Enzymatic deacetylation of galactoglucomannans

Several hemicellulolytic microorganisms were screened for their capability of liberating acetyl side groups from native softwood galactoglucomannan. All the microorganisms tested were found to produce an extracellular acetyl glucomannan esterase(s). The highest activity was detected in Schizophyllum commune culture filtrate. However, the enzyme produced by Aspergillus oryzae was most efficient in long-term hydrolysis. Acting alone, the purified esterase of A. oryzae was able to liberate most of the acetic acid from galactoglucomannan. The addition of other galactoglucomannan-degrading enzymes did not affect the action of esterase. On the other hand, the addition of esterase clearly enhanced the action of mannanase and -galactosidase. The purified acetyl esterase of Trichoderma reesei was able to liberate acetic acid from short oligomers of glucomannan, whereas the acetyl xylan esterase of T. reesei was unable to act on glucomannan oligomers of any size. Correspondence to: M. Tenkanen     

Complete and regioselective deacetylation of peracetylated uridines using a lipase

Lipase-catalysed alcoholysis and hydrolysis of 2',3',5'-tri-O-acetyluridine (1a) and 2',3',5'-tri-O-acetyl-2'-C-methyluridine (1b) were studied. Conditions for full and regioselective deacetylation of 1aand 1b are shown in the present work. New compound 2',3'-di-O-acetyl-2'-C-methyluridine (3b) was prepared by regioselective lipase-catalysed deacetylation.     

Enzymatic regioselective deprotection of peracetylated mono- and disaccharides

Selective enzymatic hydrolysis of the peracetylated disaccharides, namely cellobiose, lactose, maltose and melibiose, with lipase from Asperilligus niger in aqueous buffer and organic solvent for 30 min afforded exclusively the corresponding heptaacetates with a free hydroxyl group at C-1 in high yield. Prolonged reaction of the β-1,4 linked cellobiose and lactose peracetates afforded selectively their hexaacetates with free hydroxyl groups at C-1,2, whereas the α-1,4 linked disaccharides maltose and melibiose peracetate gave a complex mixture of products. The reaction of 2-acetamido-2-deoxy-1,3,4,6-tetra-O-acetylglucopyranose (11) for 22 h afforded as the major product the diacetate 12 with free hydroxyl groups at C-1,4.     

Enzymatic regioselective synthesis of sucrose acrylate esters

Using a protease (at 100 g l^–1) from Bacillus licheniformis, enzymatic acryloylation of sucrose (1 M) with vinyl acrylate (4 M) was carried out in anhydrous pyridine and yielded sucrose acrylate esters with more than 90% of sucrose converted in 24 h. After 5 days of reaction, the ester products consisted of 70% sucrose monoacrylate and 30% sucrose diacrylate. The monoester product was a sucrose 1-acrylate and the diester products consisted of sucrose 6,1-diacrylate and sucrose 6,1-diacrylate in the ratio of 3:2.     

Enzymatic deacetylation of N-acetylglucosamine residues in cell wall peptidoglycan

An enzyme which catalyzes the hydrolysis of acetamido groups of N-acetylglucosamine residues in cell wall peptidoglycan was found in the supernatant and 20,000 X g pellet fractions of Bacillus cereus. Autolysis of the latter fraction resulted in solubilization and activation of the deacetylase. Among various bacteria, strains of B. cereus which contain high proportions of N-unsubstituted glucosamine residues in their cell wall peptidoglycan components are particularly rich in the deacetylase. The peptidoglycan deacetylase is distinguishable from N-acetylglucosamine-6-phosphate deacetylase [EC 3.5.1.25] on the basis of their cellular distribution and chromatographic behavior. The rate of reaction of the deacetylase with (N-acetylglucosaminyl-N-acetylmuramic acid)3 [abbreviated as (GlcNAc-MurNAc)3] is less than 1/100 of that with peptidoglycan, while the enzyme is inactive towards (GlcNAc-MurNAc)2, GlcNAc-MurNAc, and monomeric N-acetylglucosamine derivatives. The enzyme also deacetylates partially O-hydroxyethylated chitin. The concentrations of peptidoglycan and partially O-hydroxyethylated chitin required for half-maximum activities were found to be 0.29 and 6.9 mg per ml (or 0.17 and 20 mM with respect to N-acetylglucosamine residues), respectively. The occurrence of this enzyme accounts for the formation of cell wall peptidoglycan N-unsubstituted at the glucosamine residues.     

