Milk Fat Globule Membrane Proteins in Aseptically Packed Ultra-heat-treated Milk: Changes during Storage

Five genes for tryptophan biosynthesis, trpE, trpD, trpC, trpB, and trpA of Brevibacterium lactofermentum, a coryne form glutamic acid-producing bacterium, were cloned as a 9.6 kb BamHl DNA fragment by colony hybridization. A previously cloned 1.2 kb Pst I DNA fragment containing a major part of the trpE gene was used as a probe. By complementation tests using the subclones of this 9.6 kb BamHl fragment and various tryptophan auxotrophs of B. lactofermentum and Escherichia coli, this fragment was found to contain a gene cluster composed of trpE, trpD, trpC, trpB, and trpA in this order. It suggests that genes for tryptophan biosynthesis in B. lactofermentum may be an operon.     

Purification and characterization of β-Mannanase from Reinekea sp. KIT-YO10 with transglycosylation activity

Marine bacterium Reinekea sp. KIT-YO10 was isolated from the seashore of Kanazawa Port in Japan as a seaweed-degrading bacterium. Homology between KIT-YO10 16S rDNA and the 16S rDNA of Reinekea blandensis and Reinekea marinisedimentorum was 96.4 and 95.4%, respectively. Endo-1,4-β-D-mannanase (β-mannanase, EC 3.2.1.78) from Reinekea sp. KIT-YO10 was purified 29.4-fold to a 21% yield using anion exchange chromatography. The purified enzyme had a molecular mass of 44.3?kDa, as estimated by SDS-PAGE. Furthermore, the purified enzyme displayed high specificity for konjac glucomannan, with no secondary agarase and arginase activity detected. Hydrolysis of konjac glucomannan and locust bean gum yielded oligosaccharides, compatible with an endo mode of substrate depolymerization. The purified enzyme possessed transglycosylation activity when mannooligosaccharides (mannotriose or mannotetraose) were used as substrates. Optimal pH and temperature were determined to be 8.0 and 70?°C, respectively. It showed thermostability at temperatures from 20 to 50?°C and alkaline stability up to pH 10.0. The current enzyme was thermostable and thermophile compared to the β-mannanase of other marine bacteria.     

High-yielding cascade enzymatic synthesis of 5-methyluridine using a novel combination of nucleoside phosphorylases

Abstract

A novel combination of Bacillus halodurans purine nucleoside phosphorylase (BhPNP1) and Escherichia coli uridine phosphorylase (EcUP) has been applied to a dual-enzyme, sequential, biocatalytic one-pot synthesis of 5-methyluridine from guanosine and thymine. A 5-methyluridine yield of >79% on guanosine was achieved in a reaction slurry at a 53 mM (1.5% w/w) guanosine concentration. 5-Methyluridine is an intermediate in synthetic routes to thymidine and the antiretroviral drugs zidovudine and stavudine.     

Restructuring of wall-bound xyloglucan by transglycosylation in living plant cells

