Expression of Cryptic β-Fructofuranosidase in Saccharomyces rouxii

Raffinose hydrolysis was studied in Saccharomyces rouxii. The responsible enzyme was identified as a beta-fructofuranosidase (EC 3.2.1.26), which has a pH optimum of 5.5 and a K(m) of 83 mM for raffinose. This enzyme was cryptic in cells from a 3-day culture. A 2-min treatment with 0.1 volume of ethyl acetate in sodium acetate buffer (pH 6) gave complete expression of the enzyme, which was still retained by the cell. Ghosts were prepared by modifying membrane structure with small basic proteins in distilled water, and after washing they showed the full complement of enzymatic activity. The enzyme remained cryptic in osmotically protected spheroplasts; however, after lysis (by dilution) release, as well as expression, was effected. Mechanical disruption of fresh cells revealed and released all of the enzyme. Cells in early stationary phase had all of their beta-fructofuranosidase in a cryptic state. Aging yielded fractional expression of activity; initially this was proportional to cell death, but later the degree of expression exceeded the death rate. Media from aged cultures or cell-free extracts of aged cells were not effective in revealing the cryptic enzyme of younger cells. S. rouxii beta-fructofuranosidase has a different autolytic-release pattern from its counterpart in S. cerevisiae. Also, high concentrations of glucose do not repress the S. rouxii enzyme. The beta-fructofuranosidase in young cells of S. rouxii must be enclosed by the protoplasmic membrane or a special vesicular structure. This system was compared with other Saccharomyces species in connection with the translocation of enzymes across the protoplasmic membrane.     

Location of Acid Phosphatase and β-Fructofuranosidase Within Yeast Cell Envelopes

After 16 hr of incubation in a low-phosphate, aerated medium, bakers' yeast was obtained with a high titer of acid phosphatase (EC 3.1.3.2) and beta-fructofuranosidase (EC 3.2.1.26). All of the beta-fructofuranosidase and 75% of the acid phosphatase were easily released by mechanical disruption in a French pressure cell. The cell wall suffered a limited number of cracks, but this was sufficient for the co-release of these enzymes. Both enzymes were subject to autolytic release, although correlation was inconclusive because of the relative instability of acid phosphatase. The data are consistent with the bulk of the two enzymes being located in the periplasmic space. Ethylacetate treatments yielded ghosts with high beta-fructofuranosidase but low acid phosphatase activities. The surviving acid phosphatase was not representative of that in live cells. It was resistant to release by mechanical disruption and showed a high susceptibility to heat inactivation. The beta-fructofuranosidase in live cells and in ethylacetatetreated cells exhibited polydispersity in heat inactivation susceptibility; but the kinetics were indistinguishable, and facile release by mechanical disruption was shown in both cases.     

Molecular characterization of totiviruses in Xanthophyllomyces dendrorhous

ABSTRACT: BACKGROUND: Occurrence of extrachromosomal dsRNA elements has been described in the red-yeast Xanthophyllomyces dendrorhous, with numbers and sizes that are highly variable among strains with different geographical origin. The studies concerning to the encapsidation of viral-like particles and dsRNA-curing have suggested that some dsRNAs are helper viruses, while others are satellite viruses. However, the nucleotide sequences and functions of these dsRNA are still unknown. In this work, the nucleotide sequences of four dsRNAs of the strain UCD 67-385 of X. dendrorhous were determined, and their identities and genome structures are proposed. Based on this molecular data, the dsRNAs of different strains of X. dendrorhous were analyzed. RESULTS: The complete sequences of L1, L2, S1 and S2 dsRNAs of X. dendrorhous UCD 67-385 were determined, finding two sequences for L1 dsRNA (L1A and L1B). Several ORFs were uncovered in both S1 and S2 dsRNAs, but no homologies were found for any of them when compared to the database. Instead, two ORFs were identified in each L1A, L1B and L2 dsRNAs, whose deduced amino acid sequences were homologous with a major capsid protein (5'-ORF) and a RNA-dependent RNA polymerase (3'-ORF) belonging to the Totivirus family. The genome structures of these dsRNAs are characteristic of Totiviruses, with two overlapped ORFs (the 3'-ORF in the -1 frame with respect to the 5'-ORF), with a slippery site and a pseudoknot in the overlapped regions. These structures are essential for the synthesis of the viral polymerase as a fusion protein with the viral capsid protein through -1 ribosomal frameshifting. In the RNase protection analysis, all the dsRNAs in the four analyzed X. dendrorhous strains were protected from enzymatic digestion. The RT-PCR analysis revealed that, similar to strain UCD 67-385, the L1A and L1B dsRNAs coexist in the strains VKM Y-2059, UCD 67-202 and VKM Y-2786. Furthermore, determinations of the relative amounts of L1 dsRNAs using two-step RT-qPCR revealed a 40-fold increment of the ratio L1A/L1B in the S2 dsRNA-cured strain compared to its parental strain. CONCLUSIONS: Three totiviruses, named as XdV-L1A, XdV-L1B and XdV-L2, were identified in the strain UCD 67-385 of X. dendrorhous. The viruses XdV-L1A and XdV-L1B were also found in other three X. dendrorhous strains. Our results suggest that the smaller dsRNAs (named XdRm-S1 and XdRm-S2) of strain UCD 67-385 are satellite viruses, and particularly that XdRm-S2 is a satellite of XdV-L1A.     

