Novel DNA-Binding Activity Exhibited by Poly(aspartic acid) Hydrolase-1 Inhibits Poly(aspartic acid) Hydrolase Activity.

Significant attention has been shifted toward the use and development of biodegradable polymeric materials to mitigate environmental accumulation and potential health impacts. One such material, poly(aspartic acid) (PAA), is a biodegradable alternative to superabsorbent poly(carboxylates), like poly(acrylate). Three enzymes are known to hydrolyze PAA: PahZ1KT-1 and PahZ2KT-1 from Sphingomonas sp. KT-1 and PahZ1KP-2 from Pedobacter sp. KP-2. We previously reported the X-ray crystal structure for PahZ1KT-1, which revealed a homodimer complex with a strongly cationic surface spanning one side of each monomer. Here, we report the first characterization of any polymer hydrolase binding to DNA, where modeling data predict binding of the polyanionic DNA near the cationic substrate binding surface. Our data reveal that PahZ1 homologues from Sphingomonas sp. KT-1 and Pedobacter sp. KP-2 bind ssDNA and dsDNA with nanomolar binding affinities. PahZ1KT-1 binds ssDNA and dsDNA with an apparent dissociation constant, KD,app = 81 ± 14 and 19 ± 1 nM, respectively, and these estimates are similar to the same behaviors exhibited by PahZ1KP-2. Gel permeation chromatography data reveal that dsDNA binding promotes inhibition of PahZ1-catalyzed PAA biodegradation for each homologue. We propose a working model wherein binding of PahZ1 to extracellular biofilm DNA aids in the localization of the hydrolase to the environment in which PAA would first be encountered, thereby providing a mechanism to degrade extracellular PAA and potentially harvest aspartic acid for nutritional uptake.

Purification, Characterization, and Structural Studies of a Sulfatase from Pedobacter yulinensis.

Sulfatases are ubiquitous enzymes that hydrolyze sulfate from sulfated organic substrates such as carbohydrates, steroids, and flavones. These enzymes can be exploited in the field of biotechnology to analyze sulfated metabolites in humans, such as steroids and drugs of abuse. Because genomic data far outstrip biochemical characterization, the analysis of sulfatases from published sequences can lead to the discovery of new and unique activities advantageous for biotechnological applications. We expressed and characterized a putative sulfatase (PyuS) from the bacterium Pedobacter yulinensis. PyuS contains the (C/S)XPXR sulfatase motif, where the Cys or Ser is post-translationally converted into a formylglycine residue (FGly). His-tagged PyuS was co-expressed in Escherichia coli with a formylglycine-generating enzyme (FGE) from Mycobacterium tuberculosis and purified. We obtained several crystal structures of PyuS, and the FGly modification was detected at the active site. The enzyme has sulfatase activity on aromatic sulfated substrates as well as phosphatase activity on some aromatic phosphates; however, PyuS did not have detectable activity on 17α-estradiol sulfate, cortisol 21-sulfate, or boldenone sulfate.

Biochemical characterization of a novel α-L-fucosidase from Pedobacter sp. and its application in synthesis of 3'-fucosyllactose and 2'-fucosyllactose.

