ABSTRACT
6'-Sialyllactose (6'-SL), the most abundant sialylated human milk oligosaccharide, has attracted attention for its potential application in supplementary infant formulas. Herein, we report a facile strategy to construct a cascade bioreactor for the enzymatic synthesis of 6'-SL by co-immobilizing an enzymatic module consisting of CMP-sialic acid synthase and α-2,6-sialyltransferase into hierarchically porous MIL-53 (HP-MIL-53). The as-prepared HP-MIL-53 showed high enzyme immobilization capacity, reaching 226 mg g-1. Furthermore, the co-immobilized enzymes exhibited higher initial catalytic efficiency, and thermal, pH and storage stability than the free ones. Finally, the 6'-SL yield remained >80% after 13 cycles of use. We expect that HP-MIL-53 would have potential industrial applications in the enzymatic modular synthesis of 6'-SL and other glycans.
Subject(s)
Enzymes, Immobilized , Sialyltransferases , Enzymes, Immobilized/chemistry , Enzymes, Immobilized/metabolism , Sialyltransferases/metabolism , Porosity , Humans , Oligosaccharides/chemistry , Oligosaccharides/metabolism , Oligosaccharides/biosynthesis , N-Acylneuraminate Cytidylyltransferase/metabolism , N-Acylneuraminate Cytidylyltransferase/chemistry , Bioreactors , Milk, Human/chemistry , Milk, Human/metabolism , Lactose/chemistry , Lactose/analogs & derivatives , Lactose/metabolism , Hydrogen-Ion Concentration , beta-D-Galactoside alpha 2-6-SialyltransferaseABSTRACT
Levan-type fructooligosaccharides (LFOS) exhibit significant biological activities and selectively promote the growth of certain beneficial bacteria. Levanase is an important enzyme for LFOS production. In this study, two isoforms of levanases, exo- and endo-type depolymerizing enzymes, from Bacillus subtilis HM7 isolated from Dynastes hercules larvae excrement were cloned, expressed, and characterized. The synergistic effect on the levan hydrolysis and kinetic properties of both isoforms were evaluated, indicating their cooperation in levan metabolism, where the endo-levanase catalyzes a rate-limiting step. In addition, homology models and molecular dynamics simulations revealed the key amino residues of the enzymes for levan binding and catalysis. It was found that both isoforms possessed distinct binding residues in the active sites, suggesting the importance of the specificity of the enzymes. Finally, we demonstrated the potential of endo-type levanase in LFOS synthesis using a one-pot reaction with levansucrase. Overall, this study fills the knowledge gap in understanding levanase's mechanism, making an important contribution to the fields of food science and biotechnology.
Subject(s)
Bacillus subtilis , Glycoside Hydrolases , Oligosaccharides , Bacillus subtilis/enzymology , Oligosaccharides/biosynthesis , Oligosaccharides/chemistry , Glycoside Hydrolases/metabolism , Glycoside Hydrolases/chemistry , Glycoside Hydrolases/genetics , Kinetics , Fructans/biosynthesis , Fructans/chemistry , Hydrolysis , Molecular Dynamics Simulation , Substrate Specificity , Hexosyltransferases/metabolism , Hexosyltransferases/chemistry , Hexosyltransferases/genetics , CatalysisABSTRACT
3'-Sialyllactose (3'-SL), one of the abundant and important sialylated human milk oligosaccharides, is an emerging food ingredient used in infant formula milk. We previously developed an efficient route for 3'-SL biosynthesis in metabolically engineered Escherichia coli BL21(DE3). Here, several promising α2,3-sialyltransferases were re-evaluated from the byproduct synthesis perspective. The α2,3-sialyltransferase from Neisseria meningitidis MC58 (NST) with great potential and the least byproducts was selected for subsequent molecular modification. Computer-assisted mutation sites combined with a semi-rational modification were designed and performed. A combination of two mutation sites (P120H/N113D) of NST was finally confirmed as the best one, which significantly improved 3'-SL biosynthesis, with extracellular titers of 24.5 g/L at 5-L fed-batch cultivations. When NST-P120H/N113D was additionally integrated into the genome of host EZAK (E. coli BL21(DE3)ΔlacZΔnanAΔnanT), the final strain generated 32.1 g/L of extracellular 3'-SL in a 5-L fed-batch fermentation. Overall, we underscored the existence of by-products and improved 3'-SL production by engineering N. meningitidis α2,3-sialyltransferase.
