Reverse Hydrolysis Research Articles

Enzymatic Synthesis of Rhamnose Containing Chemicals by Reverse Hydrolysis.

Rhamnose containing chemicals (RCCs) are widely occurred in plants and bacteria and are known to possess important bioactivities. However, few of them were available using the enzymatic synthesis method because of the scarcity of the α-L-rhamnosidases with wide acceptor specificity. In this work, an α-L-rhamnosidase from Alternaria sp. L1 was expressed in Pichia pastroris strain GS115. The recombinant enzyme was purified and used to synthesize novel RCCs through reverse hydrolysis in the presence of rhamnose as donor and mannitol, fructose or esculin as acceptors. The effects of initial substrate concentrations, reaction time, and temperature on RCC yields were investigated in detail when using mannitol as the acceptor. The mannitol derivative achieved a maximal yield of 36.1% by incubation of the enzyme with 0.4 M L-rhamnose and 0.2 M mannitol in pH 6.5 buffers at 55°C for 48 h. In identical conditions except for the initial acceptor concentrations, the maximal yields of fructose and esculin derivatives reached 11.9% and 17.9% respectively. The structures of the three derivatives were identified to be α-L-rhamnopyranosyl-(1→6')-D-mannitol, α-L-rhamnopyranosyl-(1→1')-β-D-fructopyranose, and 6,7-dihydroxycoumarin α-L-rhamnopyranosyl-(1→6')-β-D-glucopyranoside by ESI-MS and NMR spectroscopy. The high glycosylation efficiency as well as the broad acceptor specificity of this enzyme makes it a powerful tool for the synthesis of novel rhamnosyl glycosides.

Abstract 4736: Absolute quantification of BCR-ABL RNA using multiplexed digital RT-PCR

Abstract Reciprocal translocation and fusion of the genes encoding BCR (chromosome 22) and ABL1 (chromosome 9) are typical and causative for approximately 95% of chronic myeloid leukemias (CMLs). Specific and effective drugs targeting the protein products of these gene fusions have been developed and are used in current clinical practice. Molecular diagnostic methods are used for patient identification, for tracking drug effectiveness, and monitoring minimal residual disease (MRD). However, the use of quantitative PCR (qPCR) for this purpose has been problematic, particularly for determination of low limits of detection (LOD) and for comparing results between labs. International standards have been developed to ameliorate these issues, but there are significant ongoing challenges with these efforts. Here we address these challenges through the development of a multiplexed reverse transcription digital PCR (RT-dPCR) assay, and provide a direct comparison between samples run using the International Standardized qPCR and the new multiplexed RT-dPCR method. Digital PCR isolates individual single target molecules into compartments for endpoint PCR, with absolute quantification provided by simply counting the number of ‘PCR-positive’ compartments. Using the RainDrop dPCR system, we partitioned each sample's target RNA molecules into a single-molecule droplet format, together with a ‘one-step’ reverse transcription master mix (SuperScript III Platinum Taq, LifeTech) and standard qPCR hydrolysis probes/primers (IDT), for endpoint PCR and absolute quantification. We used a ‘4-plex’ multiplexed assay that counts 1) the e13:a2 isoform of BCR:ABL; 2) the e14:a2 isoform of BCR-ABL; 3) GUSB RNA as an endogenous counting control, and; 4) a synthetic RNA as an exogenous control. Initial characterization of the multiplexed assay's specificity and sensitivity were performed using RNA from cell lines (K562 for e14:a2; Invivoscribe clonal cell line for e13:a2 and e1:a2; U937 for a negative control). Samples derived from blood were split and quantified using either qPCR together with International Standard Protocols and reagents, or using the 4-plex RT-dPCR assay. Concordance and comparison of the two assay's LODs will be presented. Sensitivity assessment based on control generated data provides evidence of detection as low 1 in 200,000 without hitting a detection limit. The use of dPCR provides significant advantages over standard qPCR methods including absolute quantification which eliminates the need for standard curves, the ability to use RNA directly, the use of an exogenous control (non-ABL) and easy isoform analysis. Citation Format: Maria E. Arcila, Ryma Benayed, Iwona Kiecka, Coren Milbury, Michael Mauro, Michael L. Samuels. Absolute quantification of BCR-ABL RNA using multiplexed digital RT-PCR. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4736. doi:10.1158/1538-7445.AM2015-4736

