Simulation-guided enzyme discovery: A new microbial source of cellobiose 2-epimerase.

Cellobiose 2-epimerase (CE) is a promising industrial enzyme that can be utilized in the dairy industry. More thermostable CEs from different microorganisms are still needed for a higher lactulose productivity. This study demonstrated the feasibility to use molecular dynamics (MD) simulation as the preliminary computational filter for thermostable enzymes screening. Sequence information of eleven uncharacterized CEs were chosen to be analyzed by MD simulations. The CE from Dictyoglomus thermophilum (Dith-CE) was determined experimentally to be one of the most thermostable CEs with the highest epimerization (160 ±â€¯6.5 U mg-1) and isomerization activities (3.52 ±â€¯0.23 U mg-1) among all the reported CEs. This enzyme shows the highest isomerization activity at 85 °C and pH 7.0. The kinetic parameters (kcat and Km) of isomerization activity of this CE are 3.98 ±â€¯0.3 s-1 and 235.2 ±â€¯11.2 mM, respectively. These results suggest that the CE from Dith-CE is a promising lactulose-producing enzyme.

Continuous enzymatic synthesis of lactulose in packed-bed reactor with immobilized Aspergillus oryzae ß-galactosidase.

Lactulose synthesis from fructose and lactose in continuous packed-bed reactor operation with glyoxyl-agarose immobilized Aspergillus oryzae ß-galactosidase is reported for the first time. Alternative strategies to conventional batch synthesis have been scarcely explored for lactulose synthesis. The effect of flow rate, substrates ratio and biocatalyst-inert packing material mass ratio (MB/MIM) were studied on reactor performance. Increase in any of these variables produced an increase in lactulose yield (YLu) being higher than obtained in batch synthesis at comparable conditions. Maximum YLu of 0.6 g·g-1 was obtained at 50 °C, pH 4.5, 50% w/w total sugars, 15 mL·min-1, fructose/lactose molar ratio of 12 and MB/MIM of 1/8 g·g-1; at such conditions yield of transgalactosylated oligosaccharides (YTOS) was 0.16 g·g-1, selectivity (lactulose/TOS molar ratio) was 5.4 and lactose conversion (XLactose) was 28%. Reactor operation with recycle had no significant effect on yield, producing only some decrease in productivity.

Chemoenzymatic synthesis of sialylated lactuloses and their inhibitory effects on Staphylococcus aureus.

BACKGROUND: Sialylated glycoconjugates play important roles in physiological and pathological processes. However, available sialylated oligosaccharides source is limited which is a barrier to study their biological roles. This work reports an efficient approach to produce sialic acid-modified lactuloses and investigates their inhibitory effects on Staphylococcus aureus (S. aureus). METHODS: A one-pot two-enzyme (OPTE) sialylation system was used to efficiently synthesize sialylated lactuloses. Silica gel flash chromatography column was employed to purify the sialylated products. The purity and identity of the product structures were confirmed with mass spectrometry (MS) and nuclear magnetic resonance (NMR). The inhibitory effect of sialylated lactuloses against S. aureus was evaluated by using microplate assay, fluorescence microscopy, DAPI (4',6-diamidino-2-phenylindole) fluorescence staining and protein leakage quantification. RESULTS: Neu5Ac-containing sialylated lactuloses with either α2,3- or α2,6-linkages were efficiently synthesized via an efficient OPTE sialylation system using α-2,3-sialyltransferase or α-2,6-sialyltransferase, respectively. Neu5Ac-α2,3-lactulose and Neu5Ac-α2,6-lactulose significantly inhibited the growth of S. aureus. Fluorescence microscopy and DAPI fluorescence staining indicated that the sialylated lactuloses might disrupt nucleic acid synthesis of S. aureus. CONCLUSIONS: Neu5Ac-containing sialylated lactuloses had higher antibacterial activity against S. aureus than non-sialylated lactulose. The inhibitory effect of Neu5Ac-α2,3-lactulose was superior to that of Neu5Ac-α2,6-lactulose. The sialylated lactuloses might inhibit S. aureus by causing cell membrane leakage and disrupting nucleic acid synthesis.

Rational modification of substrate binding site by structure-based engineering of a cellobiose 2-epimerase in Caldicellulosiruptor saccharolyticus.