Subject(s)
Escherichia coli , Metabolic Engineering , Neisseria meningitidis , Sialyltransferases , Escherichia coli/genetics , Escherichia coli/metabolism , Sialyltransferases/genetics , Sialyltransferases/metabolism , Metabolic Engineering/methods , Neisseria meningitidis/genetics , Neisseria meningitidis/enzymology , Mutation , Oligosaccharides/biosynthesis , FermentationABSTRACT
A novel immobilized chitosanase was developed and utilized to produce chitosan oligosaccharides (COSs) via chitosan hydrolysis. Magnetite-agar gel particles (average particle diameter: 338 µm) were prepared by emulsifying an aqueous agar solution dispersing 200-nm magnetite particles with isooctane containing an emulsifier at 80 °C, followed by cooling the emulsified mixture. The chitosanase from Bacillus pumilus was immobilized on the magnetite-agar gel particles chemically activated by introducing glyoxyl groups with high immobilization yields (>80%), and the observed specific activity of the immobilized chitosanase was 16% of that of the free enzyme. This immobilized chitosanase could be rapidly recovered from aqueous solutions by applying magnetic force. The thermal stability of the immobilized chitosanase improved remarkably compared with that of free chitosanase: the deactivation rate constants at 35 °C of the free and immobilized enzymes were 8.1 × 10-5 and 3.9 × 10-8 s-1, respectively. This immobilized chitosanase could be reused for chitosan hydrolysis at 75 °C and pH 5.6, and 80% of its initial activity was maintained even after 10 cycles of use. COSs with a degree of polymerization (DP) of 2-7 were obtained using this immobilized chitosanase, and the product content of physiologically active COSs (DP ≥ 5) reached approximately 50%.
Subject(s)
Agar , Bacillus , Chitosan , Enzyme Stability , Enzymes, Immobilized , Glycoside Hydrolases , Oligosaccharides , Chitosan/chemistry , Chitosan/metabolism , Enzymes, Immobilized/metabolism , Enzymes, Immobilized/chemistry , Glycoside Hydrolases/metabolism , Glycoside Hydrolases/chemistry , Oligosaccharides/chemistry , Oligosaccharides/metabolism , Oligosaccharides/biosynthesis , Hydrolysis , Bacillus/enzymology , Agar/chemistry , Gels/chemistry , Bacterial Proteins/metabolism , Bacterial Proteins/chemistry , Ferrosoferric Oxide/chemistry , Biocatalysis , Hydrogen-Ion Concentration , KineticsABSTRACT
Lacto-N-difucohexaose II (LNDFH II) is a typical fucosylated human milk oligosaccharide and can be enzymatically produced from lacto-N-tetraose (LNT) by a specific α1,3/4-fucosyltransferase from Helicobacter pylori DMS 6709, referred to as FucT14. Previously, we constructed an engineered Escherichia coli BL21(DE3) with a single plasmid for highly efficient biosynthesis of LNT. In this study, two additional plasmids harboring the de novo GDP-L-fucose pathway module and FucT14, respectively, were further introduced to construct the strain for successful biosynthesis of LNDFH II. FucT14 was actively expressed, and the engineered strain produced LNDFH II as the major product, lacto-N-fucopentaose (LNFP) V as the minor product, and a trace amount of LNFP II and 3-fucosyllactose as very minor products. Additional expression of the α1,3-fucosyltransferase FutM1 from a Bacteroidaceae bacterium from the gut metagenome could obviously enhance the LNDFH II biosynthesis. After optimization of induction conditions, the maximum titer reached 3.011 g/L by shake-flask cultivation. During the fed-batch cultivation, LNDFH II was highly efficiently produced with the highest titer of 18.062 g/L and the productivity yield of 0.301 g/L·h.