Fucosyllactoses have gained much attention owing to their multiple functions, including prebiotic, immune, gut, and cognition benefits. In this study, human milk oligosaccharide (HMO) 2'-fucosyllactose (α-L-Fuc-(1,2)-D-Galß-1,4-Glu, 2'FL) and its isomer 3'-fucosyllactose (α-L-Fuc-(1,3)-D-Galß-1,4-Glu, 3'FL) with potential prebiotic effect were synthesized efficiently by a novel recombinant α-L-fucosidase. An α-L-fucosidase gene (PbFuc) from Pedobacter sp. CAU209 was successfully cloned and expressed in Escherichia coli (E. coli). The deduced amino acid sequence shared the highest identity of 36.8% with the amino sequences of other reported α-L-fucosidases. The purified α-L-fucosidase (PbFuc) had a molecular mass of 50 kDa. The enzyme exhibited specific activity (26.3 U/mg) towards 4-nitrophenyl-α-L-fucopyranoside (pNP-FUC), 3'FL (8.9 U/mg), and 2'FL (3.4 U/mg). It showed the highest activity at pH 5.0 and 35 °C, respectively. PbFuc catalyzed the synthesis of 3'FL and 2'FL through a transglycosylation reaction using pNP-FUC as donor and lactose as acceptor, and total conversion ratio was up to 85% at the optimized reaction conditions. The synthesized mixture of 2'FL and 3'FL promoted the growth of Lactobacillus delbrueckii subsp. bulgaricus NRRL B-548, L. casei subsp. casei NRRL B-1922, L. casei subsp. casei AS 1.2435, and Bifidobacterium longum NRRL B-41409. However, the growths of E. coli ATCC 11775, S. enterica AS 1.1552, L. monocytogenes CICC 21635, and S. aureus AS 1.1861 were not stimulated by the mixture of 2'FL and 3'FL. Overall, our findings suggest that PbFuc possesses a great potential for the specific synthesis of fucosylated compounds.Key Points• A novel α-L-fucosidase (PbFuc) from Pedobacter sp. was cloned and expressed.• PbFuc showed the highest hydrolysis activity at pH 5.0 and 35 °C, respectively.• It was used for synthesis of 3'-fucosyllactose (3'FL) and 2'-fucosyllactose (2'FL).• The mixture of 3'FL and 2'FL promoted the growth of some Lactobacillus sp. and Bifidobacteria sp.

Cloning, expression, and characterization of a novel heparinase I from Bacteroides eggerthii.

Heparinase I (Hep I) specifically degrades heparin to oligosaccharide or unsaturated disaccharide and has been widely used in preparation of low molecular weight heparin (LMWH). In this work, a novel Hep I from Bacteroides eggerthii VPI T5-42B-1 was cloned and overexpressed in Escherichia coli BL21 (DE3). The enzyme has specific activity of 480 IU·mg-1 at the optimal temperature and pH of 30 °C and pH 7.5, and the Km and Vmax were 3.6 mg·mL-1 and 647.93 U·mg-1, respectively. The Hep I has good stability with t1/2 values of 350 and 60 min at 30 and 37 °C, respectively. And it showed a residual relative activity of 70.8% after 21 days incubation at 4 °C. Substrate docking study revealed that Lys99, Arg101, Gln241, Lys270, Asn275, and Lys292 were mainly involved in the substrate binding of Hep I. The shorter hydrogen bonds formed between heparin and these residues suggested the higher specific activity of BeHep I. And the minimum conformational entropy value of 756 J·K-1 provides an evidence for the improved stability of this enzyme. This Hep I could be of interest in the industrial preparation of LMWH for its high specific activity and good stability.

Negative-Ion Mode Capillary Isoelectric Focusing Mass Spectrometry for Charge-Based Separation of Acidic Oligosaccharides.

Glycosaminoglycans (GAGs) are biologically and pharmacologically important linear, anionic polysaccharides containing various repeating disaccharides sequences. The analysis of these polysaccharides generally relies on their chemical or enzymatic breakdown to disaccharide units that are separated, by chromatography or electrophoresis, and detected, by UV, fluorescence, or mass spectrometry (MS). Isoelectric focusing (IEF) is an important analytical technique with high resolving power for the separation of analytes exhibiting differences in isoelectric points. One format of IEF, the capillary isoelectric focusing (cIEF), is an attractive approach in that it can be coupled with mass spectrometry (cIEF-MS) to provide online focusing and detection of complex mixtures. In the past three decades, numerous studies have applied cIEF-MS methods to the analysis of protein and peptide mixtures by positive-ion mode mass spectrometry. However, polysaccharide chemists largely rely on negative-ion mode mass spectrometry for the analysis of highly sulfated GAGs. The current study reports a negative-ion mode cIEF-MS method using an electrokinetically pumped sheath liquid nanospray capillary electrophoresis-mass spectrometry (CE-MS) coupling technology. The feasibility of this negative-ion cIEF-MS method and its potential applications are demonstrated using chondroitin sulfate and heparan sulfate oligosaccharides mixtures.