Isolation and Characterization of the Epoxide Hydrolase-Encoding Gene from Xanthophyllomyces dendrorhous

The epoxide hydrolase (EH)-encoding gene (EPH1) from the basidiomycetous yeast Xanthophyllomyces dendrorhous was isolated. The genomic sequence has a 1,236-bp open reading frame which is interrupted by eight introns that encode a 411-amino-acid polypeptide with a calculated molecular mass of 46.2 kDa. The amino acid sequence is similar to that of microsomal EH and belongs to the α/β hydrolase fold family. The EPH1 gene was not essential for growth of X. dendrorhous in rich medium under laboratory conditions. The Eph1-encoding cDNA was functionally expressed in Escherichia coli. A sixfold increase in specific activity was observed when we used resting cells rather than X. dendrorhous. The epoxides 1,2-epoxyhexane and 1-methylcyclohexene oxide were substrates for both native and recombinant Eph1. Isolation and characterization of the X. dendrorhous EH-encoding gene are essential steps in developing a yeast EH-based epoxide biotransformation system.     

Horizontal Gene Transfer and Gene Duplication of β-Fructofuranosidase Confer Lepidopteran Insects Metabolic Benefits

Horizontal gene transfer (HGT) is a potentially critical source of material for ecological adaptation and the evolution of novel genetic traits. However, reports on posttransfer duplication in organism genomes are lacking, and the evolutionary advantages conferred on the recipient are generally poorly understood. Sucrase plays an important role in insect physiological growth and development. Here, we performed a comprehensive analysis of the evolution of insect β-fructofuranosidase transferred from bacteria via HGT. We found that posttransfer duplications of β-fructofuranosidase were widespread in Lepidoptera and sporadic occurrences of β-fructofuranosidase were found in Coleoptera and Hymenoptera. β-fructofuranosidase genes often undergo modifications, such as gene duplication, differential gene loss, and changes in mutation rates. Lepidopteran β-fructofuranosidase gene (SUC) clusters showed marked divergence in gene expression patterns and enzymatic properties in Bombyx mori (moth) and Papilio xuthus (butterfly). We generated SUC1 mutations in B. mori using CRISPR/Cas9 to thoroughly examine the physiological function of SUC. BmSUC1 mutant larvae were viable but displayed delayed growth and reduced sucrase activities that included susceptibility to the sugar mimic alkaloid found in high concentrations in mulberry. BmSUC1 served as a critical sucrase and supported metabolic homeostasis in the larval midgut and silk gland, suggesting that gene transfer of β-fructofuranosidase enhanced the digestive and metabolic adaptation of lepidopteran insects. These findings highlight not only the universal function of β-fructofuranosidase with a link to the maintenance of carbohydrate metabolism but also an underexplored function in the silk gland. This study expands our knowledge of posttransfer duplication and subsequent functional diversification in the adaptive evolution and lineage-specific adaptation of organisms.     

Purification of β-acetylglucosaminase and β-galactosidase from ram testis

1. The presence of beta-galactosidase (EC 3.2.1.23) in an acetic acid extract of ram testis is reported. Some properties of the crude enzyme preparation were studied. 2. The purification of beta-acetylglucosaminase (EC 3.2.1.30) and of beta-galactosidase from the ram-testis extract by ammonium sulphate precipitation and chromatography on a CM-cellulose column is described. 3. The final purifications of the separated enzymes achieved were for the beta-acetylglucosaminase 35 times and for the beta-galactosidase 99 times. 4. The possibility of using DEAE-cellulose and Sephadex G-200 to purify the enzymes was investigated.     