Synthesis of octyl-β-d-glucopyranoside catalyzed by Thai rosewood β-glucosidase-displaying Pichia pastoris in an aqueous/organic two-phase system

We explored the ability of a Thai rosewood β-glucosidase-displaying P. pastoris whole-cell biocatalyst (Pp-DCBGL) system to synthesize alkyl β-d-glucosides. The primary investigation centered on the synthesis of octyl-β-d-glucopyranoside (octyl-glu, OG). OG could be synthesized through reverse hydrolysis reaction with very low efficiency. Then, OG was synthesized between BG and octanol by a transglycosylation reaction. In a 2-ml reaction system, OG was synthesized with a conversion rate of 51.1% in 3h when 5mg/ml BG was utilized as the glucosyl donor under optimized conditions. And, even after being reused four times, the Pp-DCBGL was relatively stable. Additionally, a 500-ml-scale reaction system was conducted in a 2-L stirred reactor with a conversion rate of 47.5% in 1.5h. Moreover, the conversion rate did not decrease after the whole-cell catalyst was reused two times. In conclusion, Pp-DCBGL has high reaction efficiency and operational stability, which is a powerful biocatalyst available for industrial synthesis.

Solvent Flux Method (SFM): A Case Study of Water Access to Candida antarctica Lipase B.

The solvent flux method (SFM) was developed to comprehensively characterize the influx of solvent molecules from the solvent environment into the active site of a protein in the framework of molecular dynamics simulations. This was achieved by introducing a solvent concentration gradient as well as partially reorienting and rescaling the velocity vector of all solvent molecules contained within a spherical volume enclosing the protein, thus inducing an accelerated solvent influx toward the active site. In addition to the detection of solvent access pathway within the protein structure, it is hereby possible to identify potential amino acid positions relevant to solvent-related enzyme engineering with high statistical significance. The method is particularly aimed at improving the reverse hydrolysis reaction rates in nonaqueous media. Candida antarctica lipase B (CALB) binds to a triglyceride-water interface with its substrate entrance channel oriented toward the hydrophobic substrate interface. The lipase-triglyceride-water system served as a model system for SFM to evaluate the influx of water molecules to the active site. As a proof of principle for SFM, a previously known water access pathway in CALB was identified as the primary water channel. In addition, a secondary water channel and two pathways for water access which contribute to water leakage between the protein and the triglyceride-water interface were identified.

Enzymatically triggered peptide hydrogels for 3D cell encapsulation and culture.

We have investigated the possibility of using enzymatically triggered peptide hydrogels for the encapsulation and culture of cells. Based on recent work done on the enzymatically triggered gelation of FEFK (F, phenylalanine; E, glutamic acid; K, lysine) using thermolysin, a protease enzyme from Bacillus Thermoproteolyticus Rokko, we have investigated the possibility of using this gelation triggering mechanism to encapsulate cells within a 3D hydrogel matrix. First, the properties of enzymatically triggered hydrogels prepared in phosphate buffer solution were investigated and compared with the properties of hydrogels prepared in HPLC grade water from our previous work. We showed that the use of phosphate buffer solution allowed the production of hydrogels with very high shear moduli (>1 MPa). The gelation kinetics was also investigated, and the mechanical properties of the system were shown to closely follow the synthesis of the octapeptide by the enzyme through reverse hydrolysis. In a second phase, we developed, on the basis of information acquired, a facile protocol for the encapsulation of cells and plating of the hydrogel. Human dermal fibroblasts were then used to exemplify the use of these materials. FEFEFKFK octapeptide hydrogels prepared under the same conditions and with the same mechanical properties were used as a control. We showed that no significant differences were observed between the two systems and that after a decrease in cell number on day 1, cells start to proliferate. After 5 days of culture, the cells can be seen to start to adopt a stretched morphology typical of fibroblasts. The results clearly show that the protocol developed minimises the potential detrimental effect that thermolysin can have on the cells and that these enzymatically triggered hydrogels can be used for the 3D encapsulation and culture of cells.

