Immobilized Baliospermum montanum hydroxynitrile lyase catalyzed synthesis of chiral cyanohydrins

Hydroxynitrile lyase (HNL) catalyzed enantioselective CC bond formation is an efficient approach to synthesize chiral cyanohydrins which are important building blocks in the synthesis of a number of fine chemicals, agrochemicals and pharmaceuticals. Immobilization of HNL is known to provide robustness, reusability and in some cases also enhances activity and selectivity.We optimized the preparation of immobilization of Baliospermium montanum HNL (BmHNL) by cross linking enzyme aggregate (CLEA) method and characterized it by SEM. Optimization of biocatalytic parameters was performed to obtain highest % conversion and ee of (S)-mandelonitrile from benzaldehyde using CLEA-BmHNL. The optimized reaction parameters were: 20 min of reaction time, 7 U of CLEA-BmHNL, 1.2 mM substrate, and 300 mM citrate buffer pH 4.2, that synthesized (S)-mandelonitrile in ∼99% ee and ∼60% conversion. Addition of organic solvent in CLEA-BmHNL biocatalysis did not improve in % ee or conversion of product unlike other CLEA-HNLs. CLEA-BmHNL could be successfully reused for eight consecutive cycles without loss of conversion or product formation and five cycles with a little loss in enantioselectivity. Eleven different chiral cyanohydrins were synthesized under optimal biocatalytic conditions in up to 99% ee and 59% conversion, however the % conversion and ee varied for different products. CLEA-BmHNL has improved the enantioselectivity of (S)-mandelonitrile synthesis compared to the use of purified BmHNL. Nine aldehydes not tested earlier with BmHNL were converted into their corresponding (S)-cyanohydrins for the first time using CLEA-BmHNL. Among the eleven (S)-cyanohydrins syntheses reported here, eight of them have not been synthesized by any CLEA-HNL. Overall, this study showed preparation, characterization of a stable, robust and recyclable biocatalyst i.e. CLEA-BmHNL and its biocatalytic application in the synthesis of different (S)-aromatic cyanohydrins.     

Structural and functional analysis of hydroxynitrile lyase from Baliospermum montanum with crystal structure,molecular dynamics and enzyme kinetics

Hydroxynitrile lyases (HNLs) catalyze degradation of cyanohydrins to hydrogen cyanide and the corresponding ketone or aldehyde. HNLs can also catalyze the reverse reaction, i.e., synthesis of cyanohydrins. Although several crystal structures of S-selective hydroxynitrile lyases (S-HNLs) have been reported, it remains unknown whether and how dynamics at the active site of S-HNLs influence their broad substrate specificity and affinity. In this study, we analyzed the structure, dynamics and function of S-HNL from Baliospermum montanum (BmHNL), which has an α/β hydrolase fold. Two crystal structures of BmHNL, apo1 and apo2, were determined at 2.55 and 1.9 Å, respectively. Structural comparison between BmHNL (apo2) and S-HNL from Hevea brasiliensis with (S)-mandelonitrile bound to the active site revealed that hydrophobic residues at the entrance region of BmHNL formed hydrophobic interactions with the benzene ring of the substrate. The flexible structures of these hydrophobic residues were confirmed by a 15 ns molecular dynamics simulation. This flexibility regulated the size of the active site cavity, enabling binding of various substrates to BmHNL. The high affinity of BmHNL toward substrates containing a benzene ring was also confirmed by comparing the kinetics of BmHNL and S-HNL from Manihot esculenta. Taken together, the results indicated that the flexibility and placement of the residues are important for the broad substrate specificity of S-HNLs.     

Effect of organic solvents on a chloroperoxidase biotransformation

The effect of organic solvents on the chlorination activity of chloroperoxidase (CPO) was identified for use in biotransformations with CPO. CPO was found to chlorinate monochlorodimedon (MCD) in the presence of organic solvents with log P values less than 0. The relative rates of chlorination with chloride ion in the presence of H₂O₂, buffer and 2.5–20% of either dimethyl sulfoxide, N,N-dimethyl formamide, methanol or acetonitrile, were in the range of 10–58% of that in buffer (pH 2.8) at the same reactant concentrations. The presence of such organic solvents was found to alter CPO catalysis by altering the protein conformation and the local environment at the active site. CPO did not display chlorination activity in the presence of organic solvents which had log P values greater than 0.     