BACKGROUND: Lactulose, a synthetic disaccharide, has received increasing interest due to its role as a prebiotic, specifically proliferating Bifidobacilli and Lactobacilli and enhancing absorption of calcium and magnesium. The use of cellobiose 2-epimerase (CE) is considered an interesting alternative for industrial production of lactulose. CE reversibly converts D-glucose residues into D-mannose residues at the reducing end of unmodified ß-1,4-linked oligosaccharides, including ß-1,4-mannobiose, cellobiose, and lactose. Recently, a few CE 3D structure were reported, revealing mechanistic details. Using this information, we redesigned the substrate binding site of CE to extend its activity from epimerization to isomerization. RESULTS: Using superimposition with 3 known CE structure models, we identified 2 residues (Tyr114, Asn184) that appeared to play an important role in binding epilactose. We modified these residues, which interact with C2 of the mannose moiety, to prevent epimerization to epilactose. We found a Y114E mutation led to increased release of a by-product, lactulose, at 65 °C, while its activity was low at 37 °C. Notably, this phenomenon was observed only at high temperature and more reliably when the substrate was increased. Using Y114E, isomerization of lactose to lactulose was investigated under optimized conditions, resulting in 86.9 g/l of lactulose and 4.6 g/l of epilactose for 2 h when 200 g/l of lactose was used. CONCLUSION: These results showed that the Y114E mutation increased isomerization of lactose, while decreasing the epimerization of lactose. Thus, a subtle modification of the active site pocket could extend its native activity from epimerization to isomerization without significantly impairing substrate binding. While additional studies are required to scale this to an industrial process, we demonstrated the potential of engineering this enzyme based on structural analysis.

Immobilization of Aspergillus oryzae ß-galactosidase in an agarose matrix functionalized by four different methods and application to the synthesis of lactulose.

Aspergillus oryzae ß-galactosidase was immobilized in monofunctional glyoxyl-agarose and heterofunctional supports (amino-glyoxyl, carboxy-glyoxyl and chelate-glyoxyl agarose), for obtaining highly active and stable catalysts for lactulose synthesis. Specific activities of the amino-glyoxyl agarose, carboxy-glyoxyl agarose and chelate-glyoxyl agarose derivatives were 3676, 430 and 454IU/g biocatalyst with half-life values at 50°C of 247, 100 and 100h respectively. Specific activities of 3490, 2559 and 1060IU/g were obtained for fine, standard and macro agarose respectively. High immobilization yield (39.4%) and specific activity of 7700IU/g was obtained with amino-glyoxyl-agarose as support. The highest yields of lactulose synthesis were obtained with monofunctional glyoxyl-agarose. Selectivity of lactulose synthesis was influenced by the support functionalization: glyoxyl-agarose and amino-glyoxyl-agarose derivatives retained the selectivity of the free enzyme, while selectivity with the carboxy-glyoxyl-agarose and chelate-glyoxyl-agarose derivatives was reduced, favoring the synthesis of transgalactosylated oligosaccharides over lactulose.

Synthesis of lactulose in batch and repeated-batch operation with immobilized ß-galactosidase in different agarose functionalized supports.

Lactulose synthesis was done in repeated-batch mode with Aspergillus oryzae ß-galactosidase immobilized in glyoxyl-agarose (GA-ßG), amino-glyoxyl-agarose (Am-GA-ßG) and chelate-glyoxyl-agarose (Che-GA-ßG), at fructose/lactose molar ratios of 4, 12 and 20. Highest yields of lactulose in batch were obtained with Che-GA-ßG (0.21, 0.29 and 0.32g·g-1) for 4, 12 and 20 fructose/lactose molar ratios respectively; when operating in 10 repeated batches highest product to biocatalyst mass ratios were obtained with Am-GA-ßG (1.82, 2.52 and 2.7g·mg-1), while the lowest were obtained with Che-GA-ßG (0.25, 0.33 and 0.39g·mg-1). Operational stability of Am-GA-ßG was higher than GA-ßG and Che-GA-ßG and much higher than that of the free enzyme, at all fructose/lactose molar ratios evaluated. Efficiency of biocatalyst use for GA-ßG were 64.4, 35.5 and 18.4kglactulose/gprotein, for fructose/lactose molar ratios of 4, 12 and 20 respectively, while for Che-GA-ßG were 1.46, 1.05 and 0.96kglactulose/gprotein.