Strategies for Stabilization of Enzymes in Organic Solvents

One of the major barriers to the use of enzymes in industrial biotechnology is their insufficient stability under processing conditions. The use of organic solvent systems instead of aqueous media for enzymatic reactions offers numerous advantages, such as increased solubility of hydrophobic substrates or suppression of water-dependent side reactions. For example, reverse hydrolysis reactions that form esters from acids and alcohols become thermodynamically favorable. However, organic solvents often inactivate enzymes. Industry and academia have devoted considerable effort into developing effective strategies to enhance the lifetime of enzymes in the presence of organic solvents. The strategies can be grouped into three main categories: (i) isolation of novel enzymes functioning under extreme conditions, (ii) modification of enzyme structures to increase their resistance toward nonconventional media, and (iii) modification of the solvent environment to decrease its denaturing effect on enzymes. Here we discuss successful examples representing each of these categories and summarize their advantages and disadvantages. Finally, we highlight some potential future research directions in the field, such as investigation of novel nanomaterials for immobilization, wider application of computational tools for semirational prediction of stabilizing mutations, knowledge-driven modification of key structural elements learned from successfully engineered proteins, and replacement of volatile organic solvents by ionic liquids and deep eutectic solvents.

Synthesis and characterisation of galactosyl glycerol by β-galactosidase catalysed reverse hydrolysis of galactose and glycerol

A study of an enzymatic method for the production of galactosylglycerol is described. The effects of enzyme sources, enzyme amount, reaction temperature, reaction time, substrate ratio of glycerol to galactose, buffer content and pH on galactosylglycerol yield (mg/ml) were investigated. Under the optimum reaction conditions of β-galactosidases from Kluyveromyces lactis 240 U/ml, temperature 40°C, time 24h, buffer amount 60% (percent volume of buffer to that of substrates, pH 6.5), and the substrate molar ratio of 10 (glycerol16mmol:galactose 1.6mmol), the yield of galactosylglycerol was up to 116.47mg/ml (galactose conversion 55.88%). The product was purified by activated charcoal and Sephadex G-15 column chromatography, up to 96%. The purified galactosylglycerol was fully characterised by MS and NMR, and identified as a mixture of (2R)- and (2S)- 3-O-β-D-galactopyranosyl-glycerol.

Effect of Enzyme Concentration of the Morphology and Properties of Enzymatically Triggered Peptide Hydrogels

We have recently shown that thermolysine, a protease enzyme obtained from Bacillus thermoproteolyticus rokko , can be used to trigger the gelation of FEFK (F, phenylalanine; E, glutamic acid; K, lysine) tetrapeptides through reverse hydrolysis and formation of longer peptide sequences, mainly octapeptides, that self-assemble readily. In this article we investigate the effect of enzyme concentration on the morphology and properties of enzymatically triggered peptide hydrogels using HPLC, FTIR, real-time SAXS, TEM, and shear rheology. We have shown that the enzyme concentration, Cenz, does not affect the final composition of the samples. Instead, this is dictated by the initial tetrapeptide concentration, C0, suggesting the existence of a chemical equilibrium. We went on to show that Cenz does not affect the self-assembly of these peptides at a molecular level either nor the structure of the fibrillar network formed at the nanometer scale. Interestingly, the mechanical properties were found to be affected by Cenz, where the shear moduli of the hydrogels were found to increase with increasing Cenz. These results suggest that morphological differences between the hydrogels at the microscale are at the origin of their difference in mechanical properties. In this paper, we propose a morphological model in which denser network regions are found around the enzymes, resulting in the creation of heterogeneous networks. These were confirmed by TEM measurements. The existence of these denser network regions will result in the reinforcement of the hydrogels, thus, explaining the high shear moduli obtained increasing Cenz.

Raffinose production using α-galactosidase from Paraphaeosphaeria sp.

