Characterization of β-Lactotensin,a Bioactive Peptide Derived from Bovine β-Lactoglobulin,as a Neurotensin Agonist

β-Lactotensin (β-LT: His-Ile-Arg-Leu) is an ileum-contracting peptide derived from residues No. 146-149 of bovine β-lactoglobulin. The ileum-contracting activity of β-LT was blocked by the NT₁ antagonist SR48692. β-LT was selective for the neurotensin NT₂ receptor while neurotensin was selective for the NT₁ receptor. β-LT is the first natural ligand showing selectivity for the NT₂ receptor. β-LT showed hypertensive activity after intravenous administration at a dose of 30 mg/kg in conscious rats, while neurotensin showed hypotensive activity. The hypertensive activity of β-LT was blocked by levocabastine (1 mg/kg, i.v.), an NT₂ antagonist. SR48692, which blocked the hypotensive activity of neurotensin, had no effect on the hypertensive activity of β-LT. These results suggest that the hypertensive activity of β-LT is mediated by the NT₂ receptor. It was concluded that the NT₁ and NT₂ receptors mediate the opposite effect on blood pressure.     

Heterologous Production of Hyaluronic Acid in an ε-Poly-l-Lysine Producer,Streptomyces albulus

Hyaluronic acid (HA) is used in a wide range of medical applications, where its performance and therapeutic efficacy are highly dependent on its molecular weight. In the microbial production of HA, it has been suggested that a high level of intracellular ATP enhances the productivity and molecular weight of HA. Here, we report on heterologous HA production in an ε-poly-l-lysine producer, Streptomyces albulus, which has the potential to generate ATP at high level. The hasA gene from Streptococcus zooepidemicus, which encodes HA synthase, was refactored and expressed under the control of a late-log growth phase-operating promoter. The expression of the refactored hasA gene, along with genes coding for UDP-glucose dehydrogenase, UDP-N-acetylglucosamine pyrophosphorylase, and UDP-glucose pyrophosphorylase, which are involved in HA precursor sugar biosynthesis, resulted in efficient production of HA in the 2.0 MDa range, which is greater than typical bacterial HA, demonstrating that a sufficient amount of ATP was provided to support the biosynthesis of the precursor sugars, which in turn promoted HA production. In addition, unlike in the case of streptococcal HA, S. albulus-derived HA was not cell associated. Based on these findings, our heterologous production system appears to have several advantages for practical HA production. We propose that the present system could be applicable to the heterologous production of a wide variety of molecules other than HA in the case their biosynthesis pathways require ATP in vivo.     

Purification and biochemical properties of a galactooligosaccharide producing β-galactosidase from Bullera singularis

A beta-galactosidase, catalyzing lactose hydrolysis and galactooligosaccharide (GalOS) synthesis from lactose, was extracted from the yeast, Bullera singularis KCTC 7534. The crude enzyme had a high transgalactosylation activity resulting in the oligosaccharide conversion of over 34% using pure lactose and cheese whey permeate as substrates. The enzyme was purified by two chromatographic steps giving 96-fold purification with a yield of 16%. The molecular weight of the purified enzyme (specific activity of 56 U mg(-1)) was approx. 53 000 Da. The hydrolytic activity was the highest at pH 5 and 50 degrees C, and was stable to 45 degrees C for 2 h. Enzyme activity was inhibited by 10 mM Ag3+ and 10 mM SDS. The Km for lactose hydrolysis was 0.58 M and the maximum reaction velocity (V(max)) was 4 mM min(-1). GalOS, including tri- and tetra-saccharides were produced with a conversion yield of 50%, corresponding to 90 g GalOS l(-1) from 180 g lactose l(-1) by the purified enzyme.     

Expression, Purification and Characterization of a Recombinant β-Glucosidase from Volvariella Volvacea

For the first time, a β-glucosidase gene from the edible straw mushroom, Volvariella volvacea V1-1, has been over-expressed in E. coli. The gene product was purified by chromatography showing a single band on SDS-PAGE. The recombinant enzyme had a molecular mass of 380 kDa with subunits of 97 kDa. The maximum activity was at pH 6.4 and 50 °C over a 5 min assay. The purified enzyme was stable from pH 5.6–8.0, had a half life of 1 h at 45 °C. The β-glucosidase had a K_m of 0.2 mM for p-nitrophenyl-β-D-glucopyranoside.     

