Recombinant Glycosyltransferase Research Articles

Green and Fast Synthesis of NiCo-MOF for Simultaneous Purification-Immobilization of Bienzyme to Catalyze the Synthesis of Ginsenoside Rh2.

Traditional metal-organic frameworks (MOFs) preparation is generally time-consuming, polluting, and lacking specificity for enzyme immobilization. This paper introduced a facile, rapid, and green method to produce three MOFs subsequently employed to purify and coimmobilize recombinant glycosyltransferase (UGT) and recombinant sucrose synthetase (SUSy) using histidine tag (His-tag) for the specific adsorption of Ni2+ and Co2+ from MOFs. This method simplified enzyme purification from crude extracts and enabled enzymes to be reused. The results demonstrated that NiCo-MOF exhibited a higher enzyme load (115.9 mg/g) than monometallic MOFs. Additionally, the NiCo-MOF@UGT&SUSy demonstrated excellent stability and efficiently produced the rare ginsenoside Rh2 by catalyzing a coupling reaction (95.6 μg/mL), solving the problem of the substrate cost of uridine diphosphate glucose (UDPG). The NiCo-MOF@UGT&SUSy retained 68.97% of the initial activity after 10 cycles. Finally, molecular docking studies elucidated the conversion mechanism of the target product Rh2. This technique is important in the industrialization of ginsenoside production and enzyme purification.

Selective Exoenzymatic Labeling of Lipooligosaccharides of Neisseria gonorrhoeae with α2,6-Sialoside Analogues.

The interactions between bacteria and their host often rely on recognition processes that involve host or bacterial glycans. Glycoengineering techniques make it possible to modify and study the glycans on the host's eukaryotic cells, but only a few are available for the study of bacterial glycans. Here, we have adapted selective exoenzymatic labeling (SEEL), a chemical reporter strategy, to label the lipooligosaccharides of the bacterial pathogen Neisseria gonorrhoeae, using the recombinant glycosyltransferase ST6Gal1, and three synthetic CMP-sialic acid derivatives. We show that SEEL treatment does not affect cell viability and can introduce an α2,6-linked sialic acid with a reporter group on the lipooligosaccharides by Western blot, flow cytometry and fluorescent microscopy. This new bacterial glycoengineering technique allows for the precise modification, here with α2,6-sialoside derivatives, and direct detection of specific surface glycans on live bacteria, which will aid in further unravelling the precise biological functions of bacterial glycans.

Enzymatic synthesis of novel unnatural phenoxodiol glycosides with a glycosyl donor flexible glycosyltransferase MeUGT1

Isoflavonoids are of great interest due to their human health-promoting properties, which have resulted in studies on exploiting these phytochemicals as hotspots in diverse bio -industries. Biocatalytic glycosylation of isoflavonoid aglycones to glycosides has attracted marked interests because it enable the biosynthesis of isoflavonoid glycosides with high selectivity under mild conditions, and also provide an environmentally friendly option for the chemical synthesis. Thus, these inspired us to exploit new flexible and effective glycosyltransferases from microbes for making glycosides attractive compounds that are in high demand in several industries. Most recently, we have reported the functional characterization of a bacterial-origin recombinant glycosyltransferase (MeUGT1). Herein, more detailed kinetic characteristics of this biocatalyst, using a number of glycosyl donor substrates, were examined for further investigation of its biocatalytic applicability, enabling it feasible to biosynthesize new glycosides; phenoxodiol-4′-O-α-glucuronide, phenoxodiol-4′-O-α-(2''-N-acetyl)glucosaminide, phenoxodiol-4′-O-α-galactoside, phenoxodiol-4′-O-α-(2''-N-acetyl)galactosaminide and phenoxodiol-4′-O-α-(2''-deoxy)glucoside. The thorough kinetic analyses revealed that while the recombinant enzyme can utilize, albeit with different substrate preference and catalytic efficiency, a total five different nucleotide sugars as glycosyl donors, exhibiting its promiscuity towards glycosyl donors. This is the first report that a recombinant glycosyltransferase MeUGT1 that can regio-specifically glycosylate C4′-hydroxyl function of semi-synthetic phenoxodiol isoflavene to biosynthesize a series of unnatural phenoxodiol-4′-O-α-glycosides.