Mass transfer limitation as a tool to enhance the enantiomeric excess in the enzymatic synthesis of chiral cyanohydrins

The enantioselective synthesis of cyanohydrins catalyzed by R-hydroxynitrile lyase in an aqueous-organic liquid two-phase system using, mass transfer limitation to enhance enantiomeric excess at 5°C and pH 5.5 is described. Benzaldehyde, a good substrate, and cinnamaldehyde, a notoriously difficult substrate, were used as model substrates and compared in order to establish the mass transfer limitation concept in a two-liquid phase system, where the non-enzymatic-racemic reaction competes. Enzyme concentration and phase volume ratio between organic and buffer phase were geared to one another to enhance the enantiomeric excess for each substrate. In both cases, after optimization, excellent chemical conversion (>99% on a 60 mmol scale), high throughput and high enantiomeric excess (benzaldehyde >99% and cinnamaldehyde >96%) were achieved.     

Hydroxynitrile Lyases from Prunus Seeds in the Preparation of Cyanohydrins

The hydroxynitrile lyase (HNL) activity of nine defatted Prunus seeds was compared for catalyzing the addition of HCN to aromatic, heteroaromatic and α,β-unsaturated aldehydes. Although the conversion and enantiomeric excess (ee) of the corresponding cyanohydrins were both influenced by the HNL source and the chemical structure of the aldehyde, Prunus HNLs were all suitable for the enantioselective preparation of cyanohydrins.     

Cloning,expression and characterization of an organic solvent tolerant lipase from Pseudomonas fluorescens JCM5963

A novel lipase gene from an organic solvent degradable strain Pseudomonas fluorescens JCM5963 was cloned, sequenced, and overexpressed as an N-terminus His-tag fusion protein in E. coli. The alignment of amino acid sequences revealed that the protein contained a lipase motif and shared a medium or high similarity with lipases from other Pseudomonas strains. It could be defined as a member of subfamily I.1 lipase. Most of the recombinant proteins expressed as enzymatically active aggregates soluble in 20 mM Tris–HCl buffer (pH 8.0) containing sodium deoxycholate are remarkably different from most subfamily I.1 and I.2 members of Pseudomonas lipases expressed as inactive inclusion body formerly described in E. coli. The recombinant lipase (rPFL) was purified to homogeneity by Ni-NTA affinity chromatography and Sephacryl S-200 gel filtration chromatography. The purified lipase was stable in broad ranges of temperatures and pH values, with the optimal temperature and pH value being 55 °C and 9.0, respectively. Its activity was found to increase in the presence of metal ions such as Ca²⁺, Sn²⁺ and some non-ionic surfactants. In addition, rPFL was activated by and remained stable in a series of water-miscible organic solvents solutions and highly tolerant to some water-immiscible organic solvents. These features render this novel lipase attraction for biotechnological applications in the field of organic synthesis and detergent additives.     

Optimization of defined medium for recombinant Komagataella phaffii expressing cyclodextrin glycosyltransferase

The cyclodextrin glycosyltransferase (CGTase) is an important enzyme for cyclodextrin (CD) production, and is also widely used in the biotechnology, food, and pharmaceuticals industries. Secretory CGTase production by recombinant Komagataella phaffii using defined medium is a promising approach because of low cost, less impurity protein. It was found that no CGTase was expressed using traditional defined medium (basal salt medium [BSM]) because of pH value decreasing significantly. CGTase was expressed by recombinant K. phaffii through pH maintenance in range of 5.5–7.0. β-CGTase activity increased to 122.0 U/mL after optimization of glycerol, phosphate buffer, pH value, ammonium sulfate, temperature, methanol, and additives based on BSM, establishing a modified defined medium. These results showed that it was necessary to establish recombinant K. phaffii-based special defined medium although the same host cell used for different heterologous protein expression.     

Effects of water-miscible solvents and polyhydroxy compounds on the structure and enzymatic activity of thermolysin

The effect of organic solvents (n-propanol, isopropanol, dimethylformamide and dimethylsulfoxide) on the structure, activity and stability of thermolysin was the focus of this investigation. Results show the ability of the solvents to cause mixed inhibition of thermolysin, which was indicated by kinetic and structural studies (near-UV CD spectra and intrinsic fluorescence). Inhibitory effect of the solvents increased with increments in solvents logP. Thermoinactivation of thermolysin was studied at 80 degrees C in 50% of solvents and showed that with the increase in solvent hydrophobicity, thermal stability of the enzyme decreased. For the stabilization of thermolysin at high temperature, additives such as glycerol, sorbitol and trehalose were employed. In the presence of DMF with a relatively low logP, trehalose was shown to be a good stabilizer, whereas glycerol had a marked stabilization effect in the presence of n-propanol and isopropanol with a relatively high logP. Consequently, it was concluded that the stabilizing effect of additives can be correlated with the logP of solvents.     