Recent advances on prebiotic lactulose production.

Lactulose, a synthetic disaccharide, has received increasing interest due to its role as a prebiotic. The production of lactulose is important in the dairy industry, as it is regarded as a high value-added derivative of whey or lactose. The industrial production of lactulose is still mainly done by chemical isomerization. Due to concerns on the environmental and tedious separation processes, the enzymatic-based lactulose synthesis has been regarded as an interesting alternative. This work aims at comparing chemical and enzyme-catalyzed lactulose synthesis. With an emphasis on the latter one, this review discusses the influences of the critical operating conditions and the suited operation mode on the transgalactosylation of lactulose using microbial enzymes. As an update and supplement to other previous reviews, this work also summarizes the recent reports that highlighted the enzymatic isomerization of lactose using cellobiose 2-epimerase to produce lactulose at elevated yields.

An Approach for Lactulose Production Using the CotX-Mediated Spore-Displayed ß-Galactosidase as a Biocatalyst.

Currently, enzymatic synthesis of lactulose, a synthetic prebiotic disaccharide, is commonly performed with glycosyl hydrolases. In this work, a new type of lactulose-producing biocatalyst was developed by displaying ß-galactosidase from Bacillus stearothermophilus IAM11001 (Bs-ß-Gal) on the surface of Bacillus subtilis 168 spores. Localization of ß-Gal on the spore surface as a fusion to CotX was verified by western blot analysis, immunofluorescence microscopy, and flow cytometry. The optimum pH and temperature for the resulting spore-displayed ß-Gal was 6.0 and 75°C, respectively. Under optimal conditions, it showed maximum activity of 0.42 U/mg spores (dry weight). Moreover, the spore-displayed CotX-ß-Gal was employed as a whole cell biocatalyst to produce lactulose, yielding 8.8 g/l from 200 g/l lactose and 100 g/l fructose. Reusability tests showed that the spore-displayed CotX-ß-Gal retained around 30.3% of its initial activity after eight successive conversion cycles. These results suggest that the CotX-mediated spore-displayed ß-Gal may provide a promising strategy for lactulose production.

Simultaneous synthesis of mixtures of lactulose and galacto-oligosaccharides and their selective fermentation.

Lactulose and galacto-oligosaccharides (GOS) are well recognized prebiotics derived from lactose. In the synthesis of lactulose with ß-galactosidases GOS are also produced, but the ratio of lactulose and GOS in the product can be tuned at will, depending on the operation conditions, so to obtain an optimal product distribution in terms of prebiotic potential. The selectivity of fermentation of each carbohydrate alone as well as mixtures of both was determined using pH-controlled anaerobic batch cultures with faecal inoculum. Within the experimental range considered, lactulose/GOS molar ratio of 4 resulted in the highest selectivity for Bifidobacterium and Lactobacillus/Enterococcus, so this ratio was selected as the target for the synthesis of lactulose from fructose and lactose with Aspergillus oryzae ß-galactosidase. Synthesis was optimized using response surface methodology, considering temperature, initial concentrations of acceptor sugars and fructose/lactose molar ratio as key variables, with the aim of maximizing lactulose yield at the optimal product distribution in terms of prebiotic potential (lactulose/GOS molar ratio of 4). Under optimal conditions (50°C, 50%w/w total initial concentrations of sugars and fructose/lactose molar ratio of 6.44), lactulose yield of 0.26g of lactulose produced per g of initial lactose was obtained at the optimal product distribution.

Continuous synthesis of lactulose in an enzymatic membrane reactor reduces lactulose secondary hydrolysis.

