In vitro synthesis of artificial polysaccharides by glycosidases and glycosynthases

Artificial polysaccharides produced by in vitro enzymatic synthesis are new biomaterials with defined structures that either mimic natural polysaccharides or have unnatural structures and functionalities. This review summarizes recent developments in the in vitro polysaccharide synthesis by endo-glycosidases, grouped in two major strategies: (a) native retaining endo-glycosidases under kinetically controlled conditions (transglycosylation with activated glycosyl donors), and (b) glycosynthases, engineered glycosidases devoid of hydrolase activity but with high transglycosylation activity. Polysaccharides are obtained by enzymatic polymerization of simple glycosyl donors by repetitive condensation. This approach not only provides a powerful methodology to produce polysaccharides with defined structures and morphologies as novel biomaterials, but is also a valuable tool to analyze the mechanisms of polymerization and packing to acquire high-order molecular assemblies.     

A new pathway for the synthesis of oligosaccharides by the use of non-Leloir glycosyltransferases

The growing recognition of the roles of carbohydrates in fundamental biological processes and their potential application as functional foods and new therapeutics have generated a need for larger amounts of different carbohydrate structures. Leloir glycosyltransferases catalyze the synthesis of complex oligosaccharides. However they are difficult or expensive to obtain, and require expensive nucleotide activated sugars. In contrast non-Leloir pathway enzymes use sucrose, which is known to be a high energy donor of d-glucose for glucosyltransferases like dextransucrase, or a donor of d-fructose for fructosyltransferases like inulin- and levansucrases for the synthesis of polysaccharides. Here we present the synthesis and kinetic studies of oligosaccharides using non-Leloir glycosyltransferases and sucrose analogues as new substrates, like β-d-fructofuranosyl-α-d-galactopyranoside (Gal-Fru) by a fructosyltransferase (FTF) from B. subtilis NCIMB 11871. The sucrose analogues carry a high binding energy in the glycosidic bond similar to that of sucrose. Thus, β-d-Fructofuranosyl-α-d-galactopyranoside (Gal-Fru) and β-d-Fructofuranosyl-α-d-fucopyranoside (d-Fuc-Fru) have been shown to be substrates for fructosyltransferases, which produce oligo- or polysaccharides, also in the presence of acceptors.     

Studies on lectins. XXXV. Water-soluble O-glycosyl polyacrylamide derivatives for specific precipitation of lectins

By copolymerization of acrylamide and allyl glycosides of various sugars, O-glycosyl derivatives of polyacrylamide copolymers were prepared. The sugar content of the copolymers can be varied in the range 0--40%, their sedimentation coefficient shows the vales of 2.5-5.7 S; the molecular weight of an O-alpha-D-mannopyranosyl polyacrylamide copolymer (29% mannose, so20,w = 2.9 S) was estimated as 44 500. Copolymers with incorporated glycosyl residues interacting specifically with lectins yield precipitates with them upon immunodiffusion in cellulose acetate. The quantitative precipitin curves obtained with these copolymers are similar to those produced by quantitative precipitation of lectins with natural polysaccharides. The copolymers may serve as model substances of natural polysaccharides.     

Glycosyl fluorides in enzymatic reactions

Glycosyl fluorides have considerable importance as substrates and inhibitors in enzymatic reactions. Their good combination of stability and reactivity has enabled their use as glycosyl donors with a variety of carbohydrate processing enzymes. Moreover, the installation of fluorine elsewhere on the carbohydrate scaffold commonly modifies the properties of the glycosyl fluoride such that the resultant compounds act as slow substrates or even inhibitors of enzyme action. This review covers the use of glycosyl fluorides as substrates for wild-type and mutant glycosidases and other enzymes that catalyze glycosyl transfer. The use of substituted glycosyl fluorides as inhibitors of enzymes that catalyze glycosyl transfer and as tools for investigation of their mechanism is discussed, including the labeling of active site residues. Synthetic applications in which glycosyl fluorides are used as glycosyl donors in enzymatic transglycosylation reactions for the synthesis of oligo- and polysaccharides are then covered, including the use of mutant glycosidases, the so-called glycosynthases, which are able to catalyze the formation of glycosides without competing hydrolysis. Finally, a short overview of the use of glycosyl fluorides as substrates and inhibitors of phosphorylases and phosphoglucomutase is given.     