Purification and Characterization of Yeast β-Glucosidases

Constitutive beta-glucosidases from Saccharomyces fragilis (Y-18) and S. dobzhanskii (Y-19) precipitated at the same concentration of ammonium sulfate. The partially purified enzymes had similar activation energies, molecular weights, affinities for certain natural and synthetic beta-glucosides, and optimal pH values for substrate hydrolysis, and they were stable over approximately the same pH range. The enzymes, however, could be clearly distinguished by other criteria. Affinities of the synthetic, sulfur-containing beta-glucosides for Y-18 enzyme were many times greater than for Y-19 enzyme. The latter enzyme was more resistant to heat. The two enzymes eluted from diethylaminoethyl cellulose at different concentrations of sodium chloride. In precipitin tests, homologous enzyme-antisera systems were highly specific. The beta-glucosidase synthesized by a hybrid, S. fragilis x S. dobzhanskii (Y-42), was unique. Characterization of this enzyme produced values which were intermediate to those for the enzymes from the parental yeast strains. Heat-inactivation slopes and Lineweaver-Burk plots for the Y-42 enzyme were anomalous. It is suggested that hydrolytic activity in Y-42 preparations is due to a spectrum of hybrid enzyme molecules composed of varying amounts of two distinct polypeptides. It is further suggested that these polypeptides may be identical to those synthesized by the parental Y-18 and Y-19 yeast strains.     

Cross Talk between β1 and αV Integrins: β1 Affects β3 mRNA Stability

There is increasing evidence that a fine-tuned integrin cross talk can generate a high degree of specificity in cell adhesion, suggesting that spatially and temporally coordinated expression and activation of integrins are more important for regulated cell adhesive functions than the intrinsic specificity of individual receptors. However, little is known concerning the molecular mechanisms of integrin cross talk. With the use of beta(1)-null GD25 cells ectopically expressing the beta(1)A integrin subunit, we provide evidence for the existence of a cross talk between beta(1) and alpha(V) integrins that affects the ratio of alpha(V)beta(3) and alpha(V)beta(5) integrin cell surface levels. In particular, we demonstrate that a down-regulation of alpha(V)beta(3) and an up-regulation of alpha(V)beta(5) occur as a consequence of beta(1)A expression. Moreover, with the use of GD25 cells expressing the integrin isoforms beta(1)B and beta(1)D, as well as two beta(1) cytoplasmic domain deletion mutants lacking either the entire cytoplasmic domain (beta(1)TR) or only its "variable" region (beta(1)COM), we show that the effects of beta(1) over alpha(V) integrins take place irrespective of the type of beta(1) isoform, but require the presence of the "common" region of the beta(1) cytoplasmic domain. In an attempt to establish the regulatory mechanism(s) whereby beta(1) integrins exert their trans-acting functions, we have found that the down-regulation of alpha(V)beta(3) is due to a decreased beta(3) subunit mRNA stability, whereas the up-regulation of alpha(V)beta(5) is mainly due to translational or posttranslational events. These findings provide the first evidence for an integrin cross talk based on the regulation of mRNA stability.     

Microbial Transformation of β-Ionone and β-Methylionone

Aspergillus niger JTS 191 was selected from many microorganisms tested as capable of converting ionones to other compounds having aromas. The individual transformation products from β-ionone were isolated and identified by comparison with synthetically derived compounds. The major products were (R)-4-hydroxy-β-ionone and (S)-2-hydroxy-β-ionone. 2-Oxo-, 4-oxo-, 3,4-dehydro-, 2,3-dehydro-4-oxo-, 3,4-dehydro-2-oxo-, (S)-2-acetoxy-, (R)-4-acetoxy-, and 5,6-epoxy-β-ionone and 4-(2,3,6-trimethylphenyl)-but-3-en-2-one were also identified. Analogous transformation products of β-methylionone also were identified. Based on gas-liquid chromatographic analysis during the fermentation, we propose two main oxidative pathways of β-ionone. The results of this study suggest that these transformations of β-ionones may be useful as tobacco-flavoring compounds.     

Partial Purification and Characterization of a Thermostable Actinomycete β-Amylase

A thermostable amylase, possibly a β-amylase from Thermoactinomyces sp. no. 2 isolated from soil, is reported. The enzyme was purified 36-fold by acetone precipitation, ion-exchange chromatography, and Sephadex G-200 gel filtration, and the molecular weight was estimated at 31,600. The enzyme was characterized by demonstration of optimum activity at 60°C and pH 7 and by retention of 70% activity at 70°C (30 min). It was stimulated by Mn²⁺ and Fe²⁺ but strongly inhibited by Hg²⁺. Maltose was the only detectable product of hydrolysis of starches and was quantitatively highest in plantain starch hydrolysate.     