Newly developed parallel small-scale enzymatic membrane reactors (EMRs) were used to enhance the synthesis of lactulose using ß-galactosidase. Under batch operation, the productivity of lactulose decreased abruptly from 2.72 down to 0.04 mg lactulose/(Uenzymeh) over 35 h of reaction. This was presumably caused by the action of ß-galactosidase which performed secondary hydrolysis upon the produced lactulose. The continuous operations of an EMR system led to continuous removal of lactulose in the reactors restricting lactulose degradation caused by secondary hydrolysis. Therefore, continuous lactulose syntheses in the EMRs yielded significantly higher specific productivities under "steady state" conditions. Approximately 0.70 and 0.50 mg lactulose/(U enzyme h) for hydraulic residence times of 5 and 7h were reached, respectively. Continuous lactulose synthesis performed in an EMR system conclusively can circumvent the drawbacks (e.g., secondary hydrolysis) of lactulose synthesis encountered in batch operation. It is, therefore, beneficial in terms of enhanced lactulose productivity and reduced enzyme consumption.

Heterofunctional hydrophilic-hydrophobic porous silica as support for multipoint covalent immobilization of lipases: application to lactulose palmitate synthesis.

Lipase-catalyzed synthesis of sugar esters, as lactulose palmitate, requires harsh conditions, making it necessary to immobilize the enzyme. Therefore, a study was conducted to evaluate the effect of different chemical surfaces of hierarchical meso-macroporous silica in the immobilization of two lipases from Pseudomonas stutzeri (PsL) and Alcaligenes sp. (AsL), which exhibit esterase activity. Porosity and chemical surface of silica supports, before and after functionalization and after immobilization, were characterized by gas adsorption and Fourier transform infrared (FTIR) spectroscopy. PsL and AsL were immobilized in octyl (OS), glyoxyl (GS), and octyl-glyoxyl silica (OGS). Hydrolytic activity, thermal and solvent stability, and sugar ester synthesis were evaluated with those catalysts. The best support in terms of expressed activity was OS in the case of PsL (100 IU g(-1)), while OS and OGS were the best for AsL with quite similar expressed activities (60 and 58 IU g(-1), respectively). At 60 °C in aqueous media the more stable biocatalysts were GS-PsL and OGS-AsL (half-lives of 566 and 248 h, respectively), showing the advantage of a heterofunctional support in the latter case. Lactulose palmitate synthesis was carried out in acetone medium (with 4% of equilibrium moisture) at 40 °C obtaining palmitic acid conversions higher than 20% for all biocatalysts, being the highest of those obtained with OGS-AsL and OS-PsL. Therefore, screening of different chemical surfaces on porous silica used as supports for lipase immobilization allowed obtaining active and stable biocatalyst to be employed in the novel synthesis of lactulose palmitate.

Expression and characterization of two ß-galactosidases from Klebsiella pneumoniae 285 in Escherichia coli and their application in the enzymatic synthesis of lactulose and 1-lactulose.

The two genes lacZ1 and lacZ2 from Klebsiella pneumoniae 285, encoding ß-galactosidase isoenzymes II and III (KpBGase-II and -III), were each cloned downstream of a T7 promoter for expression in Escherichia coli BL21(DE3), and the resulting recombinant enzymes were characterized in detail. The optimum temperature and pH value of KpBGase-II were 40 °C and 7.5, and those of KpBGase-III were 50 °C and 8.0, respectively. KpBGase-III was more stable than KpBGase-II at higher temperature (>60°C). Both ß-galactosidases were more active towards o-nitrophenyl-ß- D-galactopyranoside as compared to lactose. The enzymatic synthesis of lactulose and 1-lactulose catalyzed by KpBGase-II and KpBGase-III was investigated. Using 400 g/L lactose and 200 g/L fructose as substrates, the resulting lactulose and 1-lactulose yields with KpBGase-II were 6.2 and 42.3 g/L, while those with KpBGase-III were 5.1 and 23.8 g/L, respectively. KpBGase-II has a potential for the production of 1-lactulose from lactose and fructose. Like other ß-galactosidases, the two isozymes catalyze the transgalactosylation in the presence of fructose establishing the ß-(1→1) linkage.

Lactulose biosynthesis by ß-galactosidase from a newly isolated Arthrobacter sp.