Postnatal gender-dependent maturation of cellular cysteine uptake

Background. In view of the functional capacity of glutathione synthesis in premature infants, and because the availability of cysteine is one the rate limiting steps in glutathione synthesis, we hypothesized that the low glutathione levels in premature infants may be due to immaturity of the active cellular uptake of cysteine.

Objective. To document in cells from newborn infants the effect of maturity and gender on cysteine uptake and consequently on glutathione levels.

Methods. Incorporation of L-[35S] cysteine was measured in leukocytes from cord blood and from tracheal aspirates (TAC) of newborn infants of varying (gestational as well as postnatal) ages and gender. Cysteine uptake was correlated with glutathione in TAC.

Results. The maturity of newborn girls positively influences cysteine uptake, which is responsible for 78% of the variation in their glutathione content. However, in newborn boys, gestational and postnatal ages did not influence the cysteine uptake.

Discussion. Cysteine uptake appears to be the limiting step explaining the reported gender-related differences in glutathione as well as the low levels of this central antioxidant found in premature infants. The immature cysteine uptake found in cells from premature infants raises questions about the bioavailability of this conditionally essential amino acid in regimens of parenteral nutrition for human neonates.     

Chemical synthesis of cellulose and cello-oligomers using a hydrolysis enzyme as a catalyst

Regio- and stereo-selective synthesis of polysaccharides and oligosaccharides has been achieved by using glycosyl fluorides as substrates for cellulases. This methodology has successfully been applied to the first synthesis of cellulose via a non-biosynthetic pathway as well as to a selective preparation of cello-oligosaccharides and unnatural oligosaccharides. Using the enzymatic polymerization, it is possible to control the relative direction (parallel or anti-parallel) of each glucan chain in the synthetic cellulose in vitro. Based on these results, a new concept of ‘allos-selectivity’ in polymer synthesis has been proposed.     

Natural phosphoglycans containing glycosyl phosphate units: structural diversity and chemical synthesis

An anomeric phosphodiester linkage formed by a glycosyl phosphate unit and a hydroxyl group of another monosaccharide is found in many glycopolymers of the outer membrane in bacteria (e.g., capsular polysaccharides and lipopolysaccharides), yeasts and protozoa. The polymers (phosphoglycans) composed of glycosyl phosphate (or oligoglycosyl phosphate) repeating units could be chemically classified as poly(glycosyl phosphates). Their importance as immunologically active components of the cell wall and/or capsule of numerous microorganisms upholds the need to develop routes for the chemical preparation of these biopolymers. In this paper, we (1) present a review of the primary structures (known to date) of natural phosphoglycans from various sources, which contain glycosyl phosphate units, and (2) discuss different approaches and recent achievements in the synthesis of glycosyl phosphosaccharides and poly(glycosyl phosphates).     

New developments in the synthesis of phosphopolyprenols and their glycosyl esters.

Efficient methods were developed in our group in recent years for chemical synthesis of polyprenyl phosphates, polyprenyl monophosphate sugars, and polyprenyl diphosphate sugars, which were known to serve as important intermediates in biosynthesis of complex carbohydrates. A simple procedure was developed involving the phosphorylation of aliphatic alcohols with tetra-n-butylammonium dihydrogen phosphate and trichloroacetonitrile. Monophosphates of various natural and modified dolichols and polyprenols, as well as the derivatives of retinol, cholesterol, and nonacosanol, were prepared in high yields. First syntheses of dolichyl thiophosphate and dolichyl hydrogen phosphonate were developed, and these derivatives were of interest as analogs of dolichyl phosphate. Polyprenyl monophosphate sugars, including derivatives of alpha- and beta-anomers of D-glucopyranose, D-galactopyranose, D-mannopyranose, and 2-acetamido-2-deoxy-D-glucopyranose, were obtained smoothly from moraprenyl trichloroacetimidate and acylated glycosyl phosphates after deprotection. A method for the synthesis of polyprenyl diphosphate sugars from polyprenyl phosphoroimidazolidate and unprotected glycosyl phosphates was shown to be applicable for a wide range of the monosaccharide derivatives including hexoses, deoxyhexoses, 2-acetamido-2-deoxyhexoses, and uronic acids. A series of the oligosaccharide derivatives was also prepared by this method.     

