Cloning and functional characterization of an α-1,3-fucosyltransferase from Bacteroides fragilis

Bacteroides fragilis is a clinically important anaerobic pathogen present in the human gastrointestinal tract and is involved in a high number of anaerobic peritoneal infections. The complete genome sequence of B. fragilis NCTC 9343 revealed the presence of several putative fucosyltransferase gene homologues known as alpha-1,3-fucosyltransferases (α-1,3-FucTs). However, their expression and functional activities have not been studied. Here, we report the molecular cloning, functional expression, and characterization of the alpha-1,3-fucosyltransferase 3 (α-1,3-FucT3) enzyme from B. fragilis NCTC 9343. The polymerase chain reaction (PCR)-based approach was used to clone the 331 amino acid long (MW, ～39 kDa) PCR product encoding fucosyltransferase enzyme. The enzyme had low identity of 30–40% with other known α-1,3-FucTs from Azospirillum sp, Rickettsia bellii, and different strains of Helicobacter pylori. An in vitro enzyme reaction analysis showed the ability of the enzyme to transfer the fucose moiety from guanosine-5′-diphosphate β-l-fucose to the N-acetyllactosamine to produce Lewis X. The reaction product, Lewis X was confirmed by thin layer chromatography, liquid chromatography-mass spectroscopy, and ¹H-nuclear magnetic resonance analyses.     

Isolation and characterization of a gene encoding α-tubulin from Candida albicans

A gene encoding the α-tubulin of Candida albicans has been cloned and characterized. Nucleotide sequence analysis reveals the presence of an intron within the structural gene and predicts the synthesis of a polypeptide of 448 amino acid residues. Comparison of nucleotide and amino acid sequences with the Saccharomyces cerevisiae α-tubulin encoding genes shows a 75% homology and about 92% similarity respectively. In contrast to S. cerevisiae, C. albicans appears to possess only one gene for α-tubulin which is able to functionally complement a S. cerevisiae cold-sensitive tub1 mutant.     

Cloning,characterisation and expression analysis of α-glucuronidase from the thermophilic fungus Talaromyces emersonii

The aguA gene encoding α-glucuronidase was isolated from the thermophilic fungus Talaromyces emersonii by degenerate PCR. AguA has no introns and consists of an open reading frame of 2511 bp, encoding a putative protein of 837 amino acids. The N-terminus of the protein contains a putative signal peptide of 17 amino acids yielding a mature protein of 820 amino acids with a predicted molecular mass of 91.6 kDa. Twenty putative N-glycosylation sites and four O-glycosylation were identified. The T. emersonii α-glucuronidase falls into glycosyl hydrolase family 67, showing approximately 63% identity to similar enzymes from other fungi. Analysis of the aguA promoter revealed several possible regulatory motifs including two XlnR and a CreA binding site. Enzyme activity was optimal at 50 °C and pH 5. Enzyme production was investigated on a range of carbon sources and showed induction on beechwood, oat spelt and birchwood xylan, and repression by glucose or glucuronic acid.     

Production, purification and characterization of a constitutive intracellular α-galactosidase from the thermophilic fungus Humicola sp

The thermophilic fungus Humicola sp constitutively produces intracellular α-galactosidase (1.33 U mg⁻¹ protein) within 48 h at 45°C in shaken flasks, when grown in a medium containing 7% wheat bran extract as a carbon source and 0.5% yeast extract as a nitrogen source. The enzyme has been purified to homogeneity by ultrafiltration, ethanol precipitation, DEAE cellulose and Sephacryl S-300 chromatography with a 124-fold increase in specific activity and 29.5% recovery. The molecular weight of the enzyme is 371.5 kDa by gel filtration on Sephacryl S-300 and 87.1 kDa by SDS-polyacrylamide gel electrophoresis. The enzyme has an optimum temperature of 65°C and an optimum pH of 5.0. Humicola α-galactosidase is a glycoprotein with 8.3% carbohydrate content and is acidic in nature with a pI of 4.0. The K _m S for p-nitrophenyl-α-D-galactopyranoside, O-nitrophenyl-α-D-galactopyranoside, raffinose and stachyose are 0.279, 0.40, 1.45 and 1.42 mM respectively. The enzyme activity was strongly inhibited by Ag⁺ and Hg²⁺. D-Galactose inhibited α-galactosidase competitively and the inhibition constant (K _i) for galactose was 11 mM. Received 28 January 1999/ Accepted in revised form 07 April 1999     

Cloning,expression and functional validation of a β-fructofuranosidase from Lactobacillus plantarum