Subject(s)
Bacterial Proteins , Escherichia coli , Fucosyltransferases , Guanosine Diphosphate Fucose , Metabolic Engineering , Humans , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Biosynthetic Pathways , Escherichia coli/genetics , Escherichia coli/metabolism , Fucosyltransferases/genetics , Fucosyltransferases/metabolism , Guanosine Diphosphate Fucose/metabolism , Helicobacter pylori/genetics , Helicobacter pylori/metabolism , Helicobacter pylori/enzymology , Oligosaccharides/metabolism , Oligosaccharides/biosynthesisABSTRACT
Lacto-N-fucopentaose V (LNFP V) is a typical human milk pentasaccharide. Multi-enzymatic in vitro synthesis of LNFP V from lactose was reported, however, microbial cell factory approach to LNFP V production has not been reported yet. In this study, the biosynthetic pathway of LNFP V was examined in Escherichia coli. The previously constructed E. coli efficiently producing lacto-N-tetraose was used as the starting strain. GDP-fucose pathway module and a regio-specific glycosyltransferase with α1,3-fucosylation activity were introduced to realize the efficient synthesis of LNFP V. The α1,3/4-fucosyltransferase from Bacteroides fragilis was selected as the best enzyme for in vivo biosynthesis of LNFP V from nine candidates, with the highest titer and the lowest by-product accumulation. A beneficial variant K128D was obtained to further enhance LNFP V titer using computer-assisted site-directed mutagenesis. The final strain EW10 could produce 25.68 g/L LNFP V by fed-batch cultivation, with the productivity of 0.56 g/L·h.
Subject(s)
Bacteroides fragilis , Fucosyltransferases , Bacteroides fragilis/enzymology , Bacteroides fragilis/genetics , Fucosyltransferases/genetics , Fucosyltransferases/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Oligosaccharides/biosynthesis , Oligosaccharides/metabolism , Oligosaccharides/chemistry , Humans , Mutagenesis, Site-DirectedABSTRACT
Recently, the biosynthesis of human milk oligosaccharides (HMOs) has been attracting increasing attention. Lacto-N-neotetraose (LNnT) is one of the most important neutral-core HMOs with promising health effects for infants. It has received Generally Recognized as Safe (GRAS) status and is the second HMO commercially added in infant formula after 2'-fucosyllactose. In previous studies, a series of engineered Escherichia coli strains have been constructed and optimized to produce high titers of precursor lacto-N-triose II. On the basis of these strains, LNnT-producing strains were constructed by overexpressing the ß1,4-galactosyltransferase-encoding gene from Aggregatibacter actinomycetemcomitans NUM4039 (Aa-ß1,4-GalT). Interestingly, an appreciable LNnT titer was obtained by weakening the metabolic flux of the UDP-GlcNAc pathway and simply overexpressing the essential genes lgtA, galE, and Aa-ß1,4-GalT in lacZ-, wecB-, and nagB-deleted E. coli. Subsequently, LNnT synthesis was optimized through balancing the expression of these three biosynthetic enzymes. The optimized strain produced LNnT with an extracellular titer of 12.1 g/L in fed-batch cultivation, with the productivity and specific yield of 0.25 g/L·h and 0.27 g/g dry cell weight, respectively.