An α-galactosidase for raffinose synthesis by a reverse hydrolysis reaction was searched from intracellular of endophytic fungi. About 500 strains were screened, one strain was selected and identified as Paraphaeosphaeria sp. Partially purified α-galactosidase (1 U/ml in the reaction mixture), 100 g/L galactose, and 500 g/L sucrose at 60 °C for 48 h resulted in synthesis of 13.3 g/L raffinose and 4.6 g/L planteose at a ratio of about 3:1. The data suggest that α-galactosidase was able to synthesize raffinose at a ratio higher than that of α-galactosidases derived from other fungi.

Production of 6-kestose by the filamentous fungus Gliocladium virens as affected by sucrose concentration

The filamentous fungus Gliocladium virens is able to produce fructooligosaccharides (FOS), fructose-containing sugars, used as functional ingredients to improve nutritional and technological properties of foods. In this work we evaluated FOS production by G. virens when grown in a wide range of sucrose concentrations (10–400 g l−1). High sucrose concentrations increased both biomass and FOS production, including 6-kestose, a trisaccharide comprising β (2 → 6) linked fructosyl units, with enhanced stability and prebiotic activity when compared to the typical FOS β (2 → 1) linked. The highest 6-kestose yield (3 g l−1) was achieved in media containing 150 g l−1 sucrose after 4–5 days of culture, production being 90% greater than in media containing 10, 30, or 50 g l−1 sucrose. After 5 days, FOS production declined markedly, following complete sucrose depletion in the medium. Although most of the β-fructofuranosidases preferentially catalyze sucrose hydrolysis, FOS production in G. virens grown in high sucrose concentration, might be attributed to a reverse hydrolysis by these enzymes. In conclusion, high sucrose concentrations increase growth of G. virens whilst 6-kestose accumulation in the medium seems to be controlled both by specific properties of β-fructofuranosidases and on the sucrose concentration.

One-Step Synthesis of Cellulose from Cellobiose via Protic Acid-Assisted Enzymatic Dehydration in Aprotic Organic Media

Direct and efficient enzymatic synthesis of long-chain cellulose from cellobiose in its original form was successfully achieved via the combination of a surfactant-enveloped enzyme (SEE) and a protic acid in an aprotic organic solvent, lithium chloride/N,N-dimethylacetamide system. The SEE biocatalyst was prepared by protecting the surface of cellulase with the nonionic surfactant dioleyl-N-D-glucona-L-glutamate for keeping its enzymatic activity in nonaqueous media. Fourier transform infrared and nuclear magnetic resonance analyses elucidated the successful synthesis of cellulose, β-1,4-linked D-glucopyranose polymer, through the reverse hydrolysis of cellobiose. By using protic acid cocatalysts, a degree of polymerization of as-synthesized cellulose reached more than 120, in a ca. 26% conversion, which was 5 times higher than that obtained in an acid-free SEE system. A novel-concept biocatalysis, i.e., a protic acid-assisted SEE-mediated reaction, enables a facile, one-step chain elongation of carbohydrates without any activation via multistep organic chemistry, and can provide potential applications in the functional design of glycomaterials.

Structural analysis, enzymatic characterization, and catalytic mechanisms of β‐galactosidase from Bacillus circulans sp. alkalophilus

Crystal structures of native and α-D-galactose-bound Bacillus circulans sp. alkalophilus β-galactosidase (Bca-β-gal) were determined at 2.40 and 2.25 Å resolutions, respectively. Bca-β-gal is a member of family 42 of glycoside hydrolases, and forms a 460 kDa hexameric structure in crystal. The protein consists of three domains, of which the catalytic domain has an (α/β)(8) barrel structure with a cluster of sulfur-rich residues inside the β-barrel. The shape of the active site is clearly more open compared to the only homologous structure available in the Protein Data Bank. This is due to the number of large differences in the loops that connect the C-terminal ends of the β-strands to the N-terminal ends of the α-helices within the (α/β)(8) barrel. The complex structure shows that galactose binds to the active site as an α-anomer and induces clear conformational changes in the active site. The implications of α-D-galactose binding with respect to the catalytic mechanism are discussed. In addition, we suggest that β-galactosidases mainly utilize a reverse hydrolysis mechanism for synthesis of galacto-oligosaccharides.