Glycosylation of Ganoderic Acid A via Recombinant Glycosyltransferase of Bacillus subtilis Under Acidic Operating Condition

Ganoderic acid A (GAA) is a triterpenoid with identified effective activity, isolated from Ganoderma lucidum. Glycosylation of triterpenoid might improve its anti‐toxin bioactivity and also increase both water solubility and physical‐chemical stability. Glycosylation is carried out by glycosyltransferases (GT, EC 2.4.x.y), a type of enzyme that utilize a nucleotide‐activated sugar donor, such as uridine diphosphate (UDP)‐glucose, to transfer the sugar portion to a sugar‐linked molecule. We have utilized Bacillus sp. GA A07, isolated from intestinal bacteria of Zebrafish, to biotransform GAA to GAA‐15‐O‐β‐glucoside 1. Our previous study also showed that the Bacillus subtilis, ATCC 6633 strain, could biotransform GAA to compound, GAA‐15‐O‐b‐glucoside, and one minor compound in Fig. 1. We selected five GT genes‐BsGT110, BsGT292, BsGT296, BsGT398, and BsGT489 from the used strains, and successfully overexpressed and cloned them in Escherichia coli. Two glycosyltransferases (BsGT398 and BsGT489) have shown the capability to catalyze GAA to GAA‐15‐O‐β‐glucoside under some specific conditions. However, the chemical structure of minor compound 2 and its corresponding enzyme remain elusive. In the present study, we identified BsGT110, a GT from the used B. subtilis strain, for the biotransformation of GAA into this compound under acidic glycosylation. BsGT110 showed an optimal glycosylation activity toward GAA at pH 6 but lost most of its activity at pH 8. Through a designed operating production, this product 2 was successfully isolated using preparative high‐performance liquid chromatography and was identified to be a new triterpenoid glucoside (GAA‐26‐O‐β‐glucoside) by mass and nuclear magnetic resonance spectroscopy. We identified that BsGT110 is an unique GT that have a specific activity on the glycosylation of triterpenoid at the C‐26 position under acidic conditions (in Fig. 2) but lost most of its activity at alkaline ones, suggesting that acidic solutions may enhance the catalysis activity of this and similar types of GTs toward triterpenoids.Support or Funding InformationThis research was financially supported by the grants of the Ministry of Science and Technology, Taiwan (Project No. MOST 108‐2221‐E‐507 ‐008 ‐ and MOST 108‐2221‐E‐024 ‐008 ‐MY2).UPLC analysis of the biotranformation of GAA by BsGT110Figure 1Glycosylation of GAA by BsGT110 under the acidic conditionFigure 2

Glycosyltransformation of ginsenoside Rh2 into two novel ginsenosides using recombinant glycosyltransferase from Lactobacillus rhamnosus and its in vitro applications

BackgroundGinsenoside Rh2 is well known for many pharmacological activities, such as anticancer, antidiabetes, antiinflammatory, and antiobesity properties. Glycosyltransferases (GTs) are ubiquitous enzymes present in nature and are widely used for the synthesis of oligosaccharides, polysaccharides, glycoconjugates, and novel derivatives. We aimed to synthesize new ginsenosides from Rh2 using the recombinant GT enzyme and investigate its cytotoxicity with diverse cell lines. MethodsWe have used a GT gene with 1,224-bp gene sequence cloned from Lactobacillus rhamnosus (LRGT) and then expressed in Escherichia coli BL21 (DE3). The recombinant GT protein was purified and demonstrated to transform Rh2 into two novel ginsenosides, and they were characterized by nuclear magnetic resonance (NMR) techniques and evaluated by 3-(4, 5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide assay. ResultsTwo novel ginsenosides with an additional glucopyranosyl (6→1) and two additional glucopyranosyl (6→1) linked with the C-3 position of the substrate Rh2 were synthesized, respectively. Cell viability assay in the lung cancer (A549) cell line showed that glucosyl ginsenoside Rh2 inhibited cell viability more potently than ginsenoside Rg3 and Rh2 at a concentration of 10 μM. Furthermore, glucosyl ginsenoside Rh2 did not exhibit any cytotoxic effect in murine macrophage cells (RAW264.7), mouse embryo fibroblasts cells (3T3-L1), and skin cells (B16BL6) at a concentration of 10 μM compared with ginsenoside Rh2 and Rg3. ConclusionThis is the first report on the synthesis of two novel ginsenosides, namely, glucosyl ginsenoside Rh2 and diglucosyl ginsenoside Rh2 from Rh2 by using recombinant GT isolated from L. rhamnosus. Moreover, diglucosyl ginsenoside Rh2 might be a new candidate for treatment of inflammation, obesity, and skin whiting, and especially for anticancer.