Fusion of a Flavin-Based Fluorescent Protein to Hydroxynitrile Lyase from Arabidopsis thaliana Improves Enzyme Stability

Hydroxynitrile lyase from Arabidopsis thaliana (AtHNL) was fused to different fluorescent reporter proteins. Whereas all fusion constructs retained enzymatic activity and fluorescence in vivo and in vitro, significant differences in activity and pH stability were observed. In particular, flavin-based fluorescent reporter (FbFP) fusions showed almost 2 orders of magnitude-increased half-lives in the weakly acidic pH range compared to findings for the wild-type enzyme. Analysis of the quaternary structure of the respective FbFP-AtHNL fusion proteins suggested that this increased stability is apparently caused by oligomerization mediated via the FbFP tag. Moreover, the increased stability of the fusion proteins enabled the efficient synthesis of (R)-mandelonitrile in an aqueous-organic two-phase system at a pH of <5. Remarkably, (R)-mandelonitrile synthesis is not possible using wild-type AtHNL under the same conditions due to the inherent instability of this enzyme below pH 5. The fusion strategy presented here reveals a surprising means for the stabilization of enzymes and stresses the importance of a thorough in vitro characterization of in vivo-employed fluorescent fusion proteins.     

Effect of organic solvents on the structure and activity of moderately halophilic Bacillus sp. EMB9 protease

Halophilic enzymes have been manifested for their stability and catalytic abilities under harsh operational conditions. These have been documented to withstand denaturation in presence of high temperature, pH, presence of organic solvents and chaotropic agents. The present study aims at understanding the stability and activity of a halophilic Bacillus sp. EMB9 protease in organic solvents. The protease was uniquely stable in polar solvents. A clear correlation was evident between the protease function and conformational transitions, validated by CD and fluorescence spectral studies. The study affirms that preservation of protein structure, possibly due to charge screening of the protein surface by Ca²⁺ and Na⁺ ions provides stability against organic solvents and averts denaturation. Salt was also found to exert a protective effect on dialyzed protease against chaotropism of solvents. Presence of 1 % (w/v) NaCl restored the activity in the dialyzed protease and prevented denaturation in methanol, toluene and n-decane. The work will have further implication on discerning protein folding in saline as well as non-aqueous environments.     

Glycerol, α-glycerophosphate and other compounds as stabilizers of alcohol dehydrogenase from yeast

The storage stability of alcohol dehydrogenase from yeast has been considerably improved by the use of additives. Glycerol is an effective cryoprotectant at ?196 and ?20°C. At 4°C, glycerol and dl-α-glycerophosphate are stabilizers, while at 30°C dl-α-glycerophosphate, 3-phosphoglyceric acid, phosphocreatine, 6-phosphogluconic acid, phosphoarginine, phosphoserine and sucrose are examples of stabilizers. While no single mechanism can be adduced, stabilization by NAD⁺ and 5′-AMP may be attributed to binding at the active centre. Cryoprotection by glycerol is attributed to maintenance of the pH within the range at which the enzyme is stable. dl-α-Glycerophosphate caused a shift of 16°C in the transition temperature of the enzyme, as measured by differential scanning calorimetry.     

Lipases are soluble and active in glycerol carbonate as a novel biosolvent

Glycerol carbonate was synthesized as biosolvent for the development of soluble enzymatic system. The effects of various reaction parameters on activity and stability of lipases were investigated using the transesterification of ethyl butyrate with n-butanol as a model reaction. Enzymatic activity in glycerol carbonate was compared with that in water and in conventional organic solvents with different ionizing and dissociating abilities. The pK_a value of trichloroacetic acid and transesterification activities of Candida antarctica lipase B and Candida rugosa lipase in glycerol carbonate are similar to those in water, indicating that ionizing and dissociating powers are capable of satisfactorily predicting the biocompatibility of organic solvents for soluble enzymatic systems.     