Cloning,Expression, and Characterization of a Peculiar Choline-Binding β-Galactosidase from Streptococcus mitis

A Streptococcus mitis genomic DNA fragment carrying the SMT1224 gene encoding a putative β-galactosidase was identified, cloned, and expressed in Escherichia coli. This gene encodes a protein 2,411 amino acids long with a predicted molecular mass of 268 kDa. The deduced protein contains an N-terminal signal peptide and a C-terminal choline-binding domain consisting of five consensus repeats, which facilitates the anchoring of the secreted enzyme to the cell wall. The choline-binding capacity of the protein facilitates its purification using DEAE-cellulose affinity chromatography, although its complete purification was achieved by constructing a His-tagged fusion protein. The recombinant protein was characterized as a monomeric β-galactosidase showing a specific activity of around 2,500 U/mg of protein, with optimum temperature and pH ranges of 30 to 40°C and 6.0 to 6.5, respectively. Enzyme activity is not inhibited by glucose, even at 200 mM, and remains highly stable in solution or immobilized at room temperature in the absence of protein stabilizers. In S. mitis, the enzyme was located attached to the cell surface, but a significant activity was also detected in the culture medium. This novel enzyme represents the first β-galactosidase having a modular structure with a choline-binding domain, a peculiar property that can also be useful for some biotechnological applications.Streptococcus mitis belongs to the viridans group of streptococci and is a relevant microorganism because it is both an opportunistic pathogen and phylogenetically close to Streptococcus pneumoniae, a major respiratory human pathogen. Although S. mitis isolates usually produce only mild infections, some S. mitis strains have acquired increased virulence and are one of the main causes of infectious endocarditis (15, 36). Remarkably, S. mitis, like only a few other streptococci, displays phosphorylcholine residues in its cellular envelope (3). This aminoalcohol is used for the anchorage of proteins belonging to the so-called “choline-binding proteins” (CBPs), which fulfill important physiological functions in these bacteria. CBPs bind to phosphorylcholine residues present in the teichoic and lipoteichoic acids located at the surface of S. pneumoniae and some streptococci of the mitis group. CBPs share a modular organization consisting of a biologically active domain and a conserved choline-binding domain (CBD), which contains 6 to 18 imperfect 20-amino-acid tandem repeats each located either at the carboxy- or amino-terminal ends of the proteins (26). This CBD is able to specifically bind to choline or its structural analogues like DEAE (diethylaminoethanol), which permits purification by affinity chromatography in a single step using DEAE-cellulose supports (38). Crystallographic studies of CBPs have shown that a typical CBD consists of several β-hairpins organized as a left-handed superhelix and that the linkage of CBPs to the choline-containing cell wall substrate is carried out through the binding of choline residues to the interface of two consecutive choline-binding repeats, named choline-binding sites (9, 13, 14).β-d-Galactosidases (β-d-galactoside galactohydrolase; EC 3.2.1.23) constitute a large family of proteins that cleave the glycosidic bond between two or more carbohydrates or between a carbohydrate and a noncarbohydrate moiety, e.g., lactose and related chromogens, like o-nitrophenyl-β-d-galactopyranoside (ONPG), p-nitrophenyl-β-d-galactopyranoside (PNPG), or 6-bromo-2-naphthyl-galactopyranoside. β-d-galactosidases belong to the glycosyl hydrolase (GH) superfamily, which contains 114 families (see http://www.CAZY.org) classified on the basis of amino acid sequence similarity (12). The enzymes exhibiting β-galactosidase activity are currently classified within four different families: GH-1, GH-2, GH-35, and GH-42. β-Galactosidases are widely distributed in nature and are present in numerous microorganisms (yeasts, fungi, bacteria, and archaea), plants, and animals (34, 44). These enzymes are of great interest for several industrial or biotechnological processes; the hydrolytic activity has been applied in the food industry for decades to reduce the lactose content of milk products in order to circumvent lactose intolerance, which is prevalent in more than half of the world''s population (27). More recently, interest in β-galactosidases has increased due to their ability to synthesize β-galactosyl derivatives that have received a great deal of attention owing to their important roles in many biological processes (27).In this study, we report the purification and biochemical characterization of a peculiar β-galactosidase encoded by the SMT1224 gene of S. mitis that represents a new type of β-galactosidase within this paradigmatic enzyme family.     