Xyloglucan endotransglycosylases (XETs) cleave and then re-join xyloglucan chains and may thus contribute to both wall-assembly and wall-loosening. The present experiments demonstrate the simultaneous occurrence in vivo of two types of interpolymeric transglycosylation: "integrational" (in which a newly secreted xyloglucan reacts with a previously wall-bound one) and "restructuring" (in which one previously wall-bound xyloglucan reacts with another). Xyloglucans synthesised by cultured rose (Rosa sp.) cells in "heavy" or "light" media (with [13C,2H]glucose or [12C,1H]glucose, respectively) had buoyant densities of 1.643 and 1.585 g ml-1, respectively, estimated by isopycnic centrifugation in caesium trifluoroacetate. To detect transglycosylation, we shifted heavy rose cells into light medium, then supplied a 2-h pulse of L-[1-3H]arabinose. Light [3H]xyloglucans were thus secreted into heavy, non-radioactive walls and chased by light, non-radioactive xyloglucans. At 2 h after the start of radiolabelling, the (neutral) [3H]xyloglucans were on average 29% heavy, indicating molecular grafting during integrational transglycosylation. The [3H]xyloglucans then gradually increased in density until, by 11 h, they were 38% heavy. This density increase suggests that restructuring transglycosylation reactions occurred between the now wall-bound [3H]xyloglucan and other (mainly older, i.e. heavy) wall-bound non-radioactive xyloglucans. Brefeldin A (BFA), which blocked xyloglucan secretion, did not prevent the increase in density of wall-bound [3H]xyloglucan (2-11 h). This confirms that restructuring transglycosylation occurred between pairs of previously wall-bound xyloglucans. After 7 d in BFA, the 3H was in hybrid xyloglucans in which on average 55% of the molecule was heavy. Exogenous xyloglucan oligosaccharides (competing acceptor substrates for XETs) did not affect integrational transglycosylation whereas they inhibited restructuring transglycosylation. Possible reasons for this difference are discussed. This is the first experimental evidence for restructuring transglycosylation in vivo. We argue that both integrational and restructuring transglycosylation can contribute to both wall-assembly and -loosening.     

Microbial β-Glucosidases: Cloning,Properties, and Applications

ABSTRACT:?

β-Glucosidases constitute a major group among glycosylhydrolase enzymes. Out of the 82 families classified under glycosylhydrolase category, these belong to family 1 and family 3 and catalyze the selective cleavage of glucosidic bonds. This function is pivotal in many crucial biological pathways, such as degradation of structural and storage polysaccharides, cellular signaling, oncogenesis, host-pathogen interactions, as well as in a number of biotechnological applications. In recent years, interest in these enzymes has gained momentum owing to their biosynthetic abilities. The enzymes exhibit utility in syntheses of diverse oligosaccharides, glycoconjugates, alkyl- and amino-glucosides. Attempts are being made to understand the structure-function relationship of these versatile biocatalysts. Earlier reviews described the sources and properties of microbial β-glucosidases, yeast β-glucosidases, thermostable fungal β-glucosidase, and the physiological functions, characteristics, and catalytic action of native β-glucosidases from various plant, animal, and microbial sources. Recent efforts have been directed towards molecular cloning, sequencing, mutagenesis, and crystallography of the enzymes. The aim of the present article is to describe the sources and properties of recombinant β-glucosidases, their classification schemes based on similarity at the structural and molecular levels, elucidation of structure-function relationships, directed evolution of existing enzymes toward enhanced thermostability, substrate range, biosynthetic properties, and applications.     

New Methods for Chemo-Enzymatic Galactosidation of 2S Rapeseed Protein

Two chemo-enzymatic methodologies to synthesize neoglycoproteins from rapeseed 2S protein (napin) were developed. In the first approach, glycosidases were used to catalyse 1-O-glycosylation of serine residues, whereas in the second one, 6-N-galactosylation was examined using an amino-reduction reaction between the epsilon-NH2 of lysine residues and 6-oxogalactosides (readily available by means of the oxidation reaction of the corresponding galactosides mediated by galactose oxidase). Our results indicated that glycosidases were unable to glycosylate native proteins. Conversely, this reaction was possible, although in low yields (10%), after the introduction of a hydroxyethylene spacer. The latter modified proteins were obtained via the condensation of epsilon-NH2 of lysines with ethylene carbonate in basic medium (40% yield). The second approach was much more efficient, as 61% of the lysine residues were shown to be 6-N-galactosylated using sodium cyanoborohydride as a reduction reagent.     

Enzymatic synthesis of tyrosol and hydroxytyrosol β-d-fructofuranosides

Tyrosol β-d-fructofuranoside and hydroxytyrosol β-d-fructofuranoside have been synthesized as new compounds in 27.6 and 19.5% respective yields through transfructosylation of tyrosol and hydroxytyrosol. Yeast β-galactosidase Lactozym 3000?L comprising invertase activity was used as catalyst. Besides the main monofructosides, an equimolar mixture of tyrosol β-d-fructofuranosyl-((2→1)-β-d-fructofuranoside and tyrosol β-d-fructofuranosyl-(2→6)-β-d-fructofuranoside was isolated as additional product fraction in 14.3% yield.     

