Cloning and Expression of a β-Galactosidase Gene of Bacillus circulans

A gene of β-galactosidase from Bacillus circulans ATCC 31382 was cloned and sequenced on the basis of N-terminal and internal peptide sequences isolated from a commercial enzyme preparation, Biolacta^®. Using the cloned gene, recombinant β-galactosidase and its deletion mutants were overexpressed as His-tagged proteins in Escherichia coli cells and the enzymes expressed were characterized.     

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.     

Molecular Cloning and Characterization of a NAC-like Gene in “Navel” Orange Fruit Response to Postharvest Stresses

A cDNA subtraction library had been constructed to identify differentially expressed genes in peel pitting of citrus fruit. Based on the sequence of a cDNA fragment homologous to NAC gene family, the full-length cDNA of 1,203 nucleotides was cloned from “navel” orange by rapid amplification of cDNA ends. It was designated as CsNAC, encoding a protein of 305 amino acids. The calculated molecular weight of the CsNAC protein was 35.2 kDa, and theoretical isoelectric point was 6.72. Sequence comparison showed that the CsNAC protein had a strikingly conserved region at the N terminus, which is considered as the characteristic of the NAC protein family. CsNAC protein was orthologous to Arabidopsis thaliana ATAF1. Phylogenetic analysis confirmed CsNAC belonged to the ATAF subfamily, which plays an important role in response to stress stimuli. RNA gel blot analysis showed that the expression of CsNAC gene was rapidly and strongly induced by stresses such as wounding and no oxygen. Low temperature (4°C) and exposure to ethylene also increased the expression level of CsNAC gene. However, its expression was suppressed by high temperature (40°C) but not affected by low oxygen (3%). Our results may provide the basis for future research of NAC-like gene’s role in stress-induced citrus peel pitting. Sequence data of CsNAC from this article have been deposited at GenBank under accession number EF596736.     

Cloning,Expression, and Characterization of Rice α-Galactosidase

We have successfully cloned an α-galactosidase gene from a rice cDNA library and transformed it into Escherichia coli BL21. It was subsequently cloned to the pPIC9K vector and expressed in Pichia pastoris. A selected clone was found to result in high production yield of the galactosidase enzyme. The secreted enzyme was purified, and it revealed as a major protein band on an SDS-PAGE gel. The optimal pH value, enzyme stabilities, and substrate specificity were studied. The enzyme specificity toward the terminal α1→6, 1→4, and 1→3 linked galactosyl residue from various substrates was investigated. By determining the Michelis constant (Km) of the enzyme for melibiose, raffinose, and stachyose, our results showed that melibiose was hydrolyzed faster than raffinose, whereas the published data reported a reversed sequence, raffinose > melibiose. The enzyme also showed the ability of converting B red blood cells into O red cells. The objective of this work is to develop the Pichia system to produce a large quantity of enzyme for blood cell conversion for transfusion.     

Cloning and Characterization of a Novel Human TGF-β Activated Kinase-Like Gene

TGF-beta activated kinase (TAK1) plays a critical role in the TGF-beta signaling transduction pathway. By screening a human 18-week fetal brain library, we isolated a novel human TAK1-like (TAKL) gene. The gene encoded a putative protein of 242 amino acids, which shared a homology with human, mouse, and Xenopus TAK1. The TAKL gene was located in chromosome 21q21. Northern blot analysis revealed that the TAKL mRNA was expressed predominantly in peripheral blood leukocytes and ubiquitously in human adult and fetal tissues. TAKL was also expressed strongly in breast carcinoma GI-101, colon adenocarcinoma GI-112, and prostatic adenocarcinoma PC3.     

Molecular and Biochemical Characterization of a β-Fructofuranosidase from Xanthophyllomyces dendrorhous