Subject(s)
Escherichia coli Proteins , Escherichia coli , Oligosaccharides , Carbohydrate Epimerases/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , Infant Formula , Microorganisms, Genetically-Modified , Milk, Human/chemistry , Oligosaccharides/biosynthesisABSTRACT
Chitin refers to a natural biopolymer, which is economically significant to next-generation biorefineries. In this study, a novel high-yield method with cell surface-display chitosanase (CHI-1) was built to produce chitooligosaccharides (COS) from shrimp chaff through the co-fermentation in the presence of Bacillus subtilis and Acetobacter sp. Under the optimized co-fermentation conditions (5â¯g/L yeast extracts, 10â¯g/L KH2PO4, 6% ethanol, 50â¯g/L glucose), the final deproteinization (DP) and demineralization (DM) efficiency and the chitin yield were achieved as 94, 92 and 18%, respectively. The engineered E. coli BL21-pET23b(+)-NICHI maintained 81% of the initial enzyme activity after 40â¯days at room temperature. The crude CHI-1 was inactivated after one-day interacting with prepared chitosan. Moreover, E. coli BL21-pET23b(+)-NICHI still maintained excellent hydrolysis ability in 7â¯days, and the COS yield reached 41%. Accordingly, the proposed method exhibited excellent stability and a high hydrolysis efficiency to produce COS with whole engineered cells.
Subject(s)
Glycoside Hydrolases/metabolism , Oligosaccharides/biosynthesis , Animals , Chitosan/chemistry , Decapoda , Escherichia coli/enzymology , Fermentation , Oligosaccharides/chemistryABSTRACT
Lacto-N-neotetraose (LNnT) is a unique tetrasaccharide naturally occurring in human milk, as an important member of human milk oligosaccharides. Because of promising beneficial effects, it has been commercially added as a functional fortifier in infant formula. ß-1,4-Galactosyltransferase (ß-1,4-GalT) catalyzes LNnT biosynthesis from uridine 5'-diphospho-galactose (UDP-Gal) to lacto-N-triose II (LNT II). There have been only two LNnT-producing bacterial ß-1,4-GalTs, including the ones from Neisseria meningitidis and Histophilus somni. In this study, a novel LNnT-producing ß-1,4-GalT was identified from Aggregatibacter actinomycetemcomitans. The enzyme was easily overexpressed in E. coli in soluble form. It displayed much higher transglycosylation versus hydrolysis activity, indicating its great potential in LNnT biosynthesis. The enzyme produced 13 mM LNnT from 20 mM LNT II and 60 mM UDP-Gal, with the yield of 65 % on LNT II and very low level of UDP-Gal hydrolysis. Therefore, it could be considered as a good candidate for the practical LNnT production.
Subject(s)
Aggregatibacter actinomycetemcomitans , Bacterial Proteins , N-Acetyllactosamine Synthase , Oligosaccharides/biosynthesis , Aggregatibacter actinomycetemcomitans/enzymology , Escherichia coli/genetics , HumansABSTRACT
High costs and low availability of UDP-galactose hampers the enzymatic synthesis of valuable oligosaccharides such as human milk oligosaccharides. Here, we report the development of a platform for the scalable, biocatalytic synthesis and purification of UDP-galactose. UDP-galactose was produced with a titer of 48â mM (27.2â g/L) in a small-scale batch process (200â µL) within 24â h using 0.02 genzyme /gproduct . Through in-situ ATP regeneration, the amount of ATP (0.6â mM) supplemented was around 240-fold lower than the stoichiometric equivalent required to achieve the final product yield. Chromatographic purification using porous graphic carbon adsorbent yielded UDP-galactose with a purity of 92 %. The synthesis was transferred to 1â L preparative scale production in a stirred tank bioreactor. To further reduce the synthesis costs here, the supernatant of cell lysates was used bypassing expensive purification of enzymes. Here, 23.4â g/L UDP-galactose were produced within 23â h with a synthesis yield of 71 % and a biocatalyst load of 0.05 gtotal_protein /gproduct . The costs for substrates per gram of UDP-galactose synthesized were around 0.26â /g.
