Borate as a Phosphate Ester Mimic in Aldolase‐Catalyzed Reactions: Practical Synthesis of L‐Fructose and L‐Iminocyclitols

Dihydroxyacetone phosphate (DHAP)‐dependent aldolases have been widely used for the organic synthesis of unnatural sugars or derivatives. The practicality of using DHAP‐dependent aldolases is limited by their strict substrate specificity and the high cost and instability of DHAP. Here we report that the DHAP‐dependent aldolase L ‐rhamnulose 1‐phosphate aldolase (RhaD) accepts dihydroxyacetone (DHA) as a donor substrate in the presence of borate buffer, presumably by reversible in situ formation of DHA borate ester. The reaction appears to be irreversible, with the products thermodynamically trapped as borate complexes. We have applied this discovery to develop a practical one‐step synthesis of the non‐caloric sweetener L ‐fructose. L ‐Fructose was synthesized from racemic glyceraldehyde and DHA in the presence of RhaD and borate in 92 % yield on a gram scale. We also synthesized a series of L ‐iminocyclitols, which are potential glycosidase inhibitors, in only two steps.     

Multi‐Enzymatic Synthesis of Optically Pure β‐Hydroxy α‐Amino Acids

A novel enzymatic production system of optically pure β‐hydroxy α‐amino acids was developed. Two enzymes were used for the system: an N‐succinyl L ‐amino acid β‐hydroxylase (SadA) belonging to the iron(II)/α‐ketoglutarate‐dependent dioxygenase superfamily and an N‐succinyl L ‐amino acid desuccinylase (LasA). The genes encoding the two enzymes are part of a gene set responsible for the biosynthesis of peptidyl compounds found in the Burkholderia ambifaria AMMD genome. SadA stereoselectively hydroxylated several N‐succinyl aliphatic L ‐amino acids and produced N‐succinyl β‐hydroxy L ‐amino acids, such as N‐succinyl‐L ‐β‐hydroxyvaline, N‐succinyl‐L ‐threonine, (2S,3R)‐N‐succinyl‐L ‐β‐hydroxyisoleucine, and N‐succinyl‐L ‐threo‐β‐hydroxyleucine. LasA catalyzed the desuccinylation of various N‐succinyl‐L ‐amino acids. Surprisingly, LasA is the first amide bond‐forming enzyme belonging to the amidohydrolase superfamily, and has succinylation activity towards the amino group of L ‐leucine. By combining SadA and LasA in a preparative scale production using N‐succinyl‐L ‐leucine as substrate, 2.3 mmol of L ‐threo‐β‐hydroxyleucine were successfully produced with 93% conversion and over 99% of diastereomeric excess. Consequently, the new production system described in this study has advantages in optical purity and reaction efficiency for application in the mass production of several β‐hydroxy α‐amino acids.

     

Synthesis of silk sericin peptides–L‐asparaginase bioconjugates and their characterization

The natural silk sericin, recovered from Bombyx mori silk waste by degumming and degrading, is a water‐soluble peptide with different molecular masses, ranging from 20 to 60 kDa. It is composed of 15 sorts of amino acids, among which the polar amino acids with hydroxyl, carboxyl and amino groups such as aspartic acid, serine and lysine account for 72%. The covalent attachment of the silk sericin peptides to L ‐asparaginase (ASNase) produces silk sericin peptides–L ‐asparaginase (SS–ASNase) bioconjugates that are active, stable, have a lower immune response, and have extended half‐lives in vitro in human serum. The modified enzyme coupled with sericin protein retains 55.8% of the original activity of the native enzyme. The optimal pH of SS–ASNase derivatives shifts considerably, to 5.0 in comparison with pH 6.0–8.0 of the native form. The thermostability and resistance to trypsin digestion of the modified enzyme are greatly enhanced as compared with ASNase alone. The Michaelis constant (K_m) of SS–ASNase is 65 times lower than that of the enzyme alone. This suggests that the affinity of the enzyme to its substrate L ‐asparagine greatly increases when bioconjugated with silk sericin. The in vivo experiments also show that the silk sericin peptides have no immunogenicity, and the antigenicity of the enzyme is obviously decreased when coupled covalently with the silk sericin peptides. Copyright © 2005 Society of Chemical Industry     

