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 and characterization of a novel exo-α-1,5-L-arabinanase gene and the enzyme

A novel exo-alpha-1,5-L-arabinanase gene (arn3) was isolated, cloned, and expressed in E. coli. The recombinant enzyme (ARN3) had a pH optimum of 6.0-7.0 and a pH 3.0-7.0 stability range. The temperature optimum was 50 degrees C with a stability less than or equal to 45 degrees C. The recombinant ARN3 cleaved carboxymethyl (CM)-arabinan, debranched arabinan, and linear arabinan at a decreasing rate and is inactive on sugar beet arabinan, wheat arabinoxylan, and p-nitrophenyl-alpha-L-arabinofuranoside. The enzyme hydrolyzed debranched arabinan and synthetic arabino-oligosaccharides entirely to arabinose. The apparent K(m) and V(max) values were determined to be 6.2+/-0.3 mg/ml and 0.86+/-0.01 mg ml(-1) min(-1), respectively (pH 7.0, 37 degrees C, CM-arabinan). Multiple sequence alignment and homology modeling revealed unique short sequences of amino acids extending the loop involved in partial blocking of one end of the substrate-binding site on the surface of the molecule.     

Cloning and characterization of a novel member of human β-1, 4-galactosyltransferase gene family

By using the EST strategy for identifying novel members belonging to homologous gene families, a novel full-length cDNA encoding a protein significantly homologous to UDP-Gal: N-acetylglucosamine β-1, 4-galactosyltransferase (GAlT) was isolated from a human testis cDNA library. A nucleotide sequence of 2 173 bp long was determined to contain an open reading frame of 1032 nucleotides (344 amino acids). In view of the homology to members of the galactosyltransferase gene family and especially the closest relationship to Gallus gallus GalT type I (CK I), the predicted product of the novel cDNA was designated as human β-1, 4-galactosyltransferase homolog I (HumGT-H1). Its mRNA is present in different degrees in 16 tissues examined. Southern analysis of human genomic DNA revealed its locus on chromosome 3.     

Cloning and characterization of a novel member of human β-1,4-galactosyltransferase gene family

By using the EST strategy for identifying novel members belonging to homologous gene families, a novel fulklength cDNA encoding a protein significantly homologous to UDP-Gal: N-acetylglucosamine β-1, 4-galactosyltransferase (GalT) was isolated from a human testis cDNA library. A nucleotide sequence of 2 173 bp long was determined to contain an open reading frame of 1 032 nucleotides (344 amino acids). In view of the homology to memben of the galactosyltransferase gene family and especially the closest relationship toGallus gallus GalT type I (CK I), the predicted product of the novel cDNA was designated as human β-1,4-galactosyltransferase homolog I (HumGT-H1). Its mRNA is present in different degrees in 16 tissues examined. Southern analysis of human genomic DNA revealed its locus on chromosome 3.     

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, 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.     

Enhanced intestinal uptake of iron,zinc and calcium in rats fed pungent spice principles – Piperine,capsaicin and ginger (Zingiber officinale)

In view of the wide-spread deficiency of iron and zinc in populations dependent on plant foods, it is desirable to improve the bioavailability of the same. Specific dietary spices may alter the ultrastructure and permeability characteristics of intestines. Groups of Wistar rats were fed piperine, capsaicin and ginger containing diets for 8 weeks in order to examine their possible influence on intestinal absorption of iron, zinc and calcium. Everted segments of duodenum, jejunum and ileum portions of small intestines isolated from these rats were examined for ex vivo uptake of iron, zinc and calcium from incubations containing digesta of finger millet. Higher uptake of iron, zinc and calcium by the intestinal segments from spice-fed animals was observed. The increase in the mineral uptake was the highest for calcium with >100% in some cases. The positive influence of dietary capsaicin was more pronounced on zinc uptake as compared to that of iron. Uptake of the glutamic acid standard was 87% and 62% higher in the case of jejunal segments of rats fed piperine and ginger. The higher intestinal uptake of iron and zinc as a result of consumption of pungent spices could encourage a strategy to reduce deficiency of these trace elements prevalent in population dependent on plant based foods.     