A New Triterpenoid Glucoside from a Novel Acidic Glycosylation of Ganoderic Acid A via Recombinant Glycosyltransferase of Bacillus subtilis.

Ganoderic acid A (GAA) is a bioactive triterpenoid isolated from the medicinal fungus Ganoderma lucidum. Our previous study showed that the Bacillus subtilis ATCC (American type culture collection) 6633 strain could biotransform GAA into compound (1), GAA-15-O-β-glucoside, and compound (2). Even though we identified two glycosyltransferases (GT) to catalyze the synthesis of GAA-15-O-β-glucoside, the chemical structure of compound (2) and its corresponding enzyme remain elusive. In the present study, we identified BsGT110, a GT from the same B. subtilis strain, for the biotransformation of GAA into compound (2) through acidic glycosylation. BsGT110 showed an optimal glycosylation activity toward GAA at pH 6 but lost most of its activity at pH 8. Through a scaled-up production, compound (2) was successfully isolated using preparative high-performance liquid chromatography and identified to be a new triterpenoid glucoside (GAA-26-O-β-glucoside) by mass and nuclear magnetic resonance spectroscopy. The results of kinetic experiments showed that the turnover number (kcat) of BsGT110 toward GAA at pH 6 (kcat = 11.2 min−1) was 3-fold higher than that at pH 7 (kcat = 3.8 min−1), indicating that the glycosylation activity of BsGT110 toward GAA was more active at acidic pH 6. In short, we determined that BsGT110 is a unique GT that plays a role in the glycosylation of triterpenoid at the C-26 position under acidic conditions, but loses most of this activity under alkaline ones, suggesting that acidic solutions may enhance the catalytic activity of this and similar types of GTs toward triterpenoids.

Cyclodextrin Glycosyltransferase-Catalyzed Synthesis of Pinoresinol-α-D-glucoside Having Antioxidant and Anti-Inflammatory Activities

Enzymatic synthesis of pinoresinol-α-glucosides (PGs) was performed through the transglycosylation reaction catalyzed by the recombinant cyclodextrin glycosyltransferase (CGTase) from Bacillus circulans A11 using β-cyclodextrin as the glycosyl donor and pinoresinol as the acceptor molecule. Incubation of 0.5% (wt/vol) β-cyclodextrin with 1.5% (wt/vol) pinoresinol (P) and 80.0 U/mL of CGTase in 20 mM of Tris–HCl buffer (pH 9.0) at 50oC for 60 h was optimal for PGs synthesis. Under these conditions, two PG transfer products with molecular weights of 544 and 682 Da corresponding to pinoresinol monoglucoside (PG1-I and PG1-II) and pinoresinol diglucoside (PG2), respectively, were detected by TLC and MS. The structures of PG1 and PG2 were confirmed as pinoresinol-α-D-glucopyranoside and pinoresinol-α-D-diglucopyranoside, respectively, by 1H-NMR analysis. The free-radical scavenging and anti-inflammatory activities of PG1 and PG2 were reduced as compared with the original P using α,α-diphenyl-β-picrylhydrazyl radical scavenging reactions and the β-glucuronidase inhibition assay. The loss of antioxidant and anti-inflammatory activities might result from the addition of glucose in the position 4-OH of P that may have disturbed its electron rotation. In vivo, PGs are converted to Ps before they are absorbed and so loss of activity may be minimal. Interestingly, the α-glycosylated compounds which showed the change of physicochemical properties such as increase of solubility and sweetness could promote a positive effect on the bioavailability of original P.

