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.     

Molecular Analysis of the β-Globin Gene Cluster in the Niokholo Mandenka Population Reveals a Recent Origin of the βS Senegal Mutation

A large and ethnically well-defined Mandenka sample from eastern Senegal was analyzed for the polymorphism of the beta-globin gene cluster on chromosome 11. Five RFLP sites of the 5' region were investigated in 193 individuals revealing the presence of 10 different haplotypes. The frequency of the sickle-cell anemia causing mutation (beta(S)) in the Mandenka estimated from this sample is 11.7%. This mutation was found strictly associated with the single Senegal haplotype. Approximately 600 bp of the upstream region of the beta-globin gene were sequenced for a subset of 94 chromosomes, showing the presence of four transversions, five transitions, and a composite microsatellite polymorphism. The sequence of 22 beta(S) chromosomes was also identical to the previously defined Senegal haplotype, suggesting that this mutation is very recent. Monte Carlo simulations (allowing for a specific balancing selection model, a logistic growth of the population, and variable initial frequencies of the Senegal haplotype) were used to estimate the age of the beta(S) mutation. Resulting maximum-likelihood estimates are 45-70 generations (1,350-2,100 years) for very different demographic scenarios. Smallest confidence intervals (25-690 generations) are obtained under the hypothesis that the Mandenka population is large (N(e) >5,000) and stationary or that it has undergone a rapid demographic expansion to a current size of >5,000 reproducing individuals, which is quite likely in view of the great diversity found on beta(A) chromosomes.     

Biochemical Properties and Crystal Structure of a β-Phenylalanine Aminotransferase from Variovorax paradoxus

By selective enrichment, we isolated a bacterium that can use β-phenylalanine as a sole nitrogen source. It was identified by 16S rRNA gene sequencing as a strain of Variovorax paradoxus. Enzyme assays revealed an aminotransferase activity. Partial genome sequencing and screening of a cosmid DNA library resulted in the identification of a 1,302-bp aminotransferase gene, which encodes a 46,416-Da protein. The gene was cloned and overexpressed in Escherichia coli. The recombinant enzyme was purified and showed a specific activity of 17.5 U mg⁻¹ for (S)-β-phenylalanine at 30°C and 33 U mg⁻¹ at the optimum temperature of 55°C. The β-specific aminotransferase exhibits a broad substrate range, accepting ortho-, meta-, and para-substituted β-phenylalanine derivatives as amino donors and 2-oxoglutarate and pyruvate as amino acceptors. The enzyme is highly enantioselective toward (S)-β-phenylalanine (enantioselectivity [E], >100) and derivatives thereof with different substituents on the phenyl ring, allowing the kinetic resolution of various racemic β-amino acids to yield (R)-β-amino acids with >95% enantiomeric excess (ee). The crystal structures of the holoenzyme and of the enzyme in complex with the inhibitor 2-aminooxyacetate revealed structural similarity to the β-phenylalanine aminotransferase from Mesorhizobium sp. strain LUK. The crystal structure was used to rationalize the stereo- and regioselectivity of V. paradoxus aminotransferase and to define a sequence motif with which new aromatic β-amino acid-converting aminotransferases may be identified.     

Biochemical and Proteomic Characterization of a Novel Extracellular β-Glucosidase from Trichoderma citrinoviride

β-Glucosidases are of pivotal importance in bioconversion of carbonic biomass into fermentable and other useful metabolites, food industry, biotransformation, glyco-trimming of metabolome, etc. Trichoderma citrinoviride when grown on delignified Lantana camara produced a β-glucosidase and secreted it out in the medium. The extracellularly secreted β-glucosidase of T. citrinoviride was homogeneity purified and then characterized for its kinetic properties and proteomic characteristics. The 90 kDa enzyme was monomeric in nature, optimally active at pH 5.5 and the catalytic reaction rate was highest at 55°C. Uniquely, the enzyme was insensitive to inhibition by glucose (up to 5 mM). It also possessed catalytic ability of transglycosylation, as it could catalyze conversion of geraniol into its glucoside. MALDI-TOF assisted proteomic analysis revealed its high degree of sequence similarity with family 3 glycoside hydrolases.     