Cloning,characterization, expression analysis and inhibition studies of a novel gene encoding Bowman–Birk type protease inhibitor from rice bean

This paper presents the first study describing the isolation, cloning and characterization of a full length gene encoding Bowman–Birk protease inhibitor (RbTI) from rice bean (Vigna umbellata). A full-length protease inhibitor gene with complete open reading frame of 327 bp encoding 109 amino acids was cloned from rice bean seeds using degenerate primer set. BlastP search revealed that the RbTI encoded amino acid of approx 13.0 kDa and shared 99% homology each with BBI from Phaseolus parvulus, Vigna trilobata and Vigna vexilata. Phylogenetic tree also showed close relationship of RbTI with BBI from other members of Leguminaceae family. RbTI gene was further confirmed as intronless (GenBank accession no. KJ159908). The secondary and 3D-structural models for the RbTI were predicted with homology modeling. qRT-PCR studies revealed the highest RbTI expression in the seeds nearing maturity, whereas the low expression of the gene was noticed in young leaves. The isolated RbTI was successfully expressed in Escherichiacoli and the highest expression was recorded after 5.5 h of induction. Study on the inhibitory activity of expressed protein against the gut proteases of Hessian fly larvae revealed 87% inhibition. The novel RbTI gene will further broaden the pool of plant defense genes and could be an ideal choice for developing transgenic crops resistant to insect pests with high economic value. In addition, it has the potential to be used as a probe for selection of insect- and pathogen-resistant genotypes.     

Cloning, characterization, and antifungal activity of an endo-1,3-β-d-glucanase from Streptomyces sp. S27

An endo-1,3-β-d-glucanase gene, designated as bglS27, was cloned from Streptomyces sp. S27 and successfully expressed in Escherichia coli BL21 (DE3). The full-length gene contains 1,362 bp and encodes a protein of 453 amino acids with a calculated molecular mass of 42.7 kDa. The encoded protein comprises a catalytic module of glycosyl hydrolase family 16, a short glycine linker region, and a family 13 carbohydrate-binding module. The purified recombinant enzyme (BglS27) showed optimal activity at 65°C and pH 5.5 and preferentially catalyzed the hydrolysis of glucans with a β-1,3-linkage using an endolytic mode of action. The specific activity and K _m value of BglS27 for laminarin were 236.0 U mg^–1 and 1.89 mg ml^–1, respectively. In antifungal assay, BglS27 had the ability to inhibit the growth of phytopathogenic fungi Rhizoctonic solani and Fusarium oxysporum and some mycotoxin-producing fungi Fusarium crookwellense and Paecilomyces variotii. These favorable properties make BglS27 a good candidate for utilization in biotechnological applications such as plant protection, feed, and food preservation.     

Cloning and characterization of a new β-mannosidase from Streptomyces sp. S27

A new β-mannosidase gene, designated as man2S27, was cloned from Streptomyces sp. S27 using the colony PCR method and expressed in Escherichia coli BL21 (DE3). The full-length gene consists of 2499 bp and encodes 832 amino acids with a calculated molecular mass of 92.6 kDa. The amino acid sequence shares highest identity of 62.6% with the mannosidase Man2A from Cellulomonas fimi which belongs to the glycoside hydrolase family 2. Purified recombinant Man2S27 showed optimal activity at pH 7.0 and 50 °C. The specific activity, K_m, and k_cat values for p-nitrophenyl-β-d-mannopyranoside (p-NP-β-MP) were 35.3 U mg^-1, 0.23 mM, and 305 s^-1, respectively. Low transglycosylation activity was observed when Man2S27 was incubated with p-NP-β-MP (glycosyl donor) and methyl-α-d-mannopyranoside (p-NP-α-MP) (acceptor) at 50 °C and pH 7.0, and a small amount of methylmannobioside was synthesized. Using locust bean gum as the substrate, more reducing sugars were liberated by the synergistic action of Man2S27 and β-mannanase (Man5S27), and the synergy degree in sequential reactions with Man5S27 firstly and Man2S27 secondly was higher than that in the simultaneous reactions.     