SMUG2 DNA glycosylase from Pedobacter heparinus as a new subfamily of the UDG superfamily.

Base deamination is a common type of DNA damage that occurs in all organisms. DNA repair mechanisms are essential to maintain genome integrity, in which the base excision repair (BER) pathway plays a major role in the removal of base damage. In the BER pathway, the uracil DNA glycosylase superfamily is responsible for excising the deaminated bases from DNA and generates apurinic/apyrimidinic (AP) sites. Using bioinformatics tools, we identified a family 3 SMUG1-like DNA glycoyslase from Pedobacter heparinus (named Phe SMUG2), which displays catalytic activities towards DNA containing uracil or hypoxanthine/xanthine. Phylogenetic analyses show that SMUG2 enzymes are closely related to family 3 SMUG1s but belong to a distinct branch of the family. The high-resolution crystal structure of the apoenzyme reveals that the general fold of Phe SMUG2 resembles SMUG1s, yet with several distinct local structural differences. Mutational studies, coupled with structural modeling, identified several important amino acid residues for glycosylase activity. Substitution of G65 with a tyrosine results in loss of all glycosylase activity. The crystal structure of the G65Y mutant suggests a potential misalignment at the active site due to the mutation. The relationship between the new subfamily and other families in the UDG superfamily is discussed. The present study provides new mechanistic insight into the molecular mechanism of the UDG superfamily.

High-level expression and characterization of a new κ-carrageenase from marine bacterium Pedobacter hainanensis NJ-02.

A novel κ-carrageenase gene (CgkB) has been cloned from Pedobacter hainanensis NJ-02 and expressed heterologously in Escherichia coli BL21 (DE3). It consisted of 1935 bp and encoded 644 amino acid residues with a molecular weight of 71·61 kDa. The recombinant enzyme showed maximal activity of 2458 U mg-1 at 40°C and pH 8·0. Additionally, it could retain more than 70% of its maximal activity after being incubated at pH of 5·5-10·0 below 40°C. K+ and a broad range of NaCl can activate the enzyme. The Km and Vmax of CgkB was 2·4 mg ml-1 and 126 mmol mg-1  min-1 . The ESI-MS analysis of hydrolysates indicated that the enzyme can endolytically depolymerize the carrageenan into tetrasaccharides and hexasaccharides. The results indicated that the enzyme with high activity could be a valuable enzyme tool to produce carrageenan oligosaccharides with various activities. SIGNIFICANCE AND IMPACT OF THE STUDY: Enzymatic preparation of carrageenan oligosaccharides has drawn increased attention due to their various physiological activities. It is urgent to explore enzyme tools with higher activity and better stability. In this work, a novel κ-carrageenase was identified and characterized from marine bacterium Pedobacter hainanensis NJ-02. The enzyme with high activity could be a valuable tool to produce carrageenan oligosaccharides with various activities.

Identification, purification, and expression patterns of chitinase from psychrotolerant Pedobacter sp. PR-M6 and antifungal activity in vitro.

In this study, a novel psychrotolerant chitinolytic bacterium Pedobacter sp. PR-M6 that displayed strong chitinolytic activity on 0.5% colloidal chitin was isolated from the soil of a decayed mushroom. Chitinase activity of PR-M6 at 25 °C (C25) after 6 days of incubation with colloidal chitin increased rapidly to a maximum level (31.3 U/mg proteins). Three chitinase isozymes (chiII, chiIII, and chiIV) from the crude enzyme at 25 °C (C25) incubation were expressed on SDS-PAGE gels at 25 °C. After purification by chitin-affinity chromatography, six chitinase isozymes (chiI, chiII, chiIII, chiIV, chiV, and chiVI) from C25-fractions were expressed on SDS-PAGE gels at 25 °C. Major bands of chitinase isozymes (chiI, chiII, and chiIII) from C4-fractions were strongly expressed on SDS-PAGE gels at 25 °C. Pedobacter sp. PR-M6 showed high inhibition rate of 60.9% and 57.5% against Rhizoctonia solani and Botrytis cinerea, respectively. These results indicated that psychrotolerant Pedobacter sp. PR-M6 could be applied widely as a microorganism agent for the biocontrol of agricultural phytopathogens at low temperatures.