One‐Pot Cascade Reactions using Fructose‐6‐phosphate Aldolase: Efficient Synthesis of D‐Arabinose 5‐Phosphate,D‐Fructose 6‐Phosphate and Analogues

One‐pot multienzymatic reactions have been performed for the synthesis of 1‐deoxy‐D ‐fructose 6‐phosphate, 1,2‐dideoxy‐D ‐arabino‐hept‐3‐ulose 7‐phosphate, D ‐fructose 6‐phosphate and D ‐arabinose 5‐phosphate. The whole synthetic strategy is based on an aldol addition reaction catalysed by fructose‐6‐phosphate aldolase (FSA) as a key step of a three or four enzymes‐catalysed cascade reaction. The four known donors for FSA – dihydroxyacetone (DHA), hydroxyacetone (HA), 1‐hydroxy‐2‐butanone (HB) and glycolaldehyde (GA) – were used with D ‐glyceraldehyde 3‐phosphate as acceptor substrate. The target phosphorylated sugars were obtained in good to excellent yields and high purity.     

Kinetics of the L‐aminoacylase‐catalyzed resolution of N‐acetyl‐DL‐butyrine

The kinetics of the asymmetric hydrolysis of N‐acetyl‐DL ‐butyrine catalyzed by L ‐aminoacylase to obtain optically pure L ‐butyrine is described. Some of the constants are determined from the initial reaction rates and others from long‐term experiments in batch reactors by the numerical integration of the reactor design equation and minimization of the kinetic parameters. The methodology described can be applied to the kinetic study of other complex biocatalytic systems. Studies on enzyme activation by adding different divalent metal ions and enzymatic deactivation are also included. © 2002 Society of Chemical Industry     

One‐Pot Conversion of L‐Threonine into L‐Homoalanine: Biocatalytic Production of an Unnatural Amino Acid from a Natural One

A novel biocatalytic process for production of L ‐homoalanine from L ‐threonine has been developed using coupled enzyme reactions consisting of a threonine deaminase (TD) and an ω‐transaminase (ω‐TA). TD catalyzes the dehydration/deamination of L ‐threonine, leading to the generation of 2‐oxobutyrate which is asymmetrically converted to L ‐homoalanine via transamination with benzylamine executed by ω‐TA. To make up the coupled reaction system, we cloned and overexpressed a TD from Escherichia coli and an (S)‐specific ω‐TA from Paracoccus denitrificans. In the coupled reactions, L ‐threonine serves as a precursor of 2‐oxobutyrate for the ω‐TA reaction, eliminating the need for employing the expensive oxo acid as a starting reactant. In contrast to α‐transaminase reactions in which use of amino acids as an exclusive amino donor limits complete conversion, amines are exploited in the ω‐TA reaction and thus maximum conversion could reach 100%. The ω‐TA‐only reaction with 10 mM 2‐oxobutyrate and 20 mM benzylamine resulted in 94% yield of optically pure L ‐homoalanine (ee>99%). However, the ω‐TA‐only reaction did not produce any detectable amount of L ‐homoalanine from 10 mM L ‐threonine and 20 mM benzylamine, whereas the ω‐TA reaction coupled with TD led to 91% conversion of L ‐threonine to L ‐homoalanine.     

Phage Display on the Anti‐infective Target 1‐Deoxy‐d‐xylulose‐5‐phosphate Synthase Leads to an Acceptor–Substrate Competitive Peptidic Inhibitor

Enzymes of the 2‐C‐methyl‐d ‐erythritol‐4‐phosphate pathway for the biosynthesis of isoprenoid precursors are validated drug targets. By performing phage display on 1‐deoxy‐d ‐xylulose‐5‐phosphate synthase (DXS), which catalyzes the first step of this pathway, we discovered several peptide hits and recognized false‐positive hits. The enriched peptide binder P12 emerged as a substrate (d ‐glyceraldehyde‐3‐phosphate)‐competitive inhibitor of Deinococcus radiodurans DXS. The results indicate possible overlap of the cofactor‐ and acceptor‐substrate‐binding pockets and provide inspiration for the design of inhibitors of DXS with a unique and novel mechanism of inhibition.     