Subject(s)
Enzymes/metabolism , Uridine Diphosphate Galactose/biosynthesis , Adenosine Triphosphate/metabolism , Bioreactors , Cell-Free System , Hydrogen-Ion Concentration , Oligosaccharides/biosynthesis , Proof of Concept Study , Uridine Diphosphate Galactose/isolation & purificationABSTRACT
A variety of glucosaccharides composed of glucosyl residues can be classified into α- and ß-type and have wide application in food and medicine areas. Among these glucosaccharides, ß-type, such as cellulose and α-type, such as starch and starch derivatives, both contain 1 â 4 linkages and are well studied. Notably, in past decades also α1 â 6 glucosaccharides obtained increasing attention for unique physiochemical and biological properties. Especially in recent years, α1 â 6 glucosaccharides of different molecular weight distribution have been created and proved to be functional. However, compared to ß- type and α1 â 4 glucosaccharides, only few articles provide a systematic overview of α1 â 6 glucosaccharides. This motivated, the present first comprehensive review on structure, function and synthesis of these α1 â 6 glucosaccharides, aiming both at improving understanding of traditional α1 â 6 glucosaccharides, such as isomaltose, isomaltooligosaccharides and dextrans, and to draw the attention to newly explored α1 â 6 glucosaccharides and their derivatives, such as cycloisomaltooligosaccharides, isomaltomegalosaccharides, and isomalto/malto-polysaccharides.
Subject(s)
Glycogen Debranching Enzyme System/metabolism , Oligosaccharides/biosynthesis , Oligosaccharides/metabolism , Carbohydrate Conformation , Oligosaccharides/chemistryABSTRACT
Morinda officinalis How (MO) is one of the best-known traditional herbs and is widely cultivated in subtropical and tropical areas for many years, especially in southern China. Oligosaccharides are the major constituents in the roots of MO, which is well known for its therapeutic effects with anti-depression, anti-osteoporosis, memory-enhancing, ect. To date, the main gene families that regulate the biosynthetic pathway of MO oligosaccharides metabolism yet have been published. In our study, six cDNA libraries generated from six plants of MO were sequenced utilizing an Illumina HiSeq 4000 platform. Corresponding totals of more than 132.60 million clean reads were obtained from the six libraries and assembled into 25,812 unigenes with an average length of 1288 bp. Moreover, 6036 unigenes were found to be allocated to 26 pathways maps using several public databases, and 2538 differential expression genes (DEGs) were screened. Among them, 25 genes from three families were selected as the mainly candidate genes related to MO oligosaccharides biosynthesis. Then, the expression patterns of six DEGs closely related to MO oligosaccharides biosynthesis were verified by quantitative real-time PCR (qRT-PCR). Besides, the MO was clustered more closely to Coffea arabica of Rubiaceae. In summary, the transcriptomic analysis was used to investigate the differences in expression genes of oligosaccharides biosynthesis, with the notable outcome that several key gene families were closely linked to oligosaccharides biosynthesis.
Subject(s)
Gene Expression Profiling , Morinda/genetics , Oligosaccharides/biosynthesis , Transcriptome , Gene Expression Regulation, Plant , Gene Library , Genes, Plant , High-Throughput Nucleotide Sequencing , Molecular Sequence Annotation , Morinda/metabolism , Multigene Family , Plant RootsABSTRACT
OBJECTIVE: To develop an endo-ß-1,4-xylanase with high specificity for production of prebiotic xylooligosaccharides that optimally works at moderate temperature desirable to reduce the energy cost in the production process. RESULTS: The xylB gene, encoding for a glycosyl hydrolase family 11 xylanase from a thermoresistant fungus, Aspergillus niger BCC14405 was expressed in a methylotrophic yeast P. pastoris KM71 in a secreted form. The recombinant XylB showed a high specific activity of 3852 and 169 U mg-1 protein on beechwood xylan and arabinoxylan, respectively with no detectable side activities against different forms of cellulose (Avicel Ò PH101 microcrystalline cellulose, phosphoric acid swollen cellulose and carboxymethylcellulose). The enzyme worked optimally at 45 °C, pH 6.0. It showed a specific cleavage pattern by releasing xylobiose (X2) as the major product from xylooligosaccharides (X3 to X6) substrates. The highest XOS yield of 708 mg g-1 substrate comprising X2, X3 and X6 was obtained from beechwood xylan hydrolysis. CONCLUSION: The enzyme is potent for XOS production and for saccharification of lignocellulosic biomass.