Gene Cloning and Characterization of a Novel Cellulose-Binding β-Glucosidase from Phanerochaete chrysosporium

Analysis of a 2.4-kb cDNA of the cellulose-binding extracellular β-glucosidase (CBGL) from Phanerochaete chrysosporium suggested that CBGL is organized into two domains, an N-terminal cellulose-binding domain and a C-terminal catalytic domain. Genomic sequence analysis suggested that cbgl is encoded by 30 exons. Southern analysis of DNA from homokaryotic cultures indicated that CBGL is encoded by two alleles, cbgl-1 and cbgl-2, of a single gene.     

Staphylococcal β-Hemolysin I. Purification of β-Hemolysin

The purification of staphylococcal β-hemolysin was accomplished by the successive use of three protein fractionation methods. The first method employed was a double precipitation with the use of ammonium sulfate at 65% saturation. The second phase of purification used Sephadex G-100 column fractionation. The third phase utilized either carboxymethyl cellulose or diethylaminoethyl cellulose fractionation. The last two fractionation methods both resulted in the separation of a relatively high concentration of cationic hot-cold lysin and a low concentration of anionic hot-cold lysin. Because of the low concentration of the anionic component, its purity could not be assessed. However, the purity of the cationic component was demonstrated by immunodiffusion, microimmunoelectrophoresis, and by disc polyacrylamide gel electrophoresis. In addition, antisera against purified cationic β-hemolysin yielded one line of precipitate when tested against the original crude β-hemolysin. The purified cationic β-hemolysin was stable in the lyophilized state. Crude β-hemolysin was dermonecrotic, whereas purified cationic β-hemolysin was not dermonecrotic even after Mg⁺⁺ activation.     

Purification and Characterization of l-Methionine γ-Lyase from Brevibacterium linens BL2

l-Methionine γ-lyase (EC 4.4.1.11) was purified to homogeneity from Brevibacterium linens BL2, a coryneform bacterium which has been used successfully as an adjunct bacterium to improve the flavor of Cheddar cheese. The enzyme catalyzes the α,γ elimination of methionine to produce methanethiol, α-ketobutyrate, and ammonia. It is a pyridoxal phosphate-dependent enzyme, with a native molecular mass of approximately 170 kDa, consisting of four identical subunits of 43 kDa each. The purified enzyme had optimum activity at pH 7.5 and was stable at pHs ranging from 6.0 to 8.0 for 24 h. The pure enzyme had its highest activity at 25°C but was active between 5 and 50°C. Activity was inhibited by carbonyl reagents, completely inactivated by dl-propargylglycine, and unaffected by metal-chelating agents. The pure enzyme had catalytic properties similar to those of l-methionine γ-lyase from Pseudomonas putida. Its K_m for the catalysis of methionine was 6.12 mM, and its maximum rate of catalysis was 7.0 μmol min⁻¹ mg⁻¹. The enzyme was active under salt and pH conditions found in ripening Cheddar cheese but susceptible to degradation by intracellular proteases.