Lipase‐catalyzed synthesis and characterization of biodegradable polyester containing l‐malic acid unit in solvent system

Lipase‐catalyzed direct polycondensation of L ‐malic acid (L ‐MA), adipic acid, and 1,8‐octanediol in organic media was achieved using Novozym 435 as the biocatalyst. ¹H‐nuclear magnetic resonance spectroscopy indicated that the selectivity of Novozym 435 was unaffected by changes in the organic media. The molecular weight (M_w) of the copolymers was affected by the L ‐MA feed ratio in the diacids, hydrophobicity of the solvent, and solubility of the substrates in the solvents. The M_w reached a maximum of 17.4 kDa at 80°C in isooctane at a L ‐MA feed ratio in the diacids of 40 mol %. The M_w increased from 3.2 to 16.6 kDa when the reaction time was extended from 6 to 48 hr at 70°C, and remained relatively constant with further increases in reaction time from 48 to 72 hr. The hydrophilicity, thermal stability, and crystallizability of the copolymer were also investigated. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Solvent‐ and thermal‐induced crystallization of poly‐L‐lactic acid in supercritical CO2 medium

The effect of different annealing treatments with supercritical carbon dioxide (SCCO₂) on the structural and mechanical properties of semicrystalline poly‐L ‐lactic acid (L ‐PLA) was investigated. 2000, 27,000, 100,000, and 350,000 g mol^?1 molecular weight L ‐PLA polymers were used in the study. The solid‐state processing of L ‐PLA at temperatures lower than the effective melting point led to solvent‐ and thermal‐induced crystallization. Solvent‐induced and isothermal crystallization mechanisms could be considered similar regarding the increase of polymer chain mobility and mass‐transfer in the amorphous region; however, quite different microstructures were obtained. SCCO₂ solvent‐induced crystallization led to polymers with high crystallinity and melting point. On the contrary, SCCO₂ thermal‐induced crystallization led to polymers with high crystallinity and low melting point. For these polymers, the hardness increased and the elasticity decreased. Finally, the effect of dissolving SCCO₂ in the molten polymer (cooling from the melt) was analyzed. Cooling from the melt led to polymers with high crystallinity, low melting point, low hardness, and low elasticity. Distinctive crystal growth and nucleation episodes were identified. This work also addressed the interaction of SCCO₂‐drug (triflusal) solution with semicrystalline L ‐PLA. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2009     

Synthesis and characterization of degradable copoly(ester–amide)s: Poly(trans‐4‐hydroxy‐L‐proline‐co‐ε‐caprolactam)

Novel copolyesteramides were synthesized by reacting trans‐4‐hydroxy‐N‐benzyloxycarbonyl‐L ‐proline (N‐CBz‐Hpr) with ε‐caprolactam (CLM) in the presence of stannous octoate [Sn(II) Oct.] as a catalyst. Various techniques, including ¹H‐NMR, IR, DSC, and viscosity, were used to elucidate structural characteristics and thermal properties of the resulting copolymers. Data showed that the optimal reaction condition for the synthesis of the copolymers was obtained by using 3 wt % Sn(II) Oct. at 170°C for 24 h. The DSC analysis demonstrated amorphous structure for most of the copolymers. The glass‐transition temperature of the copolymers shifts to a higher temperature with increasing Hpr/CLM molar ratio. In vitro degradation of these poly(N‐CBz‐Hpr‐co‐CLM)s was evaluated by weight loss measurements. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 86: 1615–1621, 2002     

Synthesis of S‐Adenosyl‐L‐homocysteine Capture Compounds for Selective Photoinduced Isolation of Methyltransferases