Biotechnical modification of carbohydrates

Abstract: An overview is given of various biotechnical carbohydrate modifications. Fermentation and bioconversion processes, based on carbohydrate substrates for production of mono-, di- and oligosaccharides and the microbial synthesis of various useful polysaccharides are discussed as well as the microbial/enzymatic hydrolysis of polysaccharides into valuable oligomers or di- and monomeric sugars.     

Microbial polysaccharides with actual potential industrial applications

The microbial polysaccharides reviewed include xanthan gum, scleroglucan, PS-10, PS-21 and PS-53 gums, polysaccharides from Alcaligenes sp., PS-7 gum, gellan gum, curdlan, bacterial alginate, dextran, pullulan, Baker's Yeast Glycan, 6-deoxy-hexose-containing polysaccharides and bacterial cellulose. Factors limiting the commercial potential of certain microbial polysaccharides such as availability, rheological properties, and polyvalency are outlined. The polysaccharides are classified according to their uses as viscosity-increasing agents and as gelling agents. A third category includes polysaccharides with specific applications such as tailor-made dextran and pullulan and polysaccharides used as substrates for the preparation of rare sugars. The difficulties encountered in development of a polysaccharide at the industrial level are pointed out.     

植物蔗糖合酶的结构、功能及应用

蔗糖合酶（Sucrose synthase, EC 2.4.1.13, SuS）是植物中广泛存在的一种糖基转移酶,能催化蔗糖的分解及合成反应,是叶片光合作用产物蔗糖进入各种代谢途径所必需的关键酶之一,在植物的生长发育过程中发挥着至关重要的作用．近年研究表明,蔗糖合酶不仅在植物淀粉合成、提高植株抗逆性和影响植株生长等方面扮演着重要的角色,也能为机体提供核苷单糖供体,而这个特性也使得蔗糖合酶基因可以作为一个催化成分被用于核苷单糖的生物合成,具有广泛的应用前景．本文对蔗糖合酶家族基因的染色体定位及功能、蔗糖合酶的结构及亚细胞定位,以及其所具有的生物学功能进行了综述,旨在为蔗糖合酶的进一步研究奠定理论基础．     

Studies on lectins XXXV. Water-soluble O-glycosyl polyacrylamide derivatives for specific precipitation of lectins

By copolymerization of acrylamide and allyl glycosides of various sugars, O-glycosyl derivatives of polyacrylamide copolymers were prepared. The sugar content of the copolymers can be varied in the range 0–40%, their sedimentation coefficient shows the values of 2.5–2.7 S; the molecular weight of an $\text{O-α-}\text{d}\text{-}\text{mannopyranosyl}$ polyacrylamide copolymer (29% mannose, s_20,w⁰ = 2.9 S) was estimated as 44 500. Copolymers with incorporated glycosyl residues interacting specifically with lectins yield precipitates with them upon immunodiffusion in cellulose acetate. The quantitative precipitin curves obtained with these copolymers are similar to those produced by quantitative precipitation of lectins with natural polysaccharides. The copolymers may serve as model substances of natural polysaccharides.     

An Improvement in Staining Technic for Protozoa

A modification of Donaldson's iodine-eosin stain for staining intestinal protozoa is presented. This modification consists of using high dilutions of colloidal iodine (Chandler)² instead of Lugol's solution as well as high dilutions of eosin. A better resolution of the external and internal structures is brought about by the new method.