Lactulose, a ketose disaccharide, is used in both pharmaceutical and food industries. This study was undertaken to screen and isolate potent ß-galactosidase-producing bacteria and to evaluate their enzymatic production of lactulose. Soil samples from fruit gardens were collected. One isolate designated LAS was identified whose cell extract could convert lactose and fructose into lactulose. The 16S rDNA gene analysis of LAS revealed its phylogenetic relatedness to Arthrobacter sp. The ß-galactosidase produced by LAS was purified 15.7-fold by ammonium sulfate precipitation and subsequent Phenyl-Sepharose hydrophobic chromatography. The optimum pH and temperature for lactulose synthesis by this ß-galactosidase were 6.0 and 20 °C, respectively. The low optimum temperature of this enzyme compared to the currently used ones for lactulose production has the advantage of reducing the nonenzymatic browning in biotransformations. The results indicated that Arthrobacter could be used as a novel bacterial ß-galactosidase source for lactulose production.

Continuous production of lactulose by immobilized thermostable beta-glycosidase from Pyrococcus furiosus.

A continuous enzymatic process for the production of the prebiotic disaccharide lactulose through transgalactosylation was developed using free and immobilized beta-glycosidase from Pyrococcus furiosus. The hyperthermostable beta-glycosidase (CelB) was immobilized onto an anion-exchange resin (Amberlite IRA-93) or onto Eupergit C with immobilization yields of 72% and 83%, respectively. The immobilized biocatalysts demonstrated specific activities of 920 nkat g(-1) dry carrier and 1500 nkat g(-1) dry carrier at 75 degrees C with p-nitrophenyl-beta-D-galactopyranoside as substrate. Continuous biotransformations in packed-bed reactors using carrier bound CelB and in an enzyme membrane reactor using free CelB were carried out at 75 degrees C. Maximum lactulose yields of 43% related to the initial lactose concentration were reached with the carrier bound CelB preparations. The corresponding productivities were 52 glactulose l(-1)h(-1) (Amberlite IRA-93) and 15 glactulose l(-1)h(-1) (Eupergit C), respectively. The free enzyme tested in an enzyme membrane reactor showed a product yield of 41% and a productivity of 12 glactulose l(-1)h(-1) in the first day. While both carrier bound CelB preparations were 100% stable for at least 14 days, the half-life of the free CelB in the enzyme membrane reactor was only about 1.5 days.

Enzymatic production and complete nuclear magnetic resonance assignment of the sugar lactulose.

The enzymatic transgalactosylation from lactose to fructose leading to the prebiotic disaccharide lactulose was investigated using the beta-galactosidase from Aspergillus oryzae and the hyperthermostable beta-glycosidase from Pyrococcus furiosus (CelB). The conditions for highest lactulose yields relative to the initial lactose concentration were established on a 1 mL scale. Dependent on the initial molar ratio of lactose to fructose, more or fewer oligosaccharides other than lactulose were generated. Bioconversions on a 30 mL scale in a stirred glass reactor were performed, and lactulose yields of 46 mmol/L (44% relative to lactose) for CelB and 30 mmol/L (30% relative to lactose) for A. oryzae beta-galactosidase were achieved. Only <5% of other oligosaccharides were detectable. The corresponding productivities were 24 and 16 mmol/L/h, respectively. The molecular structure of lactulose was investigated in detail and confirmed after purification of the reaction solution by LC-MS and 1D and 2D NMR. Lactulose (4-O-beta-D-galactopyranosyl-D-fructose) was unambiguously proved to be the major transglycosylation disaccharide.

Lactulose production by beta-galactosidase in permeabilized cells of Kluyveromyces lactis.

Lactulose production from lactose and fructose was investigated with several commercial beta-galactosidases. The enzyme from Kluyveromyces lactis exhibited the highest lactulose productivity among the beta-galactosidases tested. The reaction conditions for lactulose production were optimized using cells that had been permeabilized by treatment with 50% (v/v) ethanol: cell concentration, 10.4 g l(-1); concentration of substrates, 40% (w/v) lactose and 20% (w/v) fructose; temperature, 60( degrees )C; pH 7.0. Under these conditions, the permeabilized cells produced approximately 20 g l(-1) lactulose in 3 h with a lactulose productivity of 6.8 g l(-1) h(-1). These results represent 1.3- and 2.1-fold increases in lactulose concentration and productivity compared with untreated washed cells. This is the first reported trial of enzymatic synthesis of lactulose using permeabilized yeast cells.