Understanding the interplay of different cellular proteins and their substrates is of major interest in the postgenomic era. For this purpose, selective isolation and identification of proteins from complex biological samples is necessary and targeted isolation of enzyme families is a challenging task. Over the last years, methods like activity‐based protein profiling (ABPP) and capture compound mass spectrometry (CCMS) have been developed to reduce the complexity of the proteome by means of protein function in contrast to standard approaches, which utilize differences in physical properties for protein separation. To isolate and identify the subproteome consisting of S‐adenosyl‐L ‐methionine (SAM or AdoMet)‐dependent methyltransferases (methylome), we developed and synthesized trifunctional capture compounds containing the chemically stable cofactor product S‐adenosyl‐L ‐homocysteine (SAH or AdoHcy) as selectivity function. SAH analogues with amino linkers at the N6 or C8 positions were synthesized and attached to scaffolds containing different photocrosslinking groups for covalent protein modification and biotin for affinity isolation. The utility of these SAH capture compounds for selective photoinduced protein isolation is demonstrated for various methyltransferases (MTases) acting on DNA, RNA and proteins as well as with Escherichia coli cell lysate. In addition, they can be used to determine dissociation constants for MTase–cofactor complexes.     

Purification and characterization of poly(L‐lactic acid) depolymerase from Pseudomonas sp. strain DS04‐T

Poly(L ‐lactic acid) (PLA)‐degrading Pseudomonas sp. strain DS04‐T was isolated from an activated sludge sample. A novel PLA depolymerase with a molecular weight of 34 kDa was purified to homogeneity from the culture supernatant of strain DS04‐T. The optimum pH and temperature of the PLA depolymerase are 8.5 and 50°C, respectively. The PLA depolymerase can also degrade triolein, poly(β‐hydroxybutyrate), and poly(ε‐caprolactone). Na⁺ and K⁺ enhance the enzyme activity, whereas Zn²⁺, Mg²⁺, Cu²⁺, and Fe²⁺ inhibit it. The inhibition effect of different chemicals on the PLA depolymerase activity were examined, in which ethylenediaminetetraacetic acid was found to have a significant inhibitory effect. The main degradation product of the depolymerase is identified as lactic acid monomer by mass spectrometric analysis. POLYM. ENG. SCI., 2011. © 2010 Society of Plastics Engineers.     

Zinc‐based catalyst for the ring‐opening polymerization of cyclic esters

A zinc‐based catalyst zinc bis[bis(trimethylsilyl)amide] was used for the polymerization of cyclic esters including L ‐lactide (L ‐LA) and 2‐methyl‐2‐carboxyl‐propylene carbonate (MBC). The polymerization of L ‐lactide in THF could be carried out successfully under mild conditions in very short time by using the zinc catalyst and alcohols as the initiators. Kinetic study in solution polymerization prooved the polymerization has high monomer conversion degree close to 100% and the molecular weight of the resulting polyester has linear increase with the increase of [M]₀ /[I] (molar ratio of monomer to initiator). Sequential polymerization of L ‐LA and MBC in THF also showed high MBC conversion of 94% with a narrow molecualr weight maintained, indicating a living nature of this polymerization. The zinc catalyst system has also been used for the L ‐LA bulk polymerization with a high monomer conversion. ¹³C NMR indicated the polymer possesses high regioregularity and the minor regioirregular component was owing to the D ‐LA in the monomer inserted into the polymer mainchain during the transesterifcation. Interaction between monomer and zinc catalyst has been found to be a key factor to sustain a homogenous solution during the initiating procedure. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Direct polycondensation of N‐trimellitylimido‐L‐isoleucine with aromatic diamines