Enzymatic glycosylation of oleanane-type triterpenoids

Glycosylation is an effective approach to improve the druggability of natural products by increasing their water solubility. In this work, we report the glycosylation of oleanane-type triterpenoids by a recombinant microbial glycosyltransferase YjiC1. A preliminary screening test indicated YjiC1 exhibited robust capabilities for O-glycosylation of triterpenoids, based on LC/MS analysis. Among the products, two new compounds (2a and 3a), together with a known one (1a), were isolated and characterized. These products exhibited improved water solubility, and 3a showed moderate anti-HIV activities at 100 μM. This reaction provides a facile and efficient approach to synthesize the glucosides of triterpenoids.

Synthesis, structural characterization, and biological properties of pentyl- and isopentyl-α-D-glucosides

The study was devoted to the synthesis of pentyl glucosides (PenGn) and isopentyl glucosides (Iso-PenGn) by transglycosylation using recombinant cyclodextrin glycosyltransferase from Bacillus circulans A11, β-cyclodextrin as a glucosyl donor and 1-pentanol and isopentanol as acceptors. TLC and MS analysis indicated at least 3 products which were in accordance with PenGn and IsoPenGn having glucose, maltose and maltotriose attached to the alkyl groups of both alcohols. Two products of each glucoside were purified by preparative TLC and their structures were identified by NMR technique to be pentyl-α-D-glucopyranoside (PenG1), pentyl-α-D-maltopyranoside (PenG2), isopentyl-α-D-glucopyranoside (IsoPenG1) and isopentyl- α-D-maltopyranoside (IsoPenG2). The effect of water-in-hexadecane emulsion on emulsion-forming properties showed that PenG2 had the highest emulsifying activity. Adding PenG2 to the insoluble Corynebacterium glutamicum amylomaltase from Escherichia coli transformants (A406R), helped to perform it to more soluble conformation. Moreover, it was found that PenG1,2 exhibited a higher antibacterial activity against E. coli ATCC 25922 than that of IsoPenG1,2. Hence, the biological properties of the synthesized products may be useful for their applications as emulsifying, solubilizing and antibacterial agents.

Rare ginsenoside Ia synthesized from F1 by cloning and overexpression of the UDP-glycosyltransferase gene from Bacillus subtilis: synthesis, characterization, and in vitro melanogenesis inhibition activity in BL6B16 cells

BackgroundGinsenoside F1 has been described to possess skin-whitening effects on humans. We aimed to synthesize a new ginsenoside derivative from F1 and investigate its cytotoxicity and melanogenesis inhibitory activity in B16BL6 cells using recombinant glycosyltransferase enzyme. Glycosylation has the advantage of synthesizing rare chemical compounds from common compounds with great ease. MethodsUDP-glycosyltransferase (BSGT1) gene from Bacillus subtilis was selected for cloning. The recombinant glycosyltransferase enzyme was purified, characterized, and utilized to enzymatically transform F1 into its derivative. The new product was characterized by NMR techniques and evaluated by MTT, melanin count, and tyrosinase inhibition assay. ResultsThe new derivative was identified as (20S)-3β,6α,12β,20-tetrahydroxydammar-24-ene-20-O-β-D-glucopyranosyl-3-O-β-D-glucopyranoside (ginsenoside Ia), which possesses an additional glucose linked into the C-3 position of substrate F1. Ia had been previously reported; however, no in vitro biological activity was further examined. This study focused on the mass production of arduous ginsenoside Ia from accessible F1 and its inhibitory effect of melanogenesis in B16BL6 cells. Ia showed greater inhibition of melanin and tyrosinase at 100 μmol/L than F1 and arbutin. These results suggested that Ia decreased cellular melanin synthesis in B16BL6 cells through downregulation of tyrosinase activity. ConclusionTo our knowledge, this is the first study to report on the mass production of rare ginsenoside Ia from F1 using recombinant UDP-glycosyltransferase isolated from B. subtillis and its superior melanogenesis inhibitory activity in B16BL6 cells as compared to its precursor. In brief, ginsenoside Ia can be applied for further study in cosmetics.

Vaccination of Sheep with a Methanogen Protein Provides Insight into Levels of Antibody in Saliva Needed to Target Ruminal Methanogens.