Expression and characterization of a thermostable β-xylosidase from the hyperthermophile, Thermotoga maritima

A thermostable beta-xylosidase from a hyperthermophilic bacterium, Thermotoga maritima, was over-expressed in Escherichia coli using the T7 polymerase expression system. The expressed beta-xylosidase was purified in two steps, heat treatment and immobilized metal affinity chromatography, and gave a single band on SDS-PAGE. The maximum activity on p-nitrophenyl beta-D-xylopyranoside was at 90 degrees C and pH 6.1. The purified enzyme had a half-life of over 22-min at 95 degrees C, and retained over 57% of its activity after holding a pH ranging from 5.4 to 8.5 for 1 h at 80 degrees C. Among all tested substrates, the purified enzyme had specific activities of 275, 50 and 29 U mg(-1) on pNPX, pNPAF, and pNPG, respectively. The apparent Michaelis constant of the beta-xylosidase was 0.13 mM for p NPX with a V (max) of 280 U mg(-1). When the purified beta-xylosidase was added to xylanase, corncob xylan was hydrolized completely to xylose.     

Expression of a β-galactosidase gene from Lactobacillus sake in Escherichia coli

Summary A -galactosidase gene from Lactobacillus sake coding for lactose hydrolysis was cloned and expressed in Escherichia coli. Chromosomal DNA from L. sake was partially digested with the restriction enzyme Sau3AI, and the 3–6 Kb fragment was ligated to the cloning vector pSP72 digested with BamHI. One E. coli transformant expressing -galactosidase was isolated on X-gal plates. It contained a plasmid with an insertion of approx. 4 Kb. The restriction map of the recombinant plasmid was constructed. The characteristics of the recombinant -galactosidase were compared with those of the wild type. The optima pH and temperature for both enzymes was 6.5 and 50°C, respectively. Stability of the enzymes at different temperatures and activity on lactose were determined.     

Gene Cloning and Heterologous Expression of Glycoside Hydrolase Family 55 β-1,3-Glucanase from the Basidiomycete Phanerochaete Chrysosporium

The basidiomycete Phanerochaete chrysosporium produces several β-1,3-glucanases when grown on laminarin, a β-1,3/1,6-glucan, as the sole carbon source. To characterize one of the major unknown β-1, 3-glucanases with a molecular mass of 83 kDa, identification, cloning, and heterologous over-expression were carried out using the total genomic information of P. chrysosporium. The cDNA encoding this enzyme included an ORF of 2337 bp and the deduced amino acid sequence contains a predicted signal peptide of 26 amino acids and the mature protein of 752 amino acids. The amino acid sequence showed a significant similarity with glycoside hydrolase family 55 enzymes from filamentous fungi and was named Lam55A. Since the recombinant Lam55A expressed in the methylotrophic yeast Pichia pastoris degraded branched β-1,3/1,6-glucan as well as linear β-1,3-glucan, the kinetic features of the enzyme were compared with those of other β-1,3-glucanases.     

Expression and characterization of a thermostable β-xylosidase from the hyperthermophile, Thermotoga maritima

A thermostable -xylosidase from a hyperthermophilic bacterium, Thermotoga maritima, was over-expressed in Escherichia coli using the T7 polymerase expression system. The expressed -xylosidase was purified in two steps, heat treatment and immobilized metal affinity chromatography, and gave a single band on SDS-PAGE. The maximum activity on p-nitrophenyl -d-xylopyranoside was at 90 °C and pH 6.1. The purified enzyme had a half-life of over 22-min at 95 °C, and retained over 57% of its activity after holding a pH ranging from 5.4 to 8.5 for 1 h at 80 °C. Among all tested substrates, the purified enzyme had specific activities of 275, 50 and 29 U mg^–1 on pNPX, pNPAF, and pNPG, respectively. The apparent Michaelis constant of the -xylosidase was 0.13 mm for pNPX with a V _max of 280 U mg^–1. When the purified -xylosidase was added to xylanase, corncob xylan was hydrolized completely to xylose.     

Expression in Escherichia coli of a cloned β-glucanase gene from Bacillus amyloliquefaciens