Positive and Negative Selection in the β-Esterase Gene Cluster of the Drosophila melanogaster Subgroup

We examine the pattern of molecular evolution of the β-esterase gene cluster, including the Est-6 and ψEst-6 genes, in eight species of the Drosophila melanogaster subgroup. Using maximum likelihood estimates of nonsynonymous/synonymous rate ratios, we show that the majority of Est-6 sites evolves under strong (48% of sites) or moderate (50% of sites) negative selection and a minority of sites (1.5%) is under significant positive selection. Est-6 sites likely to be under positive selection are associated with increased intraspecific variability. One positively selected site is responsible for the EST-6 F/S allozyme polymorphism; the same site is responsible for the EST-6 functional divergence between species of the melanogaster subgroup. For ψEst-6 83.7% sites evolve under negative selection, 16% sites evolve neutrally, and 0.3% sites are under positive selection. The positively selected sites of ψEst-6 are located at the beginning and at the end of the gene, where there is reduced divergence between D. melanogaster and D. simulans; these regions of ψEst-6 could be involved in regulation or some other function. Branch-site-specific analysis shows that the evolution of the melanogaster subgroup underwent episodic positive selection. Collating the present data with previous results for the β-esterase genes, we propose that positive and negative selection are involved in a complex relationship that may be typical of the divergence of duplicate genes as one or both duplicates evolve a new function. [Reviewing Editor: Dr. Martin Kreitman]     

Isolation and Identification of Tubaic Acid and β-Tubaic Acid from Derris Roots

A tobacco callus strain, OMT-53, was selected from many cultures as a desirable strain having high nicotine producing capacity. Several culture conditions were examined, aiming to get higher nicotine production with the callus strain, OMT-53. It was revealed that the nicotine production was remarkably enhanced when the callus tissues were cultured at a limited concentration of α-NAA in culture medium. The optimal concentrations of sucrose and nitrogen in the culture medium were 3 % and 840 mg N/L respectively. Some precursors in nicotine biosynthesis were examined, and only ornithine gave a slightly positive effect at 2x10^-4m concentration. Cultures at 25°C produced the highest yield for nicotine. Considerable amounts of nicotine (ca. 20% of total nicotine) were also recognized in the culture medium. Under the best culture condition mentioned above, nicotine production in tobacco callus tissues has been elevated to 2.14% on D.W, basis at 4 weeks’ culture. This value is near to that of the intact tobacco plants.     

Purification, Biochemical and Immunological Characterization of Acid Invertases from Apple Fruit

The soluble acid invertase (SAI) and cell wall-bound invertase (CWI) were purified from apple fruit to apparent electrophoretic homogeneity. Based on sequencing, substrate specificity, and immunoblotting assay, the purified enzymes were identified to be two isoforms of acid invertase (β-fructosidase; EC 3.2.1.26). The SAI and CWI have the same apparent molecular mass with a holoenzyme of molecular mass of 220 kDa composed of 50 kDa subunits. The SAI has a lower Km value for sucrose and higher Km for raffinose compared with CWI. These acid invertases differ from those in other plants in some of their biochemical properties, such as the extremely high Km value for raffinose, no hydrolytic activity for stachyose, and a mixed form of inhibition by fructose to their activity. The antibodies directed against the SAI and CWI recognized, from the crude extract, three polypeptides with a molecular mass of 50, 68, and 30 kDa, respectively.These results provide a substantial basis for the further studies of the acid invertases in apple fruit.     

Molecular Analysis of δ-Aminolevulinic Acid Dehydratase (ALAD) Gene Polymorphism in a Turkish Population

delta-Aminolevulinic acid dehydratase (ALAD) is a cytosolic enzyme in the heme biosynthetic pathway. ALAD is a polymorphic enzyme showing marked ethnic group differences. In this study, ALAD polymorphism is studied in a Turkish population. Genomic DNA extracted from 230 individuals and polymerase chain reaction (PCR) coupled with the restriction fragment length polymorphism (RFLP) technique were used to identify variants. The frequencies of the alleles ALAD1 and ALAD2 in Turkey were 0.887 and 0.113, respectively. This study provides the first analysis of the allele frequency distribution of the ALAD gene in a Turkish population. The results are compared with other world populations.     

Purification and Properties of Acid β-d-Galactosidase from Corticium rolfsii

An acid β-d-galactosidase was purified from the culture filtrate of Corticium rolfsii IFO 6146 by a combination of QAE-Sephadex A-50 and SP-Sephadex C-50 chromatography. The maximum activity of the enzyme towards p-nitrophenyl β-D-galactopyranoside was found to be at pH 2.0 to 2.5 and the enzyme was fairly active at pH 1.5 to l.8. The enzyme was quite stable over a pH range 2.0 to 8.0 at 2°C for 72 hr. The enzymic activity was clearly inhibited by Hg²⁺. Km value was determined to be 3.84 × 10^?4 m, and V_max was calculated to be 6.9 μ moles per min per mg for p-nitrophenyl β-d-galactopyranoside. Contrary to high activity on the synthetic galactoside, reaction velocity was small when the enzyme acted on lactose.     