Expanding the Catalytic Promiscuity of Heparinase†III from Pedobacter heparinus.

Glycosaminoglycans (GAG) lyases are useful biocatalysts for the preparation of oligosaccharides, but their substrate spectra are limited to the same family. Thus, the degradation activity across families of GAG lyases is advantageous and desirable for various applications. In this study, residue Lys130 at the substrate entrance of monomeric heparinase III from Pedobacter heparinus ATCC 13125 was replaced by cysteine, and the resulting mutant K130C showed novel catalytic activity in degrading hyaluronic acid without affecting its native activity toward heparin and heparan sulfate. The broadened catalytic promiscuity by mutant K130C was the result of dimerization through a disulfide bond to expand the substrate binding pocket. This bifunctional enzyme is potentially valuable in the degradation of different types of GAGs.

The Soil Microbiota Harbors a Diversity of Carbapenem-Hydrolyzing ß-Lactamases of Potential Clinical Relevance.

The origin of carbapenem-hydrolyzing metallo-ß-lactamases (MBLs) acquired by clinical bacteria is largely unknown. We investigated the frequency, host range, diversity, and functionality of MBLs in the soil microbiota. Twenty-five soil samples of different types and geographical origins were analyzed by antimicrobial selective culture, followed by phenotypic testing and expression of MBL-encoding genes in Escherichia coli, and whole-genome sequencing of MBL-producing strains was performed. Carbapenemase activity was detected in 29 bacterial isolates from 13 soil samples, leading to identification of seven new MBLs in presumptive Pedobacter roseus (PEDO-1), Pedobacter borealis (PEDO-2), Pedobacter kyungheensis (PEDO-3), Chryseobacterium piscium (CPS-1), Epilithonimonas tenax (ESP-1), Massilia oculi (MSI-1), and Sphingomonas sp. (SPG-1). Carbapenemase production was likely an intrinsic feature in Chryseobacterium and Epilithonimonas, as it occurred in reference strains of different species within these genera. The amino acid identity to MBLs described in clinical bacteria ranged between 40 and 69%. Remarkable features of the new MBLs included prophage integration of the encoding gene (PEDO-1), an unusual amino acid residue at a key position for MBL structure and catalysis (CPS-1), and overlap with a putative OXA ß-lactamase (MSI-1). Heterologous expression of PEDO-1, CPS-1, and ESP-1in E. coli significantly increased the MICs of ampicillin, ceftazidime, cefpodoxime, cefoxitin, and meropenem. Our study shows that MBL producers are widespread in soil and include four genera that were previously not known to produce MBLs. The MBLs produced by these bacteria are distantly related to MBLs identified in clinical samples but constitute resistance determinants of clinical relevance if acquired by pathogenic bacteria.

Poly(aspartic acid) (PAA) hydrolases and PAA biodegradation: current knowledge and impact on applications.

Thermally synthesized poly(aspartic acid) (tPAA) is a bio-based, biocompatible, biodegradable, and water-soluble polymer that has a high proportion of ß-Asp units and equivalent moles of D- and L-Asp units. Poly(aspartic acid) (PAA) hydrolase-1 and hydrolase-2 are tPAA biodegradation enzymes purified from Gram-negative bacteria. PAA hydrolase-1 selectively cleaves amide bonds between ß-Asp units via an endo-type process, whereas PAA hydrolase-2 catalyzes the exo-type hydrolysis of the products of tPAA hydrolysis by PAA hydrolase-1. The novel reactivity of PAA hydrolase-1 makes it a good candidate for a biocatalyst in ß-peptide synthesis. This mini-review gives an overview of PAA hydrolases with emphasis on their biochemical and functional properties, in particular, PAA hydrolase-1. Functionally related enzymes, such as poly(R-3-hydroxybutyrate) depolymerases and ß-aminopeptidases, are compared to PAA hydrolases. This mini-review also provides findings that offer an insight into the catalytic mechanisms of PAA hydrolase-1 from Pedobacter sp. KP-2.