Fructooligosaccharides (FOS) are prebiotics that selectively stimulate the growth and activity of lactobacilli and bifidobacteria. These strains metabolize FOS with endogenous β-fructofuranosidase. In this study, a β-fructofuranosidase gene from Lactobacillus plantarum ST-III designated sacA was cloned into Escherichia coli, and the properties of the recombinant protein (SacA) were examined. The sacA gene encodes a peptide of 501 amino acids with a predicted molecular weight of 56.7 kDa. Sequence alignment revealed the presence of three highly conserved motifs, NDPNG, RDP and EC, indicating that the enzyme belongs to glycoside hydrolase family 32. The predicted three-dimensional structure of the SacA enzyme was similar to β-fructofuranosidases of bifidobacteria, such that it contained a five-blade β-propeller module and a β-sandwich domain with one additional N-terminal α-helix. The optimal reaction temperature and pH of the enzyme were 37 °C and 6.0, respectively. Substrate hydrolysis and kinetic parameters demonstrated that β-fructofuranosidase from L. plantarum ST-III liberated fructosyl residues from the non-reducing terminus of fructans, such as sucrose, FOS, levan or inulin, and FOS was the preferred substrate. The expression of the sacA gene in a non-FOS-fermenting strain, Lactobacillus rhamnosus GG, enabled the recombinant strain to metabolize FOS and sucrose.     

Cloning and functional characterization of a novel endo-β-1,4-glucanase gene from a soil-derived metagenomic library

A metagenomic library consisting of 3,024 bacterial artificial chromosome clones was prepared in Escherichia coli DH10B with high-molecular-weight DNA extracted from red soil in South China. A novel cellulase gene with an open reading frame of 1,332 bp, cel5G, encoding an endo-β-1,4-glucanase was cloned using an activity-based screen. The deduced enzyme, Cel5G, belongs to the glycosyl hydrolase family 5 and shares <39% identity with endoglucanases in the GenBank database. cel5G was expressed in E. coli BL21, and the recombinant enzyme Cel5G was purified to homogeneity for enzymatic analysis. Cel5G hydrolyzed a wide range of β-1,4-, β-1,3/β-1,4-, or β-1,3/β-1,6-linked polysaccharides, amorphous cellulose, filter paper, and microcrystalline cellulose. Its highest activity was in 50 mM citrate buffer, pH 4.8, at 50°C. Cel5G is stable over a wide range of pH values (from 2.0 to 10.6) and is thermally stable under 60°C. It is highly tolerant and active in high salt concentrations and is stable in the presence of pepsin and pancreatin. The K _m and V _max values of Cel5G for carboxymethyl cellulose are 19.92 mg/ml and 1,941 μmol min⁻¹ mg⁻¹, respectively. These characteristics indicate that Cel5G has potential for industrial use.     

Cloning,characterisation and expression of the α-tubulin genes of the leech,Hirudo medicinalis

We have isolated two α-tubulin cDNAs from the leech, Hirudo medicinalis. Both encode putative proteins of 451 amino-acids which differ from each other at only two positions. Southern blotting suggests that there are only two α-tubulin genes in the leech. The genes contain two introns and, because of the extremely high homology of the nucleotide sequence from the second intron to the end of the genes, we have inferred that a gene conversion event about 9.5 million years ago has homogenised the Hirudo α-tubulin sequences. Using in situ hybridisation to tissue sections, we have shown that the two genes are probably expressed in all neurons of the leech ganglia and that their spatial distribution remains unchanged during neuronal regeneration. The deduced amino-acid sequences of the leech α-tubulins show that they have greatest similarity to those from a platyhelminth, echiuran and mollusc with rather less to arthropod α-tubulins. The protein sequences of the leech α-tubulins have been compared with representatives of those from across all phyla to determine if any specific feature labels certain isotypes of tubulin for neuronal expression.     

Cloning and characterization of a DNA polymerase β gene from Trypanosoma cruzi

A gene coding for a DNA polymerase β from the Trypanosoma cruzi Miranda clone, belonging to the TcI lineage, was cloned (Miranda Tcpolβ), using the information from eight peptides of the T. cruzi β-like DNA polymerase purified previously. The gene encodes for a protein of 403 amino acids which is very similar to the two T. cruzi CL Brener (TcIIe lineage) sequences published, but has three different residues in highly conserved segments. At the amino acid level, the identity of TcI-polβ with mitochondrial polβ and polβ-PAK from other trypanosomatids was between 68–80% and 22–30%, respectively. Miranda Tc-polβ protein has an N-terminal sequence similar to that described in the mitochondrial Crithidia fasciculata polβ, which suggests that the TcI-polβ plays a role in the organelle. Northern and Western analyses showed that this T. cruzi gene is highly expressed both in proliferative and non-proliferative developmental forms. These results suggest that, in addition to replication of kDNA in proliferative cells, this enzyme may have another function in non-proliferative cells, such as DNA repair role similar to that which has extensively been described in a vast spectrum of eukaryotic cells.     