N‐Trimellitylimido‐L ‐isoleucine (3) was prepared from the reaction of trimellitic anhydride with L ‐isoleucine [L ‐2‐amino‐3‐methylvalerianic acid or (2S,3S)‐2‐amino‐3‐methyl‐n‐valerinic acid] in an N,N‐dimethylformamide solution at the refluxing temperature. The direct polycondensation reaction of the monomer imide diacid (3) with 1,4‐phenylenediamine, 4,4′‐diaminodiphenylmethane, 4,4′‐diaminodiphenylsulfone, diaminodiphenylether, 1,5‐naphthalendiamine, 2,4‐diaminotoluene, and 1,3‐phenylenediamine was performed in a medium consisting of triphenyl phosphite, N‐methyl‐2‐pyrolidone (NMP), pyridine, and calcium chloride. The polycondensation was performed under two different conditions: in one method, the reaction mixture was heated in an NMP solution at 60, 90, and then 130°C for different periods of time, and in the other method, the reaction mixture was refluxed only for 1 min in the same solvent. The resulting poly(amide imide)s (PAIs), with inherent viscosities of 0.21–0.37 dL/g, were obtained in high yields. All of these compounds were fully characterized by IR spectra, elemental analyses, and specific rotation measurements. Some structural characterizations and physical properties of these new optically active PAIs were examined. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 89: 116–122, 2003     

Dimethylarginine‐Dimethylaminohydrolase‐2 (DDAH‐2) Does Not Metabolize Methylarginines

Free endogenous methylarginines, N^ω‐monomethyl‐L ‐arginine (L ‐NMMA) and N^ω,N^ω′‐dimethyl‐L ‐arginine (ADMA), inhibit NO synthases (NOSs) and are metabolized by dimethylargininedimethylaminohydrolase (DDAH). A postulated metabolism has been shown several times for DDAH‐1, but the involvement of DDAH‐2 in the degradation of ADMA and L ‐NMMA is still a matter of debate. Determination of the isoform‐specific DDAH protein expression profiles in various porcine tissue types shows a correlation of DDAH activity only with DDAH‐1 levels. DDAH activity (measured as L ‐citrulline formation from the conversion of methylarginines and alternative DDAH substrates) was detected in DDAH‐1‐rich porcine tissue types, that is, kidney, liver, and brain, but not in DDAH‐2‐rich porcine fractions, that is, spleen and thyroid. Furthermore, several ex vivo studies showed DDAH activity to be important for L ‐citrulline formation in porcine tissue and indicated the absence of an endogenous DDAH inhibitor in porcine tissue. This study provides new insights into tissue distributions as well as substrate selectivity for both DDAH isoforms. Although DDAH‐1 is known to metabolize the endogenous NOS inhibitors L ‐NMMA and ADMA, a physiological function for DDAH‐2 has yet to be determined. Hence, determining DDAH activity by methylarginine conversion is not suitable for analyzing isoform selectivity of DDAH‐1 inhibitors as postulated.     

Synthesis and characterization of novel optically active poly(amide–imide)s containing N,N′‐(pyromellitoyl)‐bis‐L‐valine diacid chloride and 5,5‐disubstituted hydantoin derivatives under microwave irradiation

Pyromellitic dianhydride (1,2,4,5‐benzenetetracarboxylic acid 1,2,4,5‐dianhydide) was reacted with L ‐valine in a mixture of acetic acid and pyridine (3:2) at room temperature, and then was refluxed at 90–100 °C, N,N′‐(pyromellitoyl)‐bis‐L ‐valine diacid was obtained in quantitative yield. The imide–acid was converted to N,N′‐(pyromellitoyl)‐bis‐L ‐valine diacid chloride by reaction with thionyl chloride. Rapid and highly efficient synthesis of a number of poly(amide–imide)s was achieved under microwave irradiation using a domestic microwave oven by polycondensation of N,N′‐(pyromellitoyl)‐bis‐L ‐valine diacid chloride with six different derivatives of 5,5‐disubstituted hydantoin compounds in the presence of a small amount of a polar organic medium that acts as a primary microwave absorber. A suitable organic medium was o‐cresol. The polycondensation proceeded rapidly, compared with conventional melt polycondensation and solution polycondensation and was almost completed within 8 min, giving a series of poly(amide–imide)s with inherent viscosities in the range 0.15–0.36 dl g^?1. The resulting poly(amide–imide)s were obtained in high yield and are optically active and thermally stable. All of the above compounds were fully characterized by Fourier‐transform infrared (FT‐IR) spectroscopy, elemental analysis, inherent viscosity (η_inh) measurements, solubility testing and specific rotation measurements. The thermal properties of the poly(amide–imide)s were investigated by using thermogravimetric analysis. Copyright © 2004 Society of Chemical Industry     