The procedure is as follows: A portion of the fecal material to be examined is suspended in a 0.6% salt solution; the suspension should be of a consistency so that one drop will make a satisfactory microscope mount under a cover glass. To ten parts of this suspension, in a test tube, is added one part of the stain which is prepared as follows:—

10 parts of distilled water

6 parts of a suspension of colloidal iodine (Chandler) containing 4% iodine—20% iodine suspensoid, Merck

1 part of a 10% water solution of anilin red, Merck (eosin yellowish)

Technicians will find, because iodine in the form of colloidal iodine is readily released to the organisms, that the use of this material is far superior to Lugol's solution hi carrying out the technic for staining intestinal protozoa in the study of fresh mount preparations. Not only are organisms more deeply stained with iodine but by eosin as well, even when employed in high dilutions.     

Laboratory Hints from the Literature

DYES AND THEIR BIOLOGICAL USES Burckhalter, J. H., Jones, E. M., Holcomb, W. F., and Sweet, L. A. N-substituted 2-methoxy-6-chloro-9-aminoacridines. J. Amer. Chem. Soc., 65, 2012. 1943.

Galat, Alexander. New processes for sulfanilamide. Ind. and Eng. Chem., Ind. Ed., 36, 192. 1944.

Kumler, W. D., and Daniels, T. C. The relation between chemical structure and bacteriostatic activity of sulfanilamide type compounds. J. Amer. Chem. Soc., 65, 2190. 1943.

Kwartler, C. E., and Lucas, Philip. The preparation of sutfanil-amidoindazoles. J. Amer. Chem. Soc., 65, 1804. 1943.

Mueller, A. C., and Hamilton, C. S. The synthesis of 1-substituted aminobenzo(f)quinolines. J. Amer. Chem. Soc., 65, 1017. 1943.

Popkin, A. H. Derivatives of biphenylsulfonamides. I. Preparation of p-(o-aminopnenyl)-benzenesulfonamide. J. Amer. Chem. Soc., 65, 2043. 1943.

Popkin, A. H., and Perretta, Gertrude M. Derivatives of biphenyl-sulfonamide. II. Derivatives of p-(o-aminophenyl)-benzenesulfonamide. J. Amer. Chem. Soc., 65, 2046. 1943.

Shreve, R. N., and Bennett, R. B. Studies in azo dyes. I. Preparation and bacteriostatic properties of azo derivatives of 2,6-diaminopvridine. J. Amer. Chem. Soc., 65, 2241. 1943.

Shreve, R. N., and Bennett, R. B. Studies in azo dyes. II. Preparation and bacteriostatic properties of azo derivatives of 8-quinolino. J. Amer. Chem. Soc., 65, 2243. 1943.

Siebenmann, C., and Schnitzer, R. J. Chemotherapeutic study of p-nitrobenzoyl- and related compounds. J. Amer. Chem. Soc., 65, 2127 1943.

ANIMAL MICROTECHNIC Barrett, A. M. A method for staining sections of bone marrow. J. Path & Bact., 56, 133-5. 1944.

Barrett, A. M. On the removal of formaldehyde-produced precipitate from sections. J. Path. & Bact., 56, 135-6. 1944.

Chang, Min-Chueh. Disintegration of epidymal spermatozoa by application of ice to the scrotal testis. J. Exp. Biol., 20, 16-22. 1943.

Ercoli, N., and Lewis, M. N. The age factor in response of bone tissue to alizarin dyes and the mechanism of dye fixation. Anat. Rec., 87, 67-76. 1943.

Hess, Manfred, and Hollander, Franklin. Permanent metachromatic staining of gastric mucus smears. J. Lab. and Clin. Med., 29, 321-3. 1944.

Miller, John A. A new method of staining nervous tissue. Ohio J. of Sci., 44, 31-5. 1944.     

Application of Fluorescence Microscopy and Photomicrography to Woody Tissues

A procedure is described for making preparations of woody tissues for visual observation or photography by incident-light fluorescence microscopy. The chief advantages of the technic are the following:

(1) Reliable recognition of anatomical characteristics in wood without ordinary time-consuming histological technics.

(2) Examination of relatively larger surface areas of wood blocks than by usual methods.

(3) Visual observation and, if desired, photography of tissues and cell structure in dry or in nearly natural or fresh condition.

(4) Marked color contrast without the use of stains in many tissues, including specific types of cells comprising them.

(5) Improved color contrast by use of Congo red with aspects not usually obtained by other methods.     