Cloning and functional characterization of endo-β-1,4-glucanase gene from metagenomic library of vermicompost

In the vermicomposting of paper mill sludge, the activity of earthworms is very dependent on dietetic polysaccharides including cellulose as energy sources. Most of these polymers are degraded by the host microbiota and considered potentially important source for cellulolytic enzymes. In the present study, a metagenomic library was constructed from vermicompost (VC) prepared with paper mill sludge and dairy sludge (fresh sludge, FS) and functionally screened for cellulolytic activities. Eighteen cellulase expressing clones were isolated from about 89,000 fosmid clones libraries. A short fragment library was constructed from the most active positive clone (cMGL504) and one open reading frame (ORF) of 1,092 bp encoding an endo-β-1,4-glucanase was indentified which showed 88% similarity with Cellvibrio mixtus cellulase A gene. The endo-β-1,4-glucanase cmgl504 gene was overexpressed in Escherichia coli. The purified recombinant cmgl504 cellulase displayed activities at a broad range of temperature (25–55°C) and pH (5.5–8.5). The enzyme degraded carboxymethyl cellulose (CMC) with 15.4 U, while having low activity against avicel. No detectable activity was found for xylan and laminarin. The enzyme activity was stimulated by potassium chloride. The deduced protein and three-dimensional structure of metagenome-derived cellulase cmgl504 possessed all features, including general architecture, signature motifs, and N-terminal signal peptide, followed by the catalytic domain of cellulase belonging to glycosyl hydrolase family 5 (GHF5). The cellulases cloned in this work may play important roles in the degradation of celluloses in vermicomposting process and could be exploited for industrial application in future.     

Cloning,purification and characterization of a thermostable β-galactosidase from Thermotoga naphthophila RUK-10

A novel β-galactosidase gene (Tnap1577) from the hyperthermophilic bacterium Thermotoga naphthophila RUK-10 was cloned and expressed in Escherichia coli BL21 (DE3) cells to produce β-galactosidase. The recombinant β-galactosidase was purified in three steps: heat treatment to deactivate E. coli proteins, Ni-NTA affinity chromatography and Q-sepharose chromatography. The optimum temperatures for the hydrolysis of o-nitrophenyl-β-d-galactoside (o-NPG) and lactose with the recombinant β-galactosidase were found to be 90 °C and 70 °C, respectively. The corresponding optimum pH values were 6.8 and 5.8, respectively. The molecular mass of the enzyme was estimated to be 70 kDa by SDS-PAGE analysis. Thermostability studies showed that the half-lives of the recombinant enzyme at 75 °C, 80 °C, 85 °C and 90 °C were 10.5, 4, 1, and 0.3 h, respectively. Kinetic studies on the recombinant β-galactosidase revealed K_m values for the hydrolysis of o-NPG and lactose of 1.31 mM and 1.43 mM, respectively. These values are considerably lower than those reported for other hyperthermophilic β-galactosidases, indicating high intrinsic affinity for these substrates. The recombinant β-galactosidase from Thermotoga naphthophila RUK-10 also showed transglycosylation activity in the synthesis of alkyl galactopyranoside. This additional activity suggests the enzyme has potential for broader biotechnological applications beyond the degradation of lactose.     

Cloning,expression and biochemical characterization of a GH1 β-glucosidase from Cellulosimicrobium cellulans

β-Glucosidase plays an important role in the degradation of cellulose. In this study, a novel β-glucosidase ccbgl1b gene for a glycosyl hydrolase (GH) family 1 enzyme was cloned from the genome of Cellulosimicrobium cellulans and expressed in Escherichia coli BL21 cells. The sequence contained an open reading frame of 1494?bp, encoded a polypeptide of 497?amino acid residues. The recombinant protein CcBgl1B was purified by Ni sepharose fastflow affinity chromatography and had a molecular weight of 57?kDa, as judged by SDS-PAGE. The optimum β-glucosidase activity was observed at 55?°C and pH 6.0. Recombinant CcBgl1B was found to be most active against aryl-glycosides p-nitrophenyl-β-D-glucopyranoside (pNPβGlc), followed by p-nitrophenyl-β-D-galactopyranoside (pNPβGal). Using disaccharides as substrates, the enzyme efficiently cleaved β-linked glucosyl-disaccharides, including sophorose (β-1,2-), laminaribiose (β-1,3-) and cellobiose (β-1,4-). In addition, a range of cello-oligosaccharides including cellotriose, cellotetraose and cellopentaose were hydrolysed by CcBgl1B to produce glucose. The interaction mode between the enzyme and the substrates driving the reaction was modelled using a molecular docking approach. Understanding how the GH1 enzyme CcBgl1B from C. cellulans works, particularly its activity against cello-oligosaccharides, would be potentially useful for biotechnological applications of cellulose degradation.     