Methane is produced in the rumen of ruminant livestock by methanogens and is a major contributor to agricultural greenhouse gases. Vaccination against ruminal methanogens could reduce methane emissions by inducing antibodies in saliva which enter the rumen and impair ability of methanogens to produce methane. Presently, it is not known if vaccination can induce sufficient amounts of antibody in the saliva to target methanogen populations in the rumen and little is known about how long antibody in the rumen remains active. In the current study, sheep were vaccinated twice at a 3-week interval with a model methanogen antigen, recombinant glycosyl transferase protein (rGT2) formulated with one of four adjuvants: saponin, Montanide ISA61, a chitosan thermogel, or a lipid nanoparticle/cationic liposome adjuvant (n = 6/formulation). A control group of sheep (n = 6) was not vaccinated. The highest antigen-specific IgA and IgG responses in both saliva and serum were observed with Montanide ISA61, which promoted levels of salivary antibodies that were five-fold higher than the second most potent adjuvant, saponin. A rGT2-specific IgG standard was used to determine the level of rGT2-specific IgG in serum and saliva. Vaccination with GT2/Montanide ISA61 produced a peak antibody concentration of 7 × 1016 molecules of antigen-specific IgG per litre of saliva, and it was estimated that in the rumen there would be more than 104 molecules of antigen-specific IgG for each methanogen cell. Both IgG and IgA in saliva were shown to be relatively stable in the rumen. Salivary antibody exposed for 1–2 hours to an in vitro simulated rumen environment retained approximately 50% of antigen-binding activity. Collectively, the results from measuring antibody levels and stablility suggest a vaccination-based mitigation strategy for livestock generated methane is in theory feasible.

Enzymatic synthesis of propyl-α-glycosides and their application as emulsifying and antibacterial agents

Alkyl glycosides have been effectively used in many industries because of their biodegradable, emulsification and antibacterial properties. In this study, the alkyl glycoside of propyl glycosides (PGn) was synthesized using β-cyclodextrin (β-CD) and 1-propanol through the transglycosylation reaction of recombinant cyclodextrin glycosyltransferase (CGTase) from the Bacillus circulans A11. The optimal condition for the synthesis of propyl glycosides consisted of an incubation of 1.5% (w/v) β-CD and 500 U/mL of CGTase in a water/propanol content containing 10% (v/v) 1-propanol at pH 6.0, 50°C for 96 h. Upon analysis of the product at the optimal condition by TLC, at least three products which move faster than glucose were observed. These transferred products were formed with molecular weights of 222.1, 384.1 and 546.4 daltons as determined by mass spectrometry analysis; these values were in accordance with propyl glucoside (PG1), propyl maltoside (PG2) and propyl maltotrioside (PG3), respectively. PG1 and PG2 were produced and prepared on a large scale and subsequently purified by preparative TLC. The combined 1H- and 13C-NMR analysis confirmed that the structures of PG1 and PG2 were propyl-α-D-glucopyranoside and propyl-α-D-maltopyranoside, respectively. Both PG1 and PG2 showed emulsification activity and stability in their formation in water and n-hexadecane. Furthermore, the antibacterial activity of both products was determined and it was found that PG2 had a higher antibacterial activity against Staphylococcus aureus and Escherichia coli than that of PG1.

Vaccination of cattle with a methanogen protein produces specific antibodies in the saliva which are stable in the rumen

Methane is produced in the rumen of cattle by a group of archaea (single-celled organisms forming a domain distinct from bacteria and eucarya) called methanogens. Vaccination against methanogens has the potential to reduce methane emissions by inducing antibodies in saliva which are transferred to the rumen and diminish the ability of methanogens to produce methane. Since it is likely that an effective vaccination strategy will need to produce high levels of methanogen-specific antibody in the saliva; the choice of adjuvant, route of vaccination and stability of saliva-derived antibody in the rumen all need to be considered. In this study, stability of IgA and IgG in rumen fluid was determined using an in vitro assay. IgA levels in cattle saliva were reduced by only 40% after 8h exposure to rumen contents while IgG levels were reduced by 80%. These results indicated that antibody is relatively stable in the bovine rumen. A trial was conducted in cattle to investigate induction of immune responses to a methanogen protein, recombinant glycosyl transferase protein (rGT2) from Methanobrevibacter ruminantium M1. Groups of cattle (n=6) were vaccinated subcutaneously with rGT2, formulated with Montanide ISA61 with or without the TLR4 agonist, monophosphoryl lipid A (MPL). A control group (n=6) was not vaccinated. Strong antigen-specific IgG and moderate IgA responses were measured in the serum and saliva of the vaccinated animals and antibody was also detected in the rumen.