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.     

Biochemical characterization of a novel hyperthermophilic α-l-rhamnosidase from Thermotoga petrophila and its application in production of icaritin from epimedin C with a thermostable β-glucosidase

Epimedin C, a major flavonoid extracted from Herba Epimedii, is a precursor of minor flavonoid icaritin that is a desired drug candidate with remarkable anti-cancer activities. For enhancing the biotransformation efficiency of icaritin, a novel α-l-rhamnosidase gene was cloned from hyperthermophiles Thermotoga petrophila DSM 13995. TpeRha displayed optimal activity at a pH of 4.5 and a temperature of 90 °C. The K_m and K_cat of TpeRha for p-nitrophenyl-α-l-rhamnopyranoside were 2.99 mM and 651.73 s⁻¹, respectively. It displayed broad catalytic ability in cleavage of the outer and inner rhamnopyranosyl moieties on the C-3 carbon of epimedin C. Further, this enzyme was utilized to improve the efficiency of the co-conversion system in transforming epimedin C into icaritin, in combined with a thermostable β-glucosidase Tpebgl1. In addition, a transformation pathway (epimedin C -icariin - icariside I - icaritin) with a high efficiency for icaritin production was screened. After a two-stage transformation under optimized conditions (90 °C, pH 4.5, 80 U/mL of TpeRha and 1.2 U/mL of Tpebgl1), 1 g/L of epimedin C was transformed into 0.4337 g/L of icaritin within 150 min, with a corresponding molar conversion rate of 96.9 %. This is the first report of enzymatic transformation on preparing icaritin from epimedin C by using thermostable glycosidase.     

Cloning and Biochemical Characterization of BglC, a β-Glucosidase from the Cellulolytic Actinomycete Thermobifida fusca

An operon, bglABC, that encodes two sugar permeases and a β-glucosidase was cloned from a cellulolytic actinomycete, Thermobifida fusca, into Escherichia coli and sequenced. The bglC gene encoding an intracellular β-glucosidase (β-d-glucoside glucohydrolase, EC 3.2.1.21) belonging to glycosyl hydrolase family 1 was subcloned and expressed in E. coli. The purified enzyme (MW 53,407 Da; pI 4.69) hydrolyzed substrates containing both β 1 → 4 and β 1 → 2 glycosidic bonds, and was most active against cellobiose (V_max= 29, K _m = 0.34 mm), cellotriose, cellotetraose, and sophorose. The enzyme also showed aryl-β-glucosidase activity on p-nitrophenyl-β-d-glucopyranoside and p-nitrophenyl-β-d-cellobioside. BglC had a pH optimum of 7.0 and a temperature optimum of 50°C. The enzyme was stable at 60°C, but was rapidly inactivated at 65°C. BglC was inhibited by low concentrations of gluconolactone, but was insensitive to end-product inhibition by glucose and was not affected by Ca or Mg ions or EDTA. Its properties are well suited for use in a process to hydrolyze biomass cellulose to glucose. Received: 21 August 2000 / Accepted: 4 October 2000     

Organization, Structure, and Evolution of the Nonadult Rat β-Globin Gene Cluster

The β-globin gene cluster of Wistar rat was extensively cloned and the embryonic genes were mapped and sequenced. Four overlapping λ Dash recombinant clones cover about 31 kb and contain four nonadult β-globin genes, 5′–ε1–γ1–γ2–ψγ3–3′. The ε1 and γ2 are active genes, since their protein products were detected in the fetal stage of the rat (Iwahara et al., J Biochem 119:360–366, 1996). The γ1 locus might be a pseudogene, since the ATA box in the promoter region is mutated to GTA; however, no other defect is observed. The ψγ3 locus is a truncated pseudogene because a 19-base deletion, which causes a shift of the reading frame, is observed between the second nucleotide of the putative codon 68 and codon 76. A sequence comparison suggests that the ψγ3 might be produced by a gene conversion event of the proto-γ-globin gene set. Possible histories of the evolution of rat nonadult β-globin genes are discussed. Received: 6 August 1998 / Accepted: 12 February 1999