Cloning and characterization of α-L-arabinofuranosidase and bifunctional α-L-arabinopyranosidase/β-D-galactopyranosidase from Bifidobacterium longum H-1

Aims: This study focused on the cloning, expression and characterization of recombinant α‐l ‐arabinosidases from Bifidobacterium longum H‐1. Methods and Results: α‐l ‐Arabinofuranosidase (AfuB‐H1) and bifunctional α‐l ‐arabinopyranosidase/β‐d ‐galactosidase (Apy‐H1) from B. longum H‐1 were identified by Southern blotting, and their recombinant enzymes were overexpressed in Escherichia coli BL21 (DE3). Recombinant AfuB‐H1 (rAfuB‐H1) was purified by single‐step Ni²⁺‐affinity column chromatography, whereas recombinant Apy‐H1 (rApy‐H1) was purified by serial Q‐HP and Ni²⁺‐affinity column chromatography. Enzymatic properties and substrate specificities of the two enzymes were assessed, and their kinetic constants were calculated. According to the results, rAfuB‐H1 hydrolysed p‐nitrophenyl‐α‐l ‐arabinofuranoside (pNP‐αL‐Af) and ginsenoside Rc, but did not hydrolyse p‐nitrophenyl‐α‐l ‐arabinopyranoside (pNP‐αL‐Ap). On the other hand, rApy‐H1 hydrolysed pNP‐αL‐Ap, p‐nitrophenyl‐β‐d ‐galactopyranoside (pNP‐βD‐Ga) and ginsenoside Rb2. Conclusions: Ginsenoside‐metabolizing bifidobacterial rAfuB‐H1 and rApy‐H1 were successfully cloned, expressed, and characterized. rAfuB‐H1 specifically recognized the α‐l ‐arabinofuranoside, whereas rApy‐H1 had dual functions, that is, it could hydrolyse both β‐d ‐galactopyranoside and α‐l ‐arabinopyranoside. Significance and Impact of the Study: These findings suggest that the biochemical properties and substrate specificities of these recombinant enzymes differ from those of previously identified α‐l ‐arabinosidases from Bifidobacterium breve K‐110 and Clostridium cellulovorans.     

Thermophilic α-glucan phosphorylase from Clostridium thermocellum: Cloning,characterization and enhanced thermostability

ORF Cthe0357 from the thermophilic bacterium Clostridium thermocellum ATCC 27405 that encodes a putative α-glucan phosphorylase (αGP) was cloned and expressed in Escherichia coli. The protein with a C-terminal His-tag was purified by Ni²⁺ affinity chromatography; the tag-free protein obtained from a cellulose-binding module–intein–αGP fusion protein was purified through affinity adsorption on amorphous cellulose followed by intein self-cleavage. Both purified enzymes had molecular weights of ca. 81,000 and similar specific activities. The optimal conditions were pH 6.0–6.5 and 60 °C for the synthesis direction and pH 7.0–7.5 and 80 °C for the degradation direction. This enzyme had broad substrate specificities for different chain length dextrins and soluble starch. The thermal inactivation of this enzyme strongly depended on temperature, protein concentration, and certain addictives that were shown previously to benefit the protein thermostability. The half lifetime of 0.05 mg αGP/mL at 50 °C was extended by 45-fold to 90 h through a combined addition of 0.1 mM Mg²⁺, 5 mM DTT, 1% NaCl, 0.1% Triton X-100, and 1 mg/mL BSA. The enzyme with prolonged stability would work as a building block for cell-free synthetic enzymatic pathway biotransformations, which can implement complicated biocatalysis through assembly of a number of enzymes and coenzymes.     

Cloning, sequencing, characterization, and expression of a new α-amylase isozyme gene (amy3) from Pseudomonas sp.