Stereocomplex formation between enantiomeric PLA–PEG–PLA triblock copolymers: Characterization and use as protein‐delivery microparticulate carriers

Two enantiomeric triblock ABA copolymers composed of poly(L ‐lactide)–poly(ethylene glycol)–poly(L ‐lactide) (PLLA–PEG–PLLA) and poly(D ‐lactide)–poly(ethylene glycol)–poly(D ‐lactide) (PDLA–PEG–PDLA) were synthesized with two different middle‐block PEG chain lengths by ring‐opening polymerization of L ‐lactide and D ‐lactide in the presence of PEG, respectively. A pair of enantiomeric triblock copolymers were combined to form a stereocomplex by a solvent‐casting method. The triblock copolymers and their stereocomplexes were characterized by ¹H‐ and ¹³C‐NMR spectroscopy and gel permeation chromatography. Their crystalline structures and crystalline melting behaviors were analyzed by the wide‐angle X‐ray diffraction method and differential scanning calorimetry. The stereocomplex formed between a pair of enantiomeric triblock copolymers exhibited a higher crystalline melting temperature with a distinctive 3/1 helical crystalline structure. PLLA–PEG–PLLA and its stereocomplex with PDLA–PEG–PDLA were used to fabricate a series of microspheres encapsulating a model protein drug, bovine serum albumin (BSA). They were prepared by a double‐emulsion solvent‐evaporation method. The morphological aspects of the microspheres were characterized and BSA release profiles from them were investigated. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 75: 1615–1623, 2000     

Synthesis and characterization of novel poly(amide imide)s based on bis(p‐amidobenzoic acid)‐n‐trimellitylimido‐L‐leucine

N‐Trimellitylimido‐L ‐leucine was reacted with thionyl chloride, and N‐trimellitylimido‐L ‐leucine diacid chloride was obtained in a quantitative yield. The reaction of this diacid chloride with p‐aminobenzoic acid was performed in dry tetrahydrofuran, and bis(p‐amidobenzoic acid)‐N‐trimellitylimido‐L ‐leucine (5) was obtained as a novel optically active aromatic imide–amide diacid monomer in a high yield. The direct polycondensation reaction of the monomer imide–amide diacid 5 with 4,4′‐diaminodiphenylsulfone, 4,4′‐diaminodiphenylether, 1,4‐phenylenediamine, 1,3‐phenylenediamine, 2,4‐diaminotoluene, and benzidine (4,4′‐diaminobiphenyl) was carried out in a medium consisting of triphenyl phosphite, N‐methyl‐2‐pyrolidone, pyridine, and calcium chloride. The resulting novel poly(amide imide)s (PAIs), with inherent viscosities of 0.22–0.52 dL g^?1, were obtained in high yields, were optically active, and had moderate thermal stability. All of the compounds were fully characterized with IR spectroscopy, elemental analyses, and specific rotation. Some structural characterization and physical properties of these new optically active PAIs are reported. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 84: 35–43, 2002; DOI 10.1002/app.10181     

Remarkable Activation of an Enzyme by (R)‐Pyrrolidine‐ Substituted Imidazolium Alkyl PEG Sulfate

The chiral pyrrolidine‐substituted imidazolium cetyl‐PEG10‐sulfate (D ‐ProMe) derived from D ‐proline worked as an excellent activating agent of Burkholderia cepacia lipase; it is particularly interesting that the D ‐isomer of the imidazolium salt worked better than the L ‐isomer. This suggests that the imidazolium cation group directly interacts with the enzyme protein and causes preferable modification of the reactivity.