2,3-Diamino-2,3-dideoxyuronic and 5,7-diamino-3,5,7,9-tetradeoxynonulosonic acids: new components of bacterial polysaccharides

Abstract The discovery of the derivatives of 2,3-diamino-2,3-dideoxyuronic acids and 5,7-diamino-3,5,7,9-tetradeoxynonulosonic acids in bacterial polysaccharides enlarges the list of natural monosaccharides. Many of the new sugars carry unusual N -substituents, such as formyl, ( R )-3-hydroxybutyryl, and acetimidoyl groups. They are most characteristics of O-chains of Pseudomonas aeruginosa lipopolysaccharides, composed almost exclusively of amino sugars or amphoteric amino sugars; the latter seem to play an important role as serological determinants. The identification of these sugars and the structural determination of the O-specific polysaccharides provide the chemical basis for the classification of P. aeruginosa strains. Some of the new monosaccharides enter also the polysaccharides from some other bacteria.     

Brilliant Cresyl Blue as a Stain for Plant Chromosomes

Methods are proposed for staining plant chromosomes with the dye brilliant cresyl blue, and for making these stained preparations permanent by using polyvinyl alcohol mounting medium.

The stain, which is composed of 2% brilliant cresyl blue in 45% aqueous acetic or propionic acid, is used with fixed material in making smear preparations. The technics for staining are similar to those employed in the aceto-carmine method.

The mounting medium is made by mixing 56% polyvinyl alcohol, which is diluted in water to the consistency of thick molasses, with 22% lactic acid and 22% phenol by volume. The permanent slides are made by floating off the cover slip of the temporary slide in 70% alcohol, then applying the mounting medium and replacing the cover slip.

The chief advantages of the methods described are:

1)The preparation of the stain is rapid and simple. The batch of stain will be good with the first try.

2)The staining procedure in some instances is shorter than when using aceto-carmine.

3)The stain shows a high degree of specificity for nuclear structures and gives better results than aceto-carmine when used on certain plant tissues.

4)A minimum number of cells is lost in making the slides permanent when using polyvinyl alcohol mounting medium as the slide and cover slip are run through only one solution prior to mounting.

5)The mounting medium dries rapidly and this shortens the time required before critical examination of the permanent mounts can be made.     

Specific Staining of Nucleolar Substance with Aceto-Carmine

A method is described for staining nucleoli intensely by treating tissues with formaldehyde, hydrolysing in normal HC1 at 60°C. and staining with aceto-carmine. With correct hydrolysis time, chromosomes and cytoplasm are almost colorless.

Formaldehyde increases the acidity of cell parts, especially the nucleolus, presumably by neutralizing the basic protein groups, and increases the resistance to hydrolysis, perhaps by protecting the phospholipoprotein complexes which are most abundant in the nucleolus.

Hydrolysis reduces the acidity of cell parts, chiefly by removal of nucleic acids.

Aceto-carmine stains cell structures which are weakly acid in character (about pH 4-5) probably by precipitating as large dye aggregates.

The technic appears to be highly specific for nucleoli and related cell bodies.     

合成生物学指导下芽孢杆菌合成环脂肽的研究进展*

近年来,合成生物学借助工程化在人工生命系统的设计与构建方面取得了长足进展,特别是“细胞工厂”的开发和应用为天然产物的合成带来了深刻变革。环脂肽是一类新型的天然表面活性剂,因其特殊的结构和功能亦可作为抗生素使用。目前,合成环脂肽最理想的微生物底盘是芽孢杆菌。因此,许多研究者致力于通过合成生物学技术来提升芽孢杆菌作为环脂肽细胞工厂的性能。首先,对芽孢杆菌中环脂肽的非核糖体肽合成途径进行概述;其次,重点介绍与环脂肽合成相关的调控因子;再次,从底盘细胞的选择、基因编辑工具的开发、合成路径的优化及发酵过程的优化等四个方面对合成生物学指导下环脂肽的相关研究进展进行总结;最后,讨论环脂肽合成中可能存在的挑战,并就未来研究趋势进行展望,以期为高效环脂肽细胞工厂的开发提供参考。