Cloning and characterization of a novel NK cell-specific serine protease gene and its functional 5′-flanking sequences

Rat natural killer cell Met-ase-1 (RNK-Met-1) is a 30 000 M _r serine protease (granzyme) found in the cytolytic granules of CD3- large granular lymphocytes (LGL) with natural killer (NK) activity. To characterize the genomic sequences responsible for the CD3- LGL-restricted expression of this gene, we screened a rat genomic library with RNK-Met-1 cDNA, and obtained bacteriophage clones that contained the RNK-Met-1 gene. The RNK-Met-1 gene comprises 5 exons and spans approximately 5.2 kilobases (kb), exhibiting a similar structural organization to a class of CTL-serine proteases with protease catalytic residues encoded near the borders of exons 2, 3, and 5. The 5-flanking region of the RNK-Met-1 gene contains a number of putative promoter and enhancer regulatory elements and shares several regions of homology with the 5-flanking region of the mouse perforin gene. We have prepared nested deletions from approximately 3.3 kb of the 5-flanking region of the RNK-Met-1 gene, and inserted these upstream of the chloramphenicol acetyltransferase (CAT) reporter gene. These 5-flanking RNK-Met-1-CAT constructs were transiently transfected into rat LGL leukemia, T-lymphoma, and basophilic leukemia cell lines.The nucleotide sequence data reported in this Papershave been submitted to the EMBL/GenBank nucleotide sequence database and have been assigned the accession number L38482.     

Cloning and expression of the endo-1,3(4)-β-glucanase gene from Paecilomyces sp. FLH30 and characterization of the recombinant enzyme

The cDNA encoding β-1,3(4)-glucanase, named PsBg16A, from Paecilomyces sp. FLH30 was cloned, sequenced, and over expressed in Pichia pastoris, with a yield of about 61,754 U mL?1 in a 5-L fermentor. PsBg16A has an open reading frame of 951 bp encoding 316 amino acids, and the deduced amino acid sequence of PsBg16A revealed that it belongs to glycoside hydrolase family 16. The purified recombinant PsBg16A had a pH optimum at 7.0 and a temperature optimum at 70 °C, and randomly hydrolyzed barley β-glucan, lichenin, and laminarin, suggesting that it is a typical endo-1,3(4)-β-glucanase (EC 3.2.1.6) with broad substrate specificity for β-glucans.     

Purification and characterization of a thermostable endo-β-1,4-glucanase from a novel strain of Penicillium purpurogenum

A novel endo-β-1,4-glucanase (EG)-producing strain was isolated and identified as Penicillium purpurogenum KJS506 based on its morphology and internal transcribed spacer (ITS) rDNA gene sequence. P. purpurogenum produced one of the highest levels of EG (5.6 U mg-protein^?1) with rice straw and corn steep powder as carbon and nitrogen sources, respectively. The extracellular EG was purified to homogeneity by sequential chromatography of P. purpurogenum culture supernatants on a DEAE sepharose column, a gel filtration column, and then on a Mono Q column with fast protein liquid chromatography. The purified EG was a monomeric protein with a molecular weight of 37 kDa and showed broad substrate specificity with maximum activity towards lichenan. P. purpurogenum EG showed t_1/2 value of 2 h at 70 °C and catalytic efficiency of 118 ml mg^?1 s^?1, one of the highest levels seen for EG-producing microorganisms. Although EGs have been reported elsewhere, the high catalytic efficiency and thermostability distinguish P. purpurogenum EG.