Significantly Improved Equilibrium Yield of Long-Chain Alkyl Glucosides via Reverse Hydrolysis in a Water-Poor System Using Cross-Linked Almond Meal as a Cheap and Robust Biocatalyst

An array of ten β-D-glucopyranosides with varied alkyl chain lengths were enzymatically synthesized. It was found that for longer alkyl chains a lower initial rate and final yield of glucoside was obtained except for methyl glucoside because of the severe toxicity of methanol to the enzyme. From a thermodynamics point of view, the equilibrium constant and Gibbs free energy variation of the glucoside syntheses were systematically investigated. To improve the final yields of the glucosides containing long alkyl chains the equilibrium of the enzymatic glucoside synthesis was altered. The equilibrium yield of decyl β-D-glucoside increased from 1.9% to 6.1% when the water content was reduced from 10% to 5% (v/v) using tert -butanol as a cosolvent and 0.10 mol/L of glucose as a substrate. As for the other longer alkyl chain glucosides, heptyl β-D-glucoside was found to have significant surface activity as well. 使用酶法合成了一系列不同烷基链长的 β -D-烷基葡萄糖苷. 结果表明, 除甲基糖苷外, 烷基碳链越长, 反应初速度越低, 糖苷的最终收率越低. 从热力学角度系统地研究了这些糖苷合成的平衡常数和吉布斯自由能变化. 通过改变反应的平衡, 提高了长链烷基糖苷的最终收率. 当水含量从 10% 降至 5% (v/v), 使用叔丁醇作为共溶剂, 添加 0.1 mol/L 葡萄糖作为底物时, 癸基糖苷的平衡得率从 1.9% 提高至 6.1%. 除了其他长链的烷基糖苷外, 庚基糖苷也显示了明显的表面活性剂活性. Fatty alcohols with varied chain length were enzymatically glucosylated by almond meal in a water-poor system giving β -D-glucopyranosides with significant surface activity.

A novel two-step synthesis of α-linked mannobioses based on an acid-assisted reverse hydrolysis reaction

Instead of an enzyme-assisted reverse hydrolysis reaction for the synthesis of manno-oligosaccharides, we propose here a versatile new approach. By Fischer type glycosylation, a d-mannose solution of extremely high concentration (approximately 83% (w/w)) was incubated at 60 °C for 65 h in 0.5 M HCl. After dilution and neutralization, the small amount of formed β-linked oligosaccharides was hydrolyzed by β-mannosidase. The yields of α- d-Man p-(1→2)- d-Man p (7.9%), α- d-Man p-(1→3)- d-Man p (7.9%), and α- d-Man p-(1→6)- d-Man p (29.1%) isolated by an activated carbon column chromatography were almost identical to those of the enzymatic reaction, but the yield of α- d-Man p-(1→3)- d-Man p increased enormously by the present method.

Characterization of Novel β-Glucosidases with Transglycosylation Properties from Trichosporon asahii

Two novel β-glucosidases from Trichosporon asahii, named BG1 and BG2, were purified to electrophoretic homogeneity using ammonium sulfate precipitation, hydrophobic interaction, ion exchange, and gelfiltration chromatography. The molecular weight of BG1 and BG2 were estimated as 160 kDa and 30 kDa, respectively. The K(m), V(max), K(cat), and K(cat)/K(m) values of the two β-glucosidases for p-nitrophenyl-β-D-glucopyranoside were determined. Both enzymes showed relatively high affinity to p-nitrophenyl-β-D-glucopyranoside in 4-nitrophenol glycosides and gentiobiose in saccharide substrates. The enzymes exhibited optimum activity at pH 6.0 and pH 5.5, respectively. Their respective optimum temperatures were 70 and 50 °C. Metal ions and inhibitors had different effects on the enzymes activities. Circular dichroism (CD) spectroscopy demonstrated that the purified BG1 exhibited a β-sheet-rich structure and that BG2 displayed a high random coil conformation. HPLC analysis of transglycosylation and reverse hydrolysis assays revealed that only BG1 possessed transglycosylation activity and synthesized cello-oligosaccharides by the addition of glucose. This suggested that BG1 could be used to produce complex bioactive glycosides and could be considered as a potential enzyme for industrial application.