Enzymatic method for determination of the degree of deacetylation of chitosan.

A new method was developed for the accurate determination of the degree of deacetylation of chitosan. The method involves the complete hydrolysis of chitosan to glucosamine and N-acetylglucosamine by a cooperative action of chitosanolytic enzymes exo-beta-D-glucosaminidase, beta-N-acetylhexosaminidase, and chitosanase, and subsequent determination of the monosaccharides by specific colorimetric assays or HPLC. The conditions required for the complete hydrolysis of chitosan were examined and the degree of deacetylation of several chitosan samples was determined.     

Synthesis of 2'-spiro ribo- and arabinonucleosides

Four conformationally restricted bicyclic 2'-spiro nucleosides were synthesised and incorporated into oligonucleotides. These spiro nucleotides induced decreased duplex thermostabilities.     

A pathway of chitosan formation in Mucor rouxii. Enzymatic deacetylation of chitin.

1. An enzyme that catalyzes hydrolysis of acetamido groups of chitin derivatives was found in the supernatant fraction of Mucor rouxii. 2. Partially O-hydroxyethylated chitin (glycol chitin) was used as a substrate in the purification and characterization of this enzyme. A 140-fold purification was obtained by means of ammonium sulfate fractionation followed by chromatography on carboxymethylcellulose and DEAE-cellulose. 3. The enzyme releases about 30% of the acetyl groups of glycol chitin, giving a product with a decreased sensitivity to lysozyme. The enzyme also deacetylates chitin and N-acetylchitooligoses, whereas it is inactive toward bacterial cell wall peptidoglycan, N-acetylated heparin, a polymer of N-acetylgalactosamine, di-N-acetylchitobiose and monomeric N-acetylglucosamine derivatives. 4. This enzyme shows a pH optimum of 5.5. The Km value for glycol chitin is 0.87 g/l or 2.6 mM with respect to monosaccharide residues. 5. The occurrence of this enzyme accounts for the formation of chitosan in fungi.     

New synthesis of artepillin C,a prenylated phenol,utilizing lipase-catalyzed regioselective deacetylation as the key step

We have synthesized artepillin C, a diprenylated p-hydroxycinnamate originally isolated from Brazilian propolis and exhibiting antioxidant and antitumor activities, from 2,6-diallylphenol. Replacement of the terminal vinyl with 2,2-dimethylvinyl group by olefin cross-metathesis and subsequent transformation yielded 2,6-diprenyl-1,4-hydroquinone diacetate. Candida antarctica lipase B-catalyzed deacetylation in 2-propanol regioselectively removed the less hindered acetyl group to give 2,6-diprenyl-1,4-hydroquinone 1-monoacetate. After triflation of the liberated 4-hydroxy group, a three-carbon side chain was introduced by palladium-mediated alkenylation with methyl acrylate. Final hydrolysis of the esters furnished artepillin C.     

Cloning and functional characterisation of two regioselective flavonoid glucosyltransferases from Beta vulgaris

Two full-length cDNAs encoding flavonoid-specific glucosyltransferases, UGT73A4 and UGT71F1, were isolated from a cDNA library of Beta vulgaris (Amaranthaceae) cell suspension cultures. They displayed high identity to position-specific betanidin and flavonoid glucosyltransferases from Dorotheanthus bellidiformis (Aizoaceae) and to enzymes with similar substrate specificities from various plant families. The open reading frame of the sequences encode proteins of 476 (UGT73A4) and 492 (UGT71F1) amino acids with calculated molecular masses of 54.07kDa and 54.39kDa, and isoelectric points of 5.8 and 5.6, respectively. Both enzymes were functionally expressed in Escherichia coli as His- and GST-tagged proteins, respectively. They exhibited a broad substrate specificity, but a distinct regioselectivity, glucosylating a variety of flavonols, flavones, flavanones, and coumarins. UGT73A4 showed a preference for the 4'- and 7-OH position in the flavonoids, whereas UGT71F1 preferentially glucosylated the 3- or the 7-OH position. Glucosylation of betanidin, the aglycone of the major betacyanin, betanin, in B. vulgaris was also observed to a low extent by both enzymes. Several O-glycosylated vitexin derivatives isolated from leaves of young B. vulgaris plants and rutin obtained from B. vulgaris tissue culture are discussed as potential endogenous products of UGT73A4 and UGT71F1. The results are analyzed with regard to evolution and specificity of plant natural product glucosyltransferases.     