CHoMP: a chemoenzymatic histology method using clickable probes.

The characterization of aberrant glycosylation patterns in biopsied patient samples represents a remarkable challenge for scientists and medical doctors due to the lack of specific methods for detection. Here, we report the development of a histological method, dubbed CHoMP-chemoenzymatic histology of membrane polysaccharides-for analyzing glycosylation patterns in mammalian tissues. This method exploits a recombinant glycosyltransferase to transfer a monosaccharide analogue equipped with a chemical handle to a specific cell-surface glycan target, which can then be derivatized with imaging probes by using bioorthogonal click chemistry for visualization. We applied CHoMP to survey changes in expression of N-acetyllactosamine (LacNAc) in human samples from patients afflicted with lung adenocarcinoma and observed a sharp decrease in expression levels between normal and early grade tumors, thus suggesting a potential application of this technique in early cancer diagnosis.

Extracellular expression, purification, and characterization of the recombinant cyclodextrin glycosyltransferase from Peanibacillus macerans

Extracellular expression, purification, and characterization of the recombinant cyclodextrin glycosyltransferase from Peanibacillus macerans

Crystallization and preliminary X-ray analysis of vicenisaminyltransferase VinC

A recombinant glycosyltransferase, VinC, from Streptomyces halstedii HC34 has been crystallized at 293 K using PEG 3350 as precipitant. The diffraction pattern of the crystal extends to 2.0 A resolution at 100 K using synchrotron radiation at SPring-8. The crystals are orthorhombic and belong to space group I222, with unit-cell parameters a = 98.21, b = 130.39, c = 140.11 A. The presence of two molecules per asymmetric unit gives a crystal volume per protein weight (V(M)) of 2.43 A(3) Da(-1) and a solvent content of 49.5% by volume.

Broadening the biocatalytic properties of recombinant sucrose synthase 1 from potato ( Solanum tuberosum L.) by expression in Escherichia coli and Saccharomyces cerevisiae

The present paper describes the modification of the biochemical and biocatalytic characteristics of the recombinant glycosyltransferase sucrose synthase 1 (SuSy1, EC 2.4.1.13) from Solanum tuberosum L. by expression in different hosts. SuSy1 catalyses the reversible conversion of sucrose and UDP to form UDP-α- d-glucose and d-fructose. Recombinant SuSy1 expressed in Saccharomyces cerevisiae was extensively characterized in our former studies. In contrast, the expression of SuSy1 in Escherichia coli resulted in striking differences concerning the biochemical and biocatalytic properties. The monosaccharides d-ribulose, d-tagatose, l-glucose, and l-rhamnose were identified as new acceptor substrates. Analysis of the phosphorylation status of recombinant yeast-SuSy1 and the yeast-SuSy1/S11A mutant by Fe 3+-IMAC clearly demonstrated that the wild-type enzyme is phosphorylated at the conserved phosphorylation site S11 identified in all plant sucrose synthase enzymes. The mutant enzymes, yeast-SuSy1/S11A and E. coli-SuSy1/S11D, show also an altered spectrum of specific monosaccharides as acceptor substrates. This is the first report on broadening the biocatalytic potential of sucrose synthase by expression in different hosts and furthermore by mutation of a conserved phosphorylation site. We also demonstrate for the first time that recombinant SuSy1 is phosphorylated at the conserved serine 11 when expressed in S. cerevisiae.

Genetic engineering of Escherichia coli for the economical production of sialylated oligosaccharides

We have previously described a microbiological process for the conversion of lactose into 3′sialyllactose and other ganglioside sugars by living Escherichia coli cells expressing the appropriate recombinant glycosyltransferase genes. In this system the activated sialic acid donor (CMP-Neu5Ac) was generated from exogenous sialic acid, which was transported into the cells by the permease NanT. Since sialic acid is an expensive compound, a more economical process has now been developed by genetically engineering E. coli K12 to be capable of generating CMP-Neu5Ac using its own internal metabolism. Mutant strains devoid of Neu5Ac aldolase and of ManNAc kinase were shown to efficiently produce 3′sialyllactose by coexpressing the α-2,3-sialyltransferase gene from Neisseria meningitidis with the neuC, neuB and neuA Campylobacter jejuni genes encoding N-acetylglucosamine-6-phosphate-epimerase, sialic acid synthase and CMP-Neu5Ac synthetase, respectively. A sialyllactose concentration of 25 g l −1 was obtained in long-term high cell density culture with a continuous lactose feed. This high concentration and low cost of fermentation medium should make possible to use sialylated oligosaccharides in new fields such as the food industry.