Using ionic liquid cosolvents to improve enzymatic synthesis of arylalkyl β-d-glucopyranosides

► Ethylene glycol diacetate, a green and nontoxic organic solvent, proved to be the best for enzymatic synthesis of glycosides. ► The ionic liquid [BMIm]I was applied as the solvent in biocatalysis for the first time. ► The yields were enhanced between 0.2-fold and 0.5-fold by adding 10% (v/v) [BMIm]I. Enzymatic synthesis of various arylalkyl β- d -glucopyranosides catalyzed by prune ( Prunus domestica ) seed meal via reverse hydrolysis in the mixture of organic solvent, ionic liquid (IL) and phosphate buffer was described. Among four hydrophilic organic solvents tested, ethylene glycol diacetate (EGDA) was found to be the most suitable for enzymatic synthesis of salidroside, a bioactive compound of commercial interest, from d -glucose and tyrosol. The effects of the nature of ionic liquids and their contents on the enzymatic glucosylation were studied. The addition of a suitable amount of ILs including denaturing ones was favorable to shift the reaction equilibrium toward the synthesis, thus improving the yields. Among the examined ILs, the novel IL [BMIm]I proved to be the best. And this IL was applied as the solvent in biocatalysis for the first time. The yields were found to be enhanced between 0.2-fold and 0.5-fold after the addition of 10% (v/v) [BMIm]I. In 10% (v/v) [BMIm]I-containing system, the desired arylalkyl β- d -glucopyranosides were synthesized with 15–28% yields, among which salidroside was obtained with a yield of 22%.

Preparation and enzymatic behavior of surfactant-enveloped enzymes for glycosynthesis in nonaqueous aprotic media

Abstract Surfactant-enveloped enzymes (SEEs) were prepared from pure cellulases, cellobiohydrolase I and endoglucanase I (Cel7A and Cel7B, respectively), via simply freeze-drying water-in-oil emulsions, wherein the aqueous phase containing each cellulase was stabilized with the nonionic surfactant, dioleyl- N - d -glucona- l -glutamate. The enzymatic tolerance of SEEs to various nonaqueous solvents was investigated, aiming at a novel synthetic approach in biocatalytic glycoengineering. SEE-Cel7A preserved ca . 67% of the original activity after 3 h incubation in lithium chloride (LiCl)/dimethylacetamide (DMAc) that is a good solvent for carbohydrates but completely deactivates intact enzymes. This excellent enzymatic durability depended on the preparation conditions of SEEs, e.g. pH and salt species of the aqueous phase during SEE preparation. SEE-Cel7A or SEE-Cel7B was applied as a biocatalyst to synthesize cellulose, a sugar polymer which is insoluble in common solvents but dissolves in LiCl/DMAc. Both SEEs could catalyze the direct dehydration of cellobiose without any activation of the anomeric carbon, a property that is indispensable for conventional chemo-enzymatic synthesis. The SEE-Cel7A provided short-chain cellulose with the degree of polymerization (DP) ca . 20, and longer-chain cellulose with DP ca . 60 was preferentially obtained by the SEE-Cel7B, possibly through preferential reverse hydrolysis instead of inherent hydrolysis. Nonaqueous SEE-mediated biocatalysis using inexpensive glycohydrolases and sugars that do not need to be chemically modified beforehand would have potentially wide applications in glycoengineering.

ChemInform Abstract: Enzymatic Synthesis of Phosphoric Monoesters with Alkaline Phosphatase in Reverse Hydrolysis Conditions.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

ChemInform Abstract: A New Peptidase Enzyme Useful for Peptide and Amino Acid Derivative Synthesis by Reverse Hydrolysis Reactions.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

ChemInform Abstract: Reverse Hydrolysis Reaction of Chitin Deacetylase and Enzymatic Synthesis of β-D-GlcNAc-(1→4)-GlcN from Chitobiose.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.