Enzymatic deacetylation of chitin by extracellular chitin deacetylase from a newly screened Mortierella sp. DY-52

Among more than a hundred colonies of fungi isolated from soil samples, DY-52 has been screened as an extracellular chitin deacetylase (CDA) producer. The isolate was further identified as Mortierella sp., based on the morphological properties and the nucleotide sequence of its 18S rRNA gene. The fungus exhibited maximal growth in yeast peptone glucose (YPD) liquid medium containing 2% of glucose at pH 5.0 and 28 degrees C with 150 rpm. The CDA activity of DY-52 was maximal (20 U/mg) on the 3rd day of culture in the same medium. The CDA was inducible by addition of glucose and chitin. The enzyme contained two isoforms of molecular mass 50 kDa and 59 kDa. This enzyme showed a maximal activity at pH 5.5 and 60 degrees C. In addition, it had a pH stability range of 4.5-8.0 and a temperature stability range of 4-40 degrees C. The enzyme was enhanced in the presence of Co2+ and Ca2+. Among various substrates tested, WSCT-50 (water-soluble chitin, degree of deacetylation 50%), glycol chitin, and crab chitosan (DD 71-88%) were deacetylated. Moreover, the CDA can handle N-acetylglucosamine oligomers (GlcNAc)2-7.     

Selection with two alleles and complete dominance

For a plant selection model with frequency-independent viabilities, fertilities and selfing rates, it is shown that apart from global fixation, for certain parameter combinations a protected polymorphism and facultative fixation (either allele may become fixed according to initial frequencies) may both occur. Facultative fixation requires different selling rates for the dominant and recessive type. Protection of the polymorphism requires resource allocation for male and female function. In this connection the problem of purely genetically caused population extinction is discussed.
For general frequency dependence and regular segregation, the chances for establishment of a completely recessive gene are compared to those of a completely dominant gene. It is proven that the process of establishment of the recessive gene, despite a fitness advantage, may be considerably endangered by drift effects if random mating prevails. The recessive gene may reach the same effectivity in establishment as a dominant gene, only if the recessive homozygote mates exclusively with its own type during the period of establishment.     

Enzymatic synthesis of two ginsenoside Re-beta-xylosides.

Two new beta-xylosyl derivatives of ginsenoside Re, 20(S)-protopanaxatriol 6-O-alpha-L-rhamnopyranosyl-(1 --> 2)-[beta-D-xylopyranosyl-(1 --> 4)]-beta-D-glucopyranosyl-20-O-beta-D-glucopyranoside and 20(S)-protopanaxatriol 6-O-alpha-L-rhamnopyranosyl-(1 --> 2)-[beta-D-xylopyranosyl-(1 --> 6)]-beta-D-glucopyranosyl-20-O-beta-D-glucopyranoside, were respectively synthesized from p-nitrophenyl beta-D-xylopyranoside and phenyl beta-D-xylopyranoside as donors and ginsenoside Re as the acceptor in 25% acetone and acetonitrile by a cellulase preparation from Trichoderma viride and a beta-galactosidase preparation from Aspergillus oryzae. The latter enzyme preparation also catalyzed the hydrolysis of ginsenoside Re to the minor saponin, ginsenoside Rg2.     

Continuous deacetylation of cephalosporins

Continuous deacetylation of cephalosporin C, 7-aminocephalosporanic acid, and of 2-methoxyethyl acetate in packed beds of an immobilized esterase is described by simple empirical equations relating conversion to space velocity and temperature. The choice of process conditions is discussed in relation to the effects of temperature on column efficiency, column life, growth of microbial contaminants, and the rates of thermal decomposition of the substrates. At the preferred temperature of 10°C columns were operated continuously for one month with only small losses in efficiency.