Subject(s)
Aspergillus niger/chemistry , Endo-1,4-beta Xylanases/genetics , Glucuronates/biosynthesis , Oligosaccharides/biosynthesis , Xylans/metabolism , Aspergillus niger/enzymology , Endo-1,4-beta Xylanases/isolation & purification , Enzyme Stability/genetics , Glucuronates/chemistry , Hydrogen-Ion Concentration , Hydrolysis , Oligosaccharides/chemistry , Substrate Specificity , Temperature , Xylans/geneticsABSTRACT
The xyloglucanase gene (RmXEG12A) from Rhizomucor miehei CAU432 was successfully expressed in Pichia pastoris. The highest xyloglucanase activity of 25,700 U mL-1 was secreted using high cell density fermentation. RmXEG12A was optimally active at pH 7.0 and 65 °C, respectively. The xyloglucanase exhibited the highest specific activity towards xyloglucan (7915.5 U mg-1). RmXEG12A was subjected to hydrolyze tamarind powder to produce xyloglucan oligosaccharides with the degree of polymerization (DP) 7-9. The hydrolysis ratio of xyloglucan in tamarind powder was 89.8%. Moreover, xyloglucan oligosaccharides (2.0%, w/w) improved the water holding capacity (WHC) of yoghurt by 1.1-fold and promoted the growth of Lactobacillus bulgaricus and Streptococcus thermophiles by 2.3 and 1.6-fold, respectively. Therefore, a suitable xyloglucanase for tamarind powder hydrolysis was expressed in P. pastoris at high level and xyloglucan oligosaccharides improved the quality of yoghurt.
Subject(s)
Glucans/biosynthesis , Glycoside Hydrolases/metabolism , Oligosaccharides/biosynthesis , Rhizomucor/enzymology , Saccharomycetales/metabolism , Xylans/biosynthesis , Yogurt , Enzyme Stability , Glucans/isolation & purification , Glycoside Hydrolases/isolation & purification , Hydrogen-Ion Concentration , Hydrolysis , Lactobacillus delbrueckii/growth & development , Molecular Weight , Oligosaccharides/isolation & purification , Streptococcus/growth & development , Tamarindus/chemistry , Temperature , Time Factors , Xylans/isolation & purificationABSTRACT
BACKGROUND: The Sda antigen and corresponding biosynthetic enzyme B4GALNT2 are primarily expressed in normal colonic mucosa and are down-regulated to a variable degree in colon cancer tissues. Although their expression profile is well studied, little is known about the underlying regulatory mechanisms. METHODS: To clarify the molecular basis of Sda expression in the human gastrointestinal tract, we investigated the transcriptional regulation of the human B4GALNT2 gene. The proximal promoter region was delineated using luciferase assays and essential trans-acting factors were identified through transient overexpression and silencing of several transcription factors. RESULTS: A short cis-regulatory region restricted to the -72 to +12 area upstream of the B4GALNT2 short-type transcript variant contained the essential promoter activity that drives the expression of the human B4GALNT2 regardless of the cell type. We further showed that B4GALNT2 transcriptional activation mostly requires ETS1 and to a lesser extent SP1. CONCLUSIONS: Results presented herein are expected to provide clues to better understand B4GALNT2 regulatory mechanisms.