Enzymic synthesis of lacto-N-triose II and its positional analogues

N-acetylhexosaminidase fromNocardia orientalis catalysed the synthesis of lacto-N-triose II glycoside (-d-GlcNAc-(1-3)--d-Gal-(1-4)--d-Glc-OMe,3) with its isomers -d-GlcNAc-(1-6)--d-Gal-(1-4)--d-Glc-OMe (4) and -d-Gal-(1-4)-[-d-GlcNAc-(1-6)]--d-Glc-OMe (5) throughN-acetylglucosaminyl transfer fromN,N-diacetylchitobiose (GlcNAc₂) to methyl -lactoside. The enzyme formed the mixture of trisac-charides3, 4 and5 in 17% overall yield based on GlcNAc₂, in a ratio of 20:21:59. Withp-nitrophenyl -lactoside as an acceptor, the enzyme also producedp-nitrophenyl -lacto-N-trioside II (-d-GlcNAc-(1-3)--d-Gal-(1-4)--d-Glc-OC₆H₄NO₂-p,6) with its isomers -d-GlcNAc-(1-6)--d-Gal-(1-4)--d-Glc-OC₆H₄NO₂-p (7) and -d-Gal-(1-4)-[-d-GlcNAc-(1-6)]--d-Glc-OC₆H₄NO₂-p (8). In this case, when an inclusion complex ofp-nitrophenyl lactoside acceptor with -cyclodextrin was used, the regioselectivity of glycosidase-catalysed formation of trisaccharide glycoside was substantially changed. It resulted not only in a significant increase of the overall yield of transfer products, but also in the proportion of the desired compound6.Abbreviations GlcNAc₂ 2-acetamido-2-deoxy--d-glucopyranosyl-(1-4)-2-acetamido-2-deoxy-d-glucose - NAHase N-acetylhexosaminidase - -CD -cyclodextrin     

Transglycosylation reactions by exoglycosidases from the termite Macrotermes subhyalinus

The ability of four exoglycosidases (-galactosidase, -glucosidase, -glucosidase and invertase) from the termite Macrotermes subhyalinus to catalyse tranglycosylation reactions was tested using lactose, cellobiose, maltose and sucrose as glycosyl donors and 2-phenylethanol as glycosyl acceptor. The experimental conditions were optimized in relation to the time course of the reaction, pH and concentrations of glycosyl donor and acceptor. Whereas the hydrolytic activity was largely predominant over the transferase activity with -galactosidase and -glucosidase, the transglycosylation activity represented 68% with -glucosidase. In addition, as demonstrated by the transglycosylation product formed, the hydrolysis of sucrose was catalysed by -glucosidase and not by invertase. On the basis of this work, -glucosidase from M. subhyalinus appears to be a valuable tool for the preparation of neoglycoconjugates.     

Modulation of the regioselectivity of a Bacillus alpha-galactosidase by directed evolution

-galactosidase AgaB of Bacillus stearothermophilus was subjected to directed evolution in an effort to modify its regioselectivity. The wild-type enzyme displays a major 1,6 and minor 1,3 regioselectivity. We used random mutagenesis and staggered extension process (StEP) to obtain mutant enzymes displaying modified regioselectivity. We developed a screening procedure allowing first the elimination of AgaB mutants bearing the 1,6 regioselectivity and secondly the selection of those retaining a 1,3 regioselectivity. Our results show that, among the evolved enzymes that have lost most of their activity towards the 1,6 linkage both in hydrolysis and in synthesis, one (E901) has retained its 1,3 activity. However the transglycosylation level reached by this mutant is quite low versus that of the native enzyme. This work constitutes the first example of modification of glycosylhydrolase regioselectivity by directed evolution.