A new gene encoding an -amylase has been cloned, sequenced and expressed in E. coli from an alkaliphilic Pseudomonas sp. KFCC10818. The structural gene is 1356 base pairs long and encodes a protein of 452 amino acids. The recombinant -amylase has been purified and biochemically characterized. Molecular mass of the protein deduced from SDS-PAGE was 50 kDa. The enzyme showed an activity optimum at pH 8 and at 40 °C with complete stability at pH 13 for 3 h. The enzyme released maltose and maltotriose on hydrolysis of soluble starch. Amylose was hydrolysed over 5 times faster than amylopectin by the enzyme while the hydrolysis of cyclodextrin or pullulan was negligible.     

Cloning, expression and characterization of a new ι-carrageenase from marine bacterium, Cellulophaga sp.

Purpose of work

The purpose of this study is to report a ι-carrageenase which degrades ι-carrageenan yielding neo-ι-carratetraose as the main product in the absence of NaCl. The gene for a new ι-carrageenase, CgiB_Ce, from Cellulophaga sp. QY3 was cloned and sequenced. It comprised an ORF of 1,386 bp encoding for a protein of 461 amino acid residues. From its sequence analysis, CgiB_Ce is a new member of GH family 82 and shared the highest identity of 32 % in amino acids with ι-carrageenase CgiA2 from Zobellia galactanovorans indicating that it is a hitherto uncharacterized protein. The recombinant CgiB_Ce had maximum specific activity (1,870 U/mg) at 45 °C and pH 6.5. It was stable between pH 6.0–9.6 and below 40 °C. Although its activity was enhanced by NaCl, the enzyme was active in the absence of NaCl. CgiB_Ce is an endo-type ι-carrageenase that hydrolyzes β-1,4-linkages of ι-carrageenan, yielding neo-ι-carratetraose as the main product (more than 80 % of the total product).     

Cloning,expression and characterization of Bombyx mori α1,6-fucosyltransferase

Although core α1,6-fucosylation is commonly observed in N-glycans of both vertebrates and invertebrates, the responsible enzyme, α1,6-fucosyltransferase, has been much less characterized in invertebrates compared to vertebrates. To investigate the functions of α1,6-fucosyltransferase in insects, we cloned the cDNA for the α1,6-fucosyltransferase from Bombyx mori (Bmα1,6FucT) and characterized the recombinant enzyme prepared using insect cell lines. The coding region of Bmα1,6FucT consists of 1737 bp that code for 578 amino acids of the deduced amino acid sequence, showing significant similarity to other α1,6-fucosyltransferases. Enzyme activity assays demonstrated that Bmα1,6FucT is enzymatically active in spite of being less active compared to the human enzyme. The findings also indicate that Bmα1,6FucT, unlike human enzyme, is N-glycosylated and forms a disulfide-bonded homodimer. These findings contribute to a better understanding of roles of α1,6-fucosylation in invertebrates and also to the development of the more efficient engineering of N-glycosylation of recombinant glycoproteins in insect cells.     

Cloning, expression, and characterization of a glycoside hydrolase family 50 β-agarase from a marine Agarivorans isolate

The gene for a thermostable β-agarase from Agarivorans sp. JA-1 was cloned and sequenced. It comprised an open reading frame of 2,988 base pairs, which encode a protein of 109,450 daltons consisting of 995 amino acid residues. A comparison of the entire sequence showed that the enzyme has 98.8% sequence similarities to β-agarase from Vibrio sp. JT1070, indicating that it belongs to the family glycoside hydrolase (GH)-50. The gene corresponding to a mature protein of 976 amino acids was inserted and expressed in Escherichia coli. The recombinant β-agarase was purified to homogeneity. It had maximal activity at 40°C and pH 8.0 in the presence of 1 mM NaCl and 1 mM CaCl₂. The enzyme hydrolyzed agarose as well as neoagarohexaose and neoagarotetraose to yield neoagarobiose as the main product. Thus, the enzyme would be useful for the industrial production of neoagarobiose.     

Molecular cloning and characterization of a gibberellin-inducible, putative α-glucosidase gene from barley

A putative -glucosidase clone has been isolated from a cDNA library constructed from mRNA of barley aleurones treated with gibberellin A₃ (GA). The clone is 2752 bp in length and has an uninterrupted open reading frame encoding a polypeptide of 877 amino acids. A 680 amino acid region is 43% identical to human lysosomal -glucosidase and other glycosyl hydrolases. In isolated aleurones, the levels of the corresponding mRNA increase strongly after the application of GA, similar to the pattern exhibited by low-pI -amylase mRNA. High levels are also observed in the aleurone and scutellum after germination, while low levels are found in developing seeds. The genome contains a single form of this -glucosidase gene and two additional sequences that may be related genes or pseudogenes.Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.