Subject(s)
N-Acetylgalactosaminyltransferases/genetics , Proto-Oncogene Protein c-ets-1/metabolism , Colon , HT29 Cells , Humans , Intestinal Mucosa , N-Acetylgalactosaminyltransferases/metabolism , Oligosaccharides/biosynthesis , Promoter Regions, Genetic , Sp1 Transcription Factor/metabolism , Transcriptional ActivationABSTRACT
Raffinose, stachyose and verbascose form the three major members of the raffinose family oligosaccharides (RFO) accumulated during seed development. Raffinose synthase (RS; EC 2.4.1.82) and stachyose synthase (STS; EC 2.4.1.67) have been associated with raffinose and stachyose synthesis, but the precise mechanism for verbascose synthesis is not well understood. In this study, full-length RS (2.7 kb) and STS (2.6 kb) clones were isolated by screening a cDNA library prepared from developing lentil seeds (18, 20, 22 and 24 days after flowering [DAF]) to understand the roles of RS and STS in RFO accumulation in developing lentil seeds. The nucleotide sequences of RS and STS genes were similar to those reported for Pisum sativum. Patterns of transcript accumulation, enzyme activities and RFO concentrations were also comparable to P. sativum. However, during lentil seed development raffinose, stachyose and verbascose accumulation corresponded to transcript accumulation for RS and STS, with peak transcript abundance occurring at about 22-24 DAF, generally followed by a sequential increase in raffinose, stachyose and verbascose concentrations followed by a steady level thereafter. Enzyme activities for RS, STS and verbascose synthase (VS) also indicated a sudden increase at around 24-26 DAF, but with an abrupt decline again coinciding with the subsequent steady state increase in the RFO. Galactan:galactan galactosyl transferase (GGT), the galactinol-independent pathway enzyme, however, exhibited steady increase in activity from 24 DAF onwards before abruptly decreasing at 34 DAF. Although GGT activity was detected, isolation of a GGT sequence from the cDNA library was not successful.
Subject(s)
Galactosyltransferases/genetics , Galactosyltransferases/metabolism , Lens Plant/enzymology , Lens Plant/genetics , Oligosaccharides/biosynthesis , Raffinose/biosynthesis , Seeds/growth & development , Gene Expression Regulation, Plant , Genes, Plant , Lens Plant/growth & development , Oligosaccharides/genetics , Raffinose/genetics , Seeds/enzymology , Seeds/geneticsABSTRACT
The enzyme ß-galactosidase can synthesise novel prebiotics such as oligosaccharides derived from lactulose (OsLu) which can be added as a supplement in infant food formula. In this study, the intracellular ß-galactosidase produced by the alkaliphilic bacterium Paracoccus marcusii was extracted and purified to homogeneity using hydrophobic and metal affinity chromatography. The purification resulted in 18 U/mg specific activity, with a yield of 8.86% and an 18-fold increase in purity. The purified enzyme was a monomer with an 86 kDa molecular weight as determined by SDS PAGE and Q-TOF-LC/MS. ß-Galactosidase was highly active at 50 °C and pH 6-8. The enzyme displayed an alkali tolerant nature by maintaining more than 90% of its initial activity over a pH range of 5-9 after 3 h of incubation. Furthermore, the enzyme activity was enhanced by 37% in the presence of 5 M NaCl and 3 M KCl, indicating its halophilic nature. The effects of metal ions, solvents, and other chemicals on enzyme activity were also studied. The kinetic parameters KM and Vmax of ß-galactosidase were 1 mM and 8.56 µmoles/ml/min and 72.72 mM and 11.81 µmoles/ml/min on using oNPG and lactose as substrates. P. marcusii ß-galactosidase efficiently catalysed the transgalactosylation reaction and synthesised 57 g/L OsLu from 300 g/L lactulose at 40 °C. Thus, in this study we identified a new ß-galactosidase from P. marcusii that can be used for the industrial production of prebiotic oligosaccharides.
Subject(s)
Lactulose/metabolism , Oligosaccharides/biosynthesis , Paracoccus/enzymology , Prebiotics , beta-Galactosidase/metabolism , Biocatalysis , Carbohydrate Conformation , Kinetics , Lactulose/chemistry , Oligosaccharides/chemistryABSTRACT
The Sda carbohydrate antigen and the corresponding biosynthetic enzyme B4GALNT2 are primarily expressed in human normal colonic mucosa and are down-regulated to variable degrees in colon cancer. On the other hand, the tumor associated antigen SLex is not detected in the healthy colon and is upregulated in colon cancer. High level of B4GALNT2 gene expression appears to be a good marker of prognosis in colon cancer; however, the molecular mechanisms regulating these carbohydrate antigens' expression are still poorly understood. We review here the most recent progress made towards understanding this balanced expression of blood group carbohydrate epitopes Sda and SLex . In particular in recent years, we have attained a better understanding of genetic and epigenetic regulation of the B4GALNT2 gene and of the subcellular fate of B4GALNT2 isoforms.