Determination of the stereoselective aspects in in-vitro and in-vivo metabolism of the analgesic meptazinol by high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method to separate meptazinol and its phase I metabolites has been developed using a LiChrosper 100 CN column and a mobile phase of trimethylammoniumacetate buffer (pH 5.5)-acetonitrile-methanol. Quantification of meptazinol and N-desmethylmeptazinol in biological samples was achieved by extraction with organic solvents and chromatographic analysis (detection limit 0.4 and 0.25 μg/ml, respectively). Afterwards the enantiomeric ratio of the two compounds was determined on a Chiral AGP column with a mobile phase of phosphate buffer (pH 7.0)-acetonitrile (α = 1.29 and 1.49, respectively). In-vitro metabolism data after incubation of the racemic compound and the enantiomers with liver supernatant and microsomes of different species are presented. Finally urinary data of two volunteers after oral application of the racemic drug were determined.     

Gloverins of the silkworm Bombyx mori: Structural and binding properties and activities

Gloverins are basic, glycine-rich and heat-stable antibacterial proteins (～14- kDa) in lepidopteran insects with activity against Escherichia coli, Gram-positive bacteria, fungi and a virus. Hyalophora gloveri gloverin adopts a random coil structure in aqueous solution but has α-helical structure in membrane-like environment, and it may interact with the lipid A moiety of lipopolysaccharide (LPS). Manduca sexta gloverin binds to the O-specific antigen and outer core carbohydrate of LPS. In the silkworm Bombyx mori, there are four gloverins with slightly acidic to neutral isoelectric points. In this study, we investigate structural and binding properties and activities of B. mori gloverins (BmGlvs), as well as correlations between structure, binding property and activity. Recombinant BmGlv1-4 were expressed in bacteria and purified. Circular dichroism (CD) spectra showed that all four BmGlvs mainly adopted random coli structure (>50%) in aqueous solution in regardless of pH, but contained α-helical structure in the presence of 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), smooth and rough mutants (Ra, Rc and Re) of LPS and lipid A. Plate ELISA assay showed that BmGlvs at pH 5.0 bound to rough mutants of LPS and lipid A but not to smooth LPS. Antibacterial activity assay showed that positively charged BmGlvs (at pH 5.0) were active against E. coli mutant strains containing rough LPS but inactive against E. coli with smooth LPS. Our results suggest that binding to rough LPS is the prerequisite for the activity of BmGlvs against E. coli.     

Effect of organic solvents on the activity and stability of an extracellular protease secreted by the haloalkaliphilic archaeon <Emphasis Type="Italic">Natrialba magadii</Emphasis>

The effect of various organic solvents on the activity and stability of an extracellular protease produced by the haloalkaliphilic archaeon Natrialba magadii was tested. This protease was active and stable in aqueous-organic solvent mixtures containing 1.5 M NaCl and glycerol, dimethylsulfoxide (DMSO), N,N-dimethyl formamide, propylenglycol, and dioxane. Among the solvents tested, DMSO, propylenglycol, and glycerol were effective in preserving enzyme stability in suboptimal NaCl concentrations. The stabilizing effect of DMSO on this haloalkaliphilic protease was more efficient at pH 8 than at pH 10, suggesting that DMSO may not substitute for salt to allow halophilic proteins to withstand the effect of high pH values. These results show that Nab. magadii extracellular protease is a solvent tolerant enzyme and suggest a potential application of this haloalkaliphilic protease in aqueous-organic solvent biocatalysis.     

Cross-linked crystals of hydroxynitrile lyase as catalyst for the synthesis of optically active cyanohydrins

Purified hydroxynitrile lyase (HNL) from Manihot esculenta was crystallized by the sitting-drop vapour-diffusion method. The bipyramidal crystals formed (10–20 μm) were cross-linked with different amounts of glutaraldehyde and used as biocatalyst for the synthesis of optically active cyanohydrins. The cross-linked crystals were more stable than Celite-immobilized enzymes when incubated in organic solvents, especially in polar solvents. After six consecutive batch reactions in dibutylether, the remaining activity of the cross-linked crystals was more than 70 times higher than for the immobilized enzymes. Nevertheless, the specific activity of the cross-linked crystals (per milligram protein) was reduced compared to the activity of immobilized enzymes. The product enantiopurity was independent of the type of enzyme preparation used.     