Structural and biochemical characterization of novel bacterial α-galactosidases belonging to glycoside hydrolase family 31.

Glycoside hydrolase family 31 (GH31) proteins have been reportedly identified as exo-α-glycosidases with activity for α-glucosides and α-xylosides. We focused on a GH31 subfamily, which contains proteins with low sequence identity (<24%) to the previously reported GH31 glycosidases and characterized two enzymes from Pedobacter heparinus and Pedobacter saltans. The enzymes unexpectedly exhibited α-galactosidase activity, but were not active on α-glucosides and α-xylosides. The crystal structures of one of the enzymes, PsGal31A, in unliganded form and in complexes with D-galactose or L-fucose and the catalytic nucleophile mutant in unliganded form and in complex with p-nitrophenyl-α-D-galactopyranoside, were determined at 1.85-2.30 Å (1 Å=0.1 nm) resolution. The overall structure of PsGal31A contains four domains and the catalytic domain adopts a (ß/α)8-barrel fold that resembles the structures of other GH31 enzymes. Two catalytic aspartic acid residues are structurally conserved in the enzymes, whereas most residues forming the active site differ from those of GH31 α-glucosidases and α-xylosidases. PsGal31A forms a dimer via a unique loop that is not conserved in other reported GH31 enzymes; this loop is involved in its aglycone specificity and in binding L-fucose. Considering potential genes for α-L-fucosidases and carbohydrate-related proteins within the vicinity of Pedobacter Gal31, the identified Gal31 enzymes are likely to function in a novel sugar degradation system. This is the first report of α-galactosidases which belong to GH31 family.

Crystal structure of a bacterial unsaturated glucuronyl hydrolase with specificity for heparin.

Extracellular matrix molecules such as glycosaminoglycans (GAGs) are typical targets for some pathogenic bacteria, which allow adherence to host cells. Bacterial polysaccharide lyases depolymerize GAGs in ß-elimination reactions, and the resulting unsaturated disaccharides are subsequently degraded to constituent monosaccharides by unsaturated glucuronyl hydrolases (UGLs). UGL substrates are classified as 1,3- and 1,4-types based on the glycoside bonds. Unsaturated chondroitin and heparin disaccharides are typical members of 1,3- and 1,4-types, respectively. Here we show the reaction modes of bacterial UGLs with unsaturated heparin disaccharides by x-ray crystallography, docking simulation, and site-directed mutagenesis. Although streptococcal and Bacillus UGLs were active on unsaturated heparin disaccharides, those preferred 1,3- rather than 1,4-type substrates. The genome of GAG-degrading Pedobacter heparinus encodes 13 UGLs. Of these, Phep_2830 is known to be specific for unsaturated heparin disaccharides. The crystal structure of Phep_2830 was determined at 1.35-Å resolution. In comparison with structures of streptococcal and Bacillus UGLs, a pocket-like structure and lid loop at subsite +1 are characteristic of Phep_2830. Docking simulations of Phep_2830 with unsaturated heparin disaccharides demonstrated that the direction of substrate pyranose rings differs from that in unsaturated chondroitin disaccharides. Acetyl groups of unsaturated heparin disaccharides are well accommodated in the pocket at subsite +1, and aromatic residues of the lid loop are required for stacking interactions with substrates. Thus, site-directed mutations of the pocket and lid loop led to significantly reduced enzyme activity, suggesting that the pocket-like structure and lid loop are involved in the recognition of 1,4-type substrates by UGLs.

Exploring bacterial heparinase II activities with defined substrates.