Subject(s)
Colon/metabolism , Colonic Neoplasms/metabolism , N-Acetylgalactosaminyltransferases/metabolism , Oligosaccharides/biosynthesis , Sialyl Lewis X Antigen/biosynthesis , Colonic Neoplasms/diagnosis , Humans , PrognosisABSTRACT
BACKGROUND: Saccharomyces cerevisiae var. boulardii is a representative probiotic yeast that has been widely used in the food and pharmaceutical industries. However, S. boulardii has not been studied as a microbial cell factory for producing useful substances. Agarose, a major component of red macroalgae, can be depolymerized into neoagarooligosaccharides (NAOSs) by an endo-type ß-agarase. NAOSs, including neoagarotetraose (NeoDP4), are known to be health-benefiting substances owing to their prebiotic effect. Thus, NAOS production in the gut is required. In this study, the probiotic yeast S. boulardii was engineered to produce NAOSs by expressing an endo-type ß-agarase, BpGH16A, derived from a human gut bacterium Bacteroides plebeius. RESULTS: In total, four different signal peptides were compared in S. boulardii for protein (BpGH16A) secretion for the first time. The SED1 signal peptide derived from Saccharomyces cerevisiae was selected as optimal for extracellular production of NeoDP4 from agarose. Expression of BpGH16A was performed in two ways using the plasmid vector system and the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system. The production of NeoDP4 by engineered S. boulardii was verified and quantified. NeoDP4 was produced by S. boulardii engineered using the plasmid vector system and CRISPR-Cas9 at 1.86 and 0.80 g/L in a 72-h fermentation, respectively. CONCLUSIONS: This is the first report on NAOS production using the probiotic yeast S. boulardii. Our results suggest that S. boulardii can be considered a microbial cell factory to produce health-beneficial substances in the human gut.
Subject(s)
Metabolic Engineering/methods , Oligosaccharides/biosynthesis , Probiotics/metabolism , Saccharomyces boulardii/metabolism , Bacteroides/genetics , Fermentation , Glycoside Hydrolases/chemistry , Glycoside Hydrolases/genetics , Humans , Oligosaccharides/chemistry , Oligosaccharides/genetics , Saccharomyces boulardii/genetics , Saccharomyces cerevisiae/classification , Sepharose/metabolismABSTRACT
Sialo-oligosaccharides are important products of emerging biotechnology for complex carbohydrates as nutritional ingredients. Cascade bio-catalysis is central to the development of sialo-oligosaccharide production systems, based on isolated enzymes or whole cells. Multienzyme transformations have been established for sialo-oligosaccharide synthesis from expedient substrates, but systematic engineering analysis for the optimization of such transformations is lacking. Here, we show a mathematical modeling-guided approach to 3'-sialyllactose (3SL) synthesis from N-acetyl- d-neuraminic acid (Neu5Ac) and lactose in the presence of cytidine 5'-triphosphate, via the reactions of cytidine 5'-monophosphate-Neu5Ac synthetase and α2,3-sialyltransferase. The Neu5Ac was synthesized in situ from N-acetyl- d-mannosamine using the reversible reaction with pyruvate by Neu5Ac lyase or the effectively irreversible reaction with phosphoenolpyruvate by Neu5Ac synthase. We show through comprehensive time-course study by experiment and modeling that, due to kinetic rather than thermodynamic advantages of the synthase reaction, the 3SL yield was increased (up to 75%; 10.4 g/L) and the initial productivity doubled (15 g/L/h), compared with synthesis based on the lyase reaction. We further show model-based optimization to minimize the total loading of protein (saving: up to 43%) while maintaining a suitable ratio of the individual enzyme activities to achieve 3SL target yield (61%-75%; 7-10 g/L) and overall productivity (3-5 g/L/h). Collectively, our results reveal the principal factors of enzyme cascade efficiency for 3SL synthesis and highlight the important role of engineering analysis to make multienzyme-catalyzed transformations fit for oligosaccharide production.