Parameters influencing asymmetric synthesis of (R)-mandelonitrile by a novel (R)-hydroxynitrile lyase from Eriobotrya japonica

(R)-Mandelonitrile was successfully synthesized by an enzymatic transcyanation reaction of benzaldehyde and acetone cyanohydrin catalyzed by a hydroxynitrile lyase from Eriobotrya japonica (EjHNL) in an aqueous-organic biphasic system. The effects of pH, temperature, organic solvent, substrate concentration and enzyme concentration on the initial activity and enantioselectivity of the enzyme were studied. Both pH and temperature had a large effect on the initial velocity and enantiomeric excess (e.e.) of the product, (R)-mandelonitrile. High enantiomeric purity of the product was observed at low pH and temperature because the non-enzymatic reaction producing racemates of mandelonitrile was almost suppressed. The optimum pH and temperature to obtain high e.e. were pH 4.0 and 10 °C, respectively. Surprisingly, the organic solvents had a significant influence on the initial velocity of the reaction but less influence on the enantiomeric purity of product. The EjHNL was very stable in ethyl acetate, diethyl ether, methyl-t-butyl ether, diisopropyl ether, dibutyl ether and hexane for 12 h. The best solvent for the highest initial velocity and e.e. was diethyl ether with an optimum aqueous phase content of 50% (v/v). The initial reaction rate increase as the aqueous phase content rose, but when the content was more than 50%, a reduction of e.e. was observed. Increasing the concentration of the substrates accelerated the initial velocity, but caused a slight decrease in the e.e. of the product. Under the optimized conditions, the conversion and e.e. of (R)-mandelonitrile for 3 h were 40 and 99%, respectively. The aqueous phase containing the enzyme also showed considerably efficient reusability for 4 batch reactions.     

Purification, characterization, and molecular cloning of organic-solvent-tolerant cholesterol esterase from cyclohexane-tolerant Burkholderia cepacia strain ST-200

Extracellular cholesterol esterase of Burkholderia cepacia strain ST-200 was purified from the culture supernatant. Its molecular mass was 37 kDa. The enzyme was stable at pH 5.5–12 and active at pH 5.5–6, showing optimal activity at pH 7.0 at 45°C. Relative to the commercially available cholesterol esterases, the purified enzyme was highly stable in the presence of various water-miscible organic solvents. The enzyme preferentially hydrolyzed long-chain fatty acid esters of cholesterol, except for that of cholesteryl palmitate. The enzyme exhibited lipolytic activity toward various p-nitrophenyl esters. The hydrolysis rate of p-nitrophenyl caprylate was enhanced 3.5- to 7.2-fold in the presence of 5–20% (vol/vol) water-miscible organic solvents relative to that in the absence of organic solvents. The structural gene encoding the cholesterol esterase was cloned and sequenced. The primary translation product was predicted to be 365 amino acid residues. The mature product is composed of 325 amino acid residues. The amino acid sequence of the product showed the highest similarity to the lipase LipA (87%) from B. cepacia DSM3959.     

Characterization of two bacterial hydroxynitrile lyases with high similarity to cupin superfamily proteins

Hydroxynitrile lyases (HNLs) catalyze the cleavage of cyanohydrins. In the reverse reaction, they catalyze the formation of carbon-carbon bonds by enantioselective condensation of hydrocyanic acid with carbonyls. In this study, we describe two proteins from endophytic bacteria that display activity in the cleavage and the synthesis reaction of (R)-mandelonitrile with up to 74% conversion of benzaldehyde (enantiopreference ee 89%). Both showed high similarity to proteins of the cupin superfamily which so far were not known to exhibit HNL activity.     

Conversion of Glycerol to Dihydroxyacetone by Immobilized Whole Cells of Acetobacter xylinum

Enzymatic production of dihydroxyacetone (DHA) was studied by immobilization of the whole cells of acetic acid bacteria capable of oxidizing glycerol to DHA. Acetobacter xylinum A-9 cells immobilized in a polyacrylamide gel were selected as the most favorable enzyme preparation. The enzymatic properties of immobilized cells converting glycerol to DHA were investigated and compared with those of intact cells. The optimum pH for the immobilized cells was broad (4.0 to 5.5), whereas the intact cells had a narrow pH optimum at 5.5. The thermal stability of the immobilized cells was somewhat higher than that of the intact cells. Apparent K_m values for glycerol with both intact and immobilized cells were about equal, 6.3 × 10⁻² to 6.5 × 10⁻² M. The complete conversion of glycerol to DHA was achieved within 40 h under optimum conditions, and pure crystalline DHA was readily isolated from the reaction mixture with over 80% yield.