Methanethiol is associated with desirable Cheddar-type sulfur notes in good-quality Cheddar cheese (2, 27). The mechanism for the production of methanethiol in cheese is unknown, but it is linked to the catabolism of methionine (1, 15). l-Methionine γ-lyase (EC 4.4.1.11; MGL), also known as methionase, l-methionine γ-demethiolase, and l-methionine methanethiollyase (deaminating), is a pyridoxal phosphate (PLP)-dependent enzyme that catalyzes the direct conversion of l-methionine to α-ketobutyrate, methanethiol, and ammonia by an α,γ-elimination reaction (26). It does not catalyze the conversion of d enantiomers (24–26). MGL in Pseudomonas putida is a multifunctional enzyme system since it catalyzes the α,γ- and α,β-elimination reactions of methionine and its derivatives (24). In addition, the enzyme also catalyzes the β-replacement reactions of sulfur amino acids (24). Since its discovery in Escherichia coli and Proteus vulgaris by Onitake (19), this enzyme has been found in various bacteria and is regarded as a key enzyme in the bacterial metabolism of methionine. However, this enzyme has not been purified to homogeneity from any food-grade microorganisms.MGL is widely distributed in bacteria, especially in pseudomonads, and is induced by the addition of l-methionine to the culture medium (9, 28). The enzyme has been purified from Pseudomonas putida (25), Aeromonas sp. (26), Clostridium sporogenes (11), and Trichomonas vaginalis (16) and partially purified from and characterized for Brevibacterium linens NCDO 739 (4).B. linens is a nonmotile, non-spore-forming, non-acid-fast, gram-positive coryneform bacterium normally found on the surfaces of Limburger and other Trappist-type cheeses. This organism tolerates salt concentrations ranging between 8 and 20% and is capable of growing in a broad pH range from 5.5 to 9.5, with an optimum pH of 7.0 (20). In Trappist-type cheeses, brevibacteria depend on Saccharomyces cerevisiae to metabolize lactate, which increases the pH of the curd, as well as to produce growth factors that are important for their growth (20). Interest in B. linens has focused around its ability to produce an extracellular protease, which has recently been isolated (21), and its ability to produce high levels of methanethiol (3, 9, 10, 22).B. linens produces various sulfur compounds, including methanethiol, that are thought to be important in Cheddar-like flavor and aroma (3, 9, 10, 22). Ferchichi et al. (9) suggested that MGL is responsible for the methanethiol-producing capability of B. linens but did not provide definitive evidence. Weimer et al. (28) proposed that B. linens BL2 is responsible for Cheddar-type flavor development in low-fat cheese, but again conclusive evidence was lacking. In this study, MGL was purified to homogeneity from B. linens BL2 and its physical and chemical properties were examined.     

Protection from Free β-Tubulin by the β-Tubulin Binding Protein Rbl2p

Free beta-tubulin not in heterodimers with alpha-tubulin can be toxic, disrupting microtubule assembly and function. We are interested in the mechanisms by which cells protect themselves from free beta-tubulin. This study focused specifically on the function of Rbl2p, which, like alpha-tubulin, can rescue cells from free beta-tubulin. In vitro studies of the mammalian homolog of Rbl2p, cofactor A, have suggested that Rbl2p/cofactor A may be involved in tubulin folding. Here we show that Rbl2p becomes essential in cells containing a modest excess of beta-tubulin relative to alpha-tubulin. However, this essential activity of Rbl2p/cofactorA does not depend upon the reactions described by the in vitro assay. Rescue of beta-tubulin toxicity requires a minimal but substoichiometric ratio of Rbl2p to beta-tubulin. The data suggest that Rbl2p binds transiently to free beta-tubulin, which then passes into an aggregated form that is not toxic.     

Production of Epoxides from α,β-Halohydrins by Flavobacterium sp

The relative activity of Flavobacterium whole cells on the enzymatic synthesis of epoxides from α,β-chlorohydrins, -bromohydrins, and -iodohydrins is described.     

Construction and Characterization of Salmonella typhimurium Strains That Accumulate and Excrete α- and β- Isopropylmalate

Two Salmonella typhimurium strains, which could be used as sources for the leucine biosynthetic intermediates α- and β-isopropylmalate were constructed by a series of P22-mediated transductions. One strain, JK527 [flr-19 leuA2010 Δ(leuD-ara)798 fol-162], accumulated and excreted α-isopropylmalate, whereas the second strain, JK553 (flr-19 leuA2010 leuB698), accumulated and excreted α- and β-isopropylmalate. The yield of α-isopropylmalate isolated from the culture medium of JK527 was more than five times the amount obtained from a comparable volume of medium in which Neurospora crassa strain FLR₉₂-1-216 (normally used as the source for α- and β-isopropylmalate) was grown. Not only was the yield greater, but S. typhimurium strains are much easier to handle and grow to saturation much faster than N. crassa strains. The combination of the two regulatory mutations flr-19, which results in constitutive expression of the leucine operon, and leuA2010, which renders the first leucine-specific biosynthetic enzyme insensitive to feedback inhibition by leucine, generated limitations in the production of valine and pantothenic acid. The efficient, irreversible, and unregulated conversion of α-ketoisovaleric acid into α-isopropylmalate (α-isopropylmalate synthetase K_m for α-ketoisovaleric acid, 6 × 10⁻⁵ M) severely restricted the amount of α-ketoisovaleric acid available for conversion into valine and pantothenic acid (ketopantoate hydroxymethyltransferase K_m for α-ketoisovaleric acid, 1.1 × 10⁻³ M; transaminase B K_m for α-ketoisovaleric acid, 2 × 10⁻³ M).