An extracellular β-fructofuranosidase from the yeast Xanthophyllomyces dendrorhous was characterized biochemically, molecularly, and phylogenetically. This enzyme is a glycoprotein with an estimated molecular mass of 160 kDa, of which the N-linked carbohydrate accounts for 60% of the total mass. It displays optimum activity at pH 5.0 to 6.5, and its thermophilicity (with maximum activity at 65 to 70°C) and thermostability (with a T₅₀ in the range 66 to 71°C) is higher than that exhibited by most yeast invertases. The enzyme was able to hydrolyze fructosyl-β-(2→1)-linked carbohydrates such as sucrose, 1-kestose, or nystose, although its catalytic efficiency, defined by the k_cat/K_m ratio, indicates that it hydrolyzes sucrose approximately 4.2 times more efficiently than 1-kestose. Unlike other microbial β-fructofuranosidases, the enzyme from X. dendrorhous produces neokestose as the main transglycosylation product, a potentially novel bifidogenic trisaccharide. Using a 41% (wt/vol) sucrose solution, the maximum fructooligosaccharide concentration reached was 65.9 g liter⁻¹. In addition, we isolated and sequenced the X. dendrorhous β-fructofuranosidase gene (Xd-INV), showing that it encodes a putative mature polypeptide of 595 amino acids and that it shares significant identity with other fungal, yeast, and plant β-fructofuranosidases, all members of family 32 of the glycosyl-hydrolases. We demonstrate that the Xd-INV could functionally complement the suc2 mutation of Saccharomyces cerevisiae and, finally, a structural model of the new enzyme based on the homologous invertase from Arabidopsis thaliana has also been obtained.The basidiomycetous yeast Xanthophyllomyces dendrorhous (formerly Phaffia rhodozyma) produces astaxanthin (3-3′-dihydroxy-β,β-carotene-4,4 dione [17, 25]). Different industries have displayed great interest in this carotenoid pigment due to its attractive red-orange color and antioxidant properties, which has intensified the molecular and genetic study of this yeast. As a result, several genes involved in the astaxanthin biosynthetic pathway have been cloned and/or characterized, as well as some other genes such as those encoding actin (60), glyceraldehyde-3-phosphate dehydrogenase (56), endo-β-1,3-glucanase, and aspartic protease (4). In terms of the use of carbon sources, a β-amylase (9), and an α-glucosidase (33) with glucosyltransferase activity (12), as well as a yeast cell-associated invertase (41), have also been reported.Invertases or β-fructofuranosidases (EC 3.2.1.26) catalyze the release of β-fructose from the nonreducing termini of various β-d-fructofuranoside substrates. Yeast β-fructofuranosidases have been widely studied, including that of Saccharomyces cerevisiae (11, 14, 45, 46), Schizosaccharomyces pombe (36), Pichia anomala (40, 49), Candida utilis (5, 8), or Schwanniomyces occidentalis (2). They generally exhibit strong similarities where sequences are available, and they have been classified within family 32 of the glycosyl-hydrolases (GH) on the basis of their amino acid sequences. The catalytic mechanism proposed for the S. cerevisiae enzyme implies that an aspartate close to the N terminus (Asp-23) acts as a nucleophile, and a glutamate (Glu-204) acts as the acid/base catalyst (46). In addition, the three-dimensional structures of some enzymes in this family have been resolved, such as that of an exoinulinase from Aspergillus niger (var. awamori; 37) and the invertase from Arabidopsis thaliana (55).As well as hydrolyzing sucrose, β-fructofuranosidases from microorganisms may also catalyze the synthesis of short-chain fructooligosaccharides (FOS), in which one to three fructosyl moieties are linked to the sucrose skeleton by different glycosidic bonds depending on the source of the enzyme (3, 52). FOS are one of the most promising ingredients for functional foods since they act as prebiotics (44), and they exert a beneficial effect on human health, participating in the prevention of cardiovascular diseases, colon cancer, or osteoporosis (28). Currently, Aspergillus fructosyltransferase is the main industrial producer of FOS (15, 52), producing a mixture of FOS with an inulin-type structure, containing β-(2→1)-linked fructose-oligomers (¹F-FOS: 1-kestose, nystose, or ¹F-fructofuranosylnystose). However, there is certain interest in the development of novel molecules that may have better prebiotic and physiological properties. In this context, β-(2→6)-linked FOS, where this link exits between two fructose units (⁶F-FOS: 6-kestose) or between fructose and the glucosyl moiety (⁶G-FOS: neokestose, neonystose, and neofructofuranosylnystose), may have enhanced prebiotic properties compared to commercial FOS (29, 34, 54). The enzymatic synthesis of 6-kestose and other related β-(2→6)-linked fructosyl oligomers has already been reported in yeasts such as S. cerevisiae (11) or Schwanniomyces occidentalis (2) and in fungi such as Thermoascus aurantiacus (26) or Sporotrichum thermophile (27). However, the production of FOS included in the ⁶G-FOS series has not been widely reported in microorganisms, probably because they are not generally produced (2, 15) or because they represent only a minor biosynthetic product (e.g., with baker''s yeast invertase) (11). Most research into neo-FOS production has been carried out with Penicillium citrinum cells (19, 31, 32, 39). In this context, neokestose is the main transglycosylation product accumulated by whole X. dendrorhous cells from sucrose (30), although the enzyme responsible for this reaction remained uncharacterized.Here, we describe the molecular, phylogenetic, and biochemical characterization of an extracellular β-fructofuranosidase from X. dendrorhous. Kinetic studies of its hydrolytic activity were performed using different substrates, and we investigated its fructosyltransferase capacity. The functionality of the gene analyzed was verified through its heterologous expression, and a structural model of this enzyme based on the homologous invertase from A. thaliana has also been obtained.     