Summary The recombinant phage G1 has been identified by screening 700 plaques of a Charon 4A library, containing DNA of Bacillus amyloliquefaciens, for phage clones directing the hydrolysis of lichenan in Escherichia coli, as indicated by haloes surrounding plaques on lichenan agar. The gene coding for an endo--1.3–1.4-glucanase was recloned within a 3.6 kb EcoRI fragment into the EcoRI site of plasmid pBR322, in both orientations.The location and extent of the bgl gene on the 3.6 kb Bacillus DNA insert was estimated by insertion mutagenesis with transposon Tn5 and restriction mapping of Tn5 insertions within or near to the bgl gene.The -glucanase synthesized by E. coli harbouring plasmids pEG1 or pEG2 was shown to accumulate mainly in the periplasmic space but -glucanase activities were also detected extracellulary and in the cytoplasm. The molecular weight of the enzyme synthesized in E. coli harbouring pEG1 was estimated by SDS-polyacrylamide gel electrophoresis to be about 24000. It was shown that the level of bgl gene expression in E. coli varies about 10-fold, depending on the orientation of the 3.6 kb DNA-fragment cloned within the EcoRI site of pBR322. After insertion of HindIII-DNA fragments from phage into the HindIII site of the -glucanase-high-expression plasmid pEG1, we obtained clones also showing an approximately 10-fold reduction in -glucanase activites. It was thus concluded that on plasmid pEG1 the leftward acting Ap^r (PI) promotor of plasmid pBR322 strongly increases the expression in E. coli of the cloned B. amyloliquefaciens bgl gene.Abbreviations Ap ampicillin, Km, kanamycin - kd kilodalton - kb kilobase pairs - moi multiplicity of infection - pfu plaque forming units - SDS sodium dodecylsulphate - Tc tetracycline     

Cloning,Sequencing, and Expression of the Gene Encoding an Intracellular β-D-Xylosidase from Streptomyces thermoviolaceus OPC-520

The intracellular β-xylosidase was induced when Streptomyces thermoviolaceus OPC-520 was grown at 50°C in a minimal medium containing xylan or xylooligosaccharides. The 82-kDa protein with β-xylosidase activity was partially purified and its N-terminal amino acid sequence was analyzed. The gene encoding the enzyme was cloned, sequenced, and expressed in Escherichia coli. The bxlA gene consists of a 2,100-bp open reading frame encoding 770 amino acids. The deduced amino acid sequence of the bxlA gene product had significant similarity with β-xylosidases classified into family 3 of glycosyl hydrolases. The bxlA gene was expressed in E. coli, and the recombinant protein was purified to homogeneity. The enzyme was a monomer with a molecular mass of 82 kDa. The purified enzyme showed hydrolytic activity towards only p-nitrophenyl-β-D-xylopyranoside among the synthetic glycosides tested. Thin-layer chromatography analysis showed that the enzyme is an exo-type enzyme that hydrolyze xylooligosaccharides, but had no activity toward xylan. High activity against pNPX occurred in the pH range 6.0-7.0 and temperature range 40-50°C.     

Heterologous expression of β-xylosidase gene from Paecilomyces thermophila in Pichia pastoris

β-xylosidase from thermophilic fungi Paecilomyces thermophila was functionally expressed in Pichia pastoris with a his tag in the C-terminal under the alcohol oxidase 1 (AOX1) promoter and secreted into the medium at 0.22 mg l^?1. Its molecular mass was estimated to be 52.3 kDa based on the SDS-PAGE analysis, which is 1.3 times higher than the predicted 39.31 kDa from its amino acid compositions, although no potential N- or O- glycosylation sites were predicted from its amino acid sequence. This is presumed to be caused by some unpredictable posttranslational modifications based on mass spectrum analysis of the recombinant protein. The enzyme was most active at 60 °C and pH 7. It showed not only a β-xylosidase activity with a K_m of 8 mM and a V_max of 54 μmol min^?1 mg^?1 for hydrolysis of p-nitrophenyl β-d-xylopyranoside but also an arabinofuranosidase activity (6.2 U mg^?1) on p-nitrophenyl arabinofuranoside.     

Production of Bioactive Human β-defensin-3 in Escherichia coli by Soluble Fusion Expression

A codon optimized mature human β-defensin-3 gene (smHBD3) was synthesized and fused with TrxA to construct pET32-smHBD3 vector, which was transformed into E. coli BL21(DE3) and cultured in MBL medium. The volumetric productivity of fusion protein reached 0.99 g fusion protein l⁻¹, i.e. 0.21 g mature HBD3 l⁻¹. Ninety-six percentage of the fusion protein was in a soluble form and constituted about 45% of the total soluble protein. After cell disruption, the soluble fusion protein was separated by affinity chromatography and cleaved by enterokinase, and then the mature HBD3 was purified by cationic ion exchange chromatography. The overall recovery ratio of HBD3 was 43%. The purified mature HBD3 demonstrated antimicrobial activity against E. coli. Revisions requested 13 December 2005; Revisions received 24 January 2006