Bacterial heparinases that cleave heparan sulfate (HS) and heparin are widely used to generate low-molecular-weight heparins (LMWHs) and to structurally and functionally characterise heparin and HS biomolecules. We provide novel insights into the substrate specificity of heparinase II from two different bacteria: Pedobacter heparinus (formerly Flavobacterium heparinum) and Bacteroides eggerthii. The activity towards various well-defined HS oligosaccharides was investigated by (1) H NMR spectroscopy; this revealed distinct specificities for the two heparinases. Heparinase II from P. heparinus appears to be more active and displays a broader substrate specificity than B. eggerthii heparinase II. Furthermore, HS di- and tetrasaccharides inhibited B. eggerthii heparinase II activity. A better understanding of heparinase substrate specificity will contribute to the production of homogenous LMWHs, provide better characterisation of heparin and HS and assist therapeutic applications.

Crystal structure of Pedobacter heparinus heparin lyase Hep III with the active site in a deep cleft.

Pedobacter heparinus (formerly known as Flavobacterium heparinum) is a typical glycosaminoglycan-degrading bacterium that produces three heparin lyases, Hep I, Hep II, and Hep III, which act on heparins with 1,4-glycoside bonds between uronate and amino sugar residues. Being different from Hep I and Hep II, Hep III is specific for heparan sulfate. Here we describe the crystal structure of Hep III with the active site located in a deep cleft. The X-ray crystallographic structure of Hep III was determined at 2.20 Å resolution using single-wavelength anomalous diffraction. This enzyme comprised an N-terminal α/α-barrel domain and a C-terminal antiparallel ß-sheet domain as its basic scaffold. Overall structures of Hep II and Hep III were similar, although Hep III exhibited an open form compared with the closed form of Hep II. Superimposition of Hep III and heparin tetrasaccharide-bound Hep II suggested that an active site of Hep III was located in the deep cleft at the interface between its two domains. Three mutants (N240A, Y294F, and H424A) with mutations at the active site had significantly reduced enzyme activity. This is the first report of the structure-function relationship of P. heparinus Hep III.

Epilactose production by 2 cellobiose 2-epimerases in natural milk.

It was reported recently that cellobiose 2-epimerases (CE) from various aerobic microorganisms convert lactose to epilactose in defined buffer systems. In this study, we showed that CE from 2 mesophilic microorganisms, Flavobacterium johnsoniae and Pedobacter heparinus, were capable of converting lactose to prebiotic epilactose not only in buffer but also in a complex milk system. First, the 2 enzymes were separately cloned, recombinantly expressed in Escherichia coli, and purified by column chromatography. The production of F. johnsoniae CE was carried out in a stirred-tank reactor, indicating that future upscaling is possible. The bioconversions of milk lactose were carried out at an industrially relevant low temperature of 8°C to avoid undesired microbial contaminations or chemical side reactions. Both enzymes were reasonably active at this low temperature, because of their origin from mesophilic organisms. The enzymes showed different operational stabilities over a 24-h time-course. A conversion yield of about 30 to 33% epilactose was achieved with both enzymes. No side products were detected other than epilactose. Therefore, CE may introduce an added value for particular dairy products by in situ production of the prebiotic sugar epilactose.

Production of unsulfated chondroitin and associated chondro-oligosaccharides in recombinant Escherichia coli.

We designed metabolically engineered non-pathogenic strains of Escherichia coli to produce unsulfated chondroitin with and without chondroitin lyase to produce the chondroitin polymer or its related oligosaccharides. Chondroitin was synthesized using chondroitin synthase KfoC and chondroitin was degraded using Pl35, a chondroitin lyase from Pedobacter heparinus. Pl35 behaved as a true endo-enzyme generating a large panel of oligosaccharides ranging from trimers to 18-mers instead of the di- and tetramers obtained with most chondroitin lyases. Two series of oligosaccharides were characterized, sharing an unsaturated uronic acid (4-deoxy-α-L-threo-hex-4-enepyranosyluronic acid, △UA) residue at their non-reducing end. The major "even-numbered" series was characterized by a terminal reducing N-acetylgalactosaminyl residue. The minor "odd-numbered" series oligosaccharides carried a terminal reducing glucuronic acid residue instead. Cultures were conducted in fed-batch conditions, and led to the production of up to 10 g L-1 chondroitin or chondroitin oligosaccharides. All products were purified and fully characterized using NMR and mass spectrometry analyses. This is the first report of the microbial production of large chondro-oligosaccharides.