Functional Identification of a Putative β-Galactosidase Gene in the Special lac Gene Cluster of Lactobacillus acidophilus

The putative β-galactosidase gene (lacZ) of Lactobacillus acidophilus has a very low degree of homology to the Escherichia coli β-galactosidase gene (lacZ) and locates in a special lac gene cluster which contains two β-galactosidase genes. No functional characteristic of the putative β-galactosidase has been described so far. In this study, the lacZ gene of L. acidophilus was hetero-expressed in E. coli and the recombinant protein was purified by a three-step procedure. The product of the lacZ gene was also extracted from L. acidophilus ATCC 4356 and active staining was carried out. The enzymatic properties of the purified recombinant LacZ were assayed. The results of hetero-expression showed the recombinant LacZ without tag had β-galactosidase activity. The purified recombinant LacZ had a specific activity of 43.2 U/mg protein. The result of active staining showed that the functional product of the lacZ gene did exist in L. acidophilus. The L. acidophilus β-galactosidase (LacZ) had an optimal pH of 6, an optimal temperature of 37°C and could hydrolyze 73% of lactose in milk in 30 h at 10°C. The L. acidophilus β-galactosidase (LacZ) was identified as cold-adapted β-galactosidase in this study for the first time, and may be useful for lactose removal from dairy products at low temperatures.     

Cloning,Sequencing, and Expression of a β-Amylase Gene from Bacillus cereus var. mycoides and Characterization of Its Products

The cloned gene was composed of 1638 bp for coding plus promoter like and SD-like sequences ahead of it. The deduced amino acid sequence had high similarity with known β-amylases. The N-terminal sequence of the cloned β-amylase seemed to be a signal peptide. The gene was introduced into Bacillus subtilis 1A289 using pHY300PLK as a vector and the expressed protein was recovered from the culture media. The enzyme fraction produced was divided into two components upon the DEAE column chromatography. The amino acid sequence of one fraction (FrI) was the same as the mature enzyme, and the other (FrII) lacked the N-terminal amino acid residue (Ala) of the mature enzyme. The kinetic parameters of the hydrolysis catalyzed by the enzyme component FrI were measured, and the subsite affinities of the enzyme were evaluated. In conclusion, it was shown that the recombinant enzyme was the same as the mature enzyme functionally and proteochemically.     

Cloning and Characterization of the nagA Gene that Encodes β-N-Acetylglucosaminidase from Aspergillus nidulans and Its Expression in Aspergillus oryzae

We isolated a β-N-acetylglucosaminidase encoding gene and its cDNA from the filamentous fungus Aspergillus nidulans, and designated it nagA. The nagA gene contained no intron and encoded a polypeptide of 603 amino acids with a putative 19-amino acid signal sequence. The deduced amino acid sequence was very similar to the sequence of Candida albicans Hex1 and Trichoderma harzianum Nag1. Yeast cells containing the nagA cDNA under the control of the GAL1 promoter expressed β-N-acetylglucosaminidase activity. The chromosomal nagA gene of A. nidulans was disrupted by replacement with the argB marker gene. The disruptant strains expressed low levels of β-N-acetylglucosaminidase activity and showed poor growth on a medium containing chitobiose as a carbon source. Aspergillus oryzae strain carrying the nagA gene under the control of the improved glaA promoter produced large amounts of β-N-acetylglucosaminidase in a wheat bran solid culture.     

Cloning and Molecular Characterization of the First Innexin of the Phylum Annelida—Expression of the Gene During Development

A novel member of the innexin family (cv-inx) has been isolated from the annelid polychaete worm Chaetopterus variopedatus using a PCR approach on genomic DNA and sequence analysis on genomic DNA clones. The gene is present in a HindIII-HindIII segment of 2250 bp containing an uninterrupted open reading frame of 1196 bp encoding a protein of 399 amino acids. The predicted protein shows the typical structural features of innexins and consensus sites for phosphorylation. Analyses on genomic DNA demonstrate that cv-inx is a single copy gene with no introns in the coding region, exactly corresponding to the cDNA sequence. The gene expression is regulated during development as shown by Northern blots analyses of the RNA and by immunoreaction with antibodies against the protein at several embryonic stages. The finding of an innexin in the phylum Annelida, outside of the Ecdysozoa clade, and its peculiar gene structure suggest the necessity to reconsider the current hypothesis on the origin and evolution of gap junctional proteins. Received: 15 December 2000 / Accepted: 27 August 2001