An Enzyme Module System for in situ Regeneration of Deoxythymidine 5′‐Diphosphate (dTDP)‐Activated Deoxy Sugars

AbstractA highly flexible enzyme module system (EMS) was developed which allows for the first time the in situ regeneration of deoxythymidine 5′‐diphosphate (dTDP)‐activated deoxy sugars and furthermore enables us to produce novel sorangiosides in a combinatorial biocatalytic approach using three enzyme modules. The SuSy module with the recombinant plant enzyme sucrose synthase (SuSy) and the deoxy sugar module consisting of the enzymes RmlB (4,6‐dehydratase), RmlC (3,5‐epimerase) and RmlD (4‐ketoreductase) from the biosynthetic pathway of dTDP‐β‐L‐rhamnose were combined with the glycosyltransferase module containing the promiscuous recombinant glycosyltransferase SorF from Sorangium cellulosum So ce12. Kinetic data and the catalytic efficiency were determined for the donor substrates of SorF: dTDP‐α‐D‐glucose, dTDP‐β‐L‐rhamnose, uridine diphosphate (UDP)‐α‐D‐glucose (Glc), and dTDP‐6‐deoxy‐4‐keto‐α‐D‐glucose. The synthesis of glucosyl‐sorangioside with in situ regeneration of dTDP‐Glc was accomplished by combination of SuSy and SorF. The potential of the EMS is demonstrated by combining SuSy, RmlB, RmlC, RmlD with SorF in one‐pot for the in situ regeneration of dTDP‐activated (deoxy) sugars. The HPLC/MS analysis revealed the formation of rhamnosyl‐sorangioside and glucosyl‐sorangioside, demonstrating the in situ regeneration of dTDP‐β‐L‐rhamnose and dTDP‐α‐D‐glucose and a cycle number for dTDP higher than 9. Furthermore, NADH (reduced form of nicotinamdie adenine dinucleotide) regeneration with formate dehydrogenase in the reduction step catalyzed by the 4‐ketoreductase RmlD could be integrated in the one‐pot synthesis yielding similar conversion rates and cycle numbers. In summary, we have established the first in situ regeneration cycle for dTDP‐activated (deoxy) sugars by a highly flexible EMS which allows simple exchange of enzymes in the deoxy sugar module and exchange of glycosyltransferases as well as aglycones in the glycosyltransferase module to synthesize new hybrid glycosylated natural products in one‐pot.

A recombinant cyclodextrin glycosyltransferase cloned from Paenibacillus sp. strain RB01 showed improved catalytic activity in coupling reaction between cyclodextrins and disaccharides

Recombinant cyclodextrin glycosyltransferase (CGTase) was obtained by cloning the PCR gene fragment from thermotolerant Paenibacillus sp. strain RB01 screened from hot spring area in Thailand and cloned into the Escherichia coli expression vector. The nucleotide sequence was analyzed and aligned. Nucleotide sequence of the recombinant CGTase contained an open reading frame of 2139 bp encoding 713 amino acid residues. The recombinant required one-third of culture time and neutral pH to produce CGTase compared to wild type. CGTases from both wild type and transformant were purified in parallel by starch adsorption and DEAE cellulose column. Their biochemical properties such as molecular weight, optimum pH and temperature were quite similar. However, the recombinant enzyme showed improved catalytic activity in the coupling reaction between cyclodextrins (CDs) and some disaccharides. Among several sugars tested with excess βCD, cellobiose was the best substrate followed by leucrose. Very low activity was observed with trehalose, lactose and mellibiose. Sucrose and raffinose showed no activity. The K m and other kinetic parameters of recombinant enzyme were determined for cellobiose and several cyclodextrin derivatives. Recombinant CGTase showed lower K m for βCD and its derivatives, with improved activity compared to wild type enzyme.