A Combination of Structural, Genetic, Phenotypic and Enzymatic Analyses Reveals the Importance of a Predicted Fucosyltransferase to Protein O-Glycosylation in the Bacteroidetes.

Diverse members of the Bacteroidetes phylum have general protein O-glycosylation systems that are essential for processes such as host colonization and pathogenesis. Here, we analyzed the function of a putative fucosyltransferase (FucT) family that is widely encoded in Bacteroidetes protein O-glycosylation genetic loci. We studied the FucT orthologs of three Bacteroidetes species-Tannerella forsythia, Bacteroides fragilis, and Pedobacter heparinus. To identify the linkage created by the FucT of B. fragilis, we elucidated the full structure of its nine-sugar O-glycan and found that l-fucose is linked ß1,4 to glucose. Of the two fucose residues in the T. forsythia O-glycan, the fucose linked to the reducing-end galactose was shown by mutational analysis to be l-fucose. Despite the transfer of l-fucose to distinct hexose sugars in the B. fragilis and T. forsythia O-glycans, the FucT orthologs from B. fragilis, T. forsythia, and P. heparinus each cross-complement the B. fragilis ΔBF4306 and T. forsythia ΔTanf_01305 FucT mutants. In vitro enzymatic analyses showed relaxed acceptor specificity of the three enzymes, transferring l-fucose to various pNP-α-hexoses. Further, glycan structural analysis together with fucosidase assays indicated that the T. forsythia FucT links l-fucose α1,6 to galactose. Given the biological importance of fucosylated carbohydrates, these FucTs are promising candidates for synthetic glycobiology.

Enzymatic depolymerization of streptococcus pneumoniae type 8 polysaccharide.

Although there have been decades of research on streptococcus pneumoniae, it is still among the leading cause of infectious disease in the world. As a type of capsular polysaccharide (CPS) of streptococcus pneumoniae, pneumococcal polysaccharides are essential components for colonization and virulence in mammalian hosts. This study aimed to characterize the CPS structure of type 8 streptococcus pneumoniae, which is one of the most fatal serotypes. In this work, heparinase I&III was used to successfully digest pneumococcal type 8 polysaccharide (Pn8P). We characterized the oligosaccharide generated from the enzymatic depolymerization of Pn8P by size exclusion chromatography, mass spectrometry and nuclear magnetic resonance. This is the first study to enzymatically depolymerize and characterize Pn8P.

Engineering the heparin-binding pocket to enhance the catalytic efficiency of a thermostable heparinase III from Bacteroides thetaiotaomicron.

Heparinase has attracted much attention because of its applications in pharmaceutical industry. Herein, the heparinases III from Flavobacterium heparinum (FhepIII) and Bacteroides thetaiotaomicron (BhepIII) were firstly comparatively characterized. BhepIII showed higher catalytic activity and thermostability toward heparin comparing to FhepIII. To further upgraded BhepIII, a protein engineering approach based on B-factor was performed. By site-saturated mutagenesis of the flexible residues within an 8 Šradius around the catalytic residue, Asp321 and Ser264 were identified as essential residues for catalytic efficiency and thermostability, respectively. D321Q mutation enhanced catalytic efficiency (kcat/Km) with a 68.4% increase by increasing the surface potential while S264 F mutation increased thermostability with a half-time at 50℃ (t1/250℃) of 3.8 h versus 2.7 h of the wild-type by increasing rigidity and interactions within the active pocket. Double mutation of S264 F and D321Q resulted in a 245% increase in kcat/Km but with a decreased t1/250℃ (2.0 h). E105R mutation that generated a 348% increase in kcat/Km was further identified by electric potential engineering of the pocket tunnel. Eventually, the variant E105R/S264 F that showed a 418% increase in kcat/Km without compromise of thermostability was constructed. The engineered E105R/S264 F has a great potential for the commercial production of low molecular weight heparin in the future.