Cycloalkyl β-D-glucopyranosides were synthesized by transglucosylation of a β-glucosidase from fungi. When cyclopropanemethanol (CPAM), cyclopentanol (CPE) and cyclopentanemethanol (CPEM) were used as the acceptors, the enzyme stereoselectively synthesized cyclopropylmethyl, cyclopentyl and cyclopentylmethyl β-D-glucopyranosides (abbreviated to CPAM-β-G, CPE-β-G and CPEM-β-G, respectively) from cellobiose as a glucosyl donor. Among the above three β-glucopyranosides, only CPEM-β-G was found to inhibit sweet almond enzyme activity (Ki=0.15± 0.02 mM). Other cycloalkyl β-D-glucopyranosides had little/or no inhibitory activity toward the β-glucosidases examined. CPEM itself had a weak inhibitory activity for sweet almond enzyme, with an uncompetitive type. However, the introduction of a glucose molecule to CPEM as a glycon converted its inhibition type into a competitive one. The Ki value of CPEM-β-G for the enzyme was reduced to about 1/7 compared with the corresponding cyclic alcohol. As it is expected that CPEM-β-G is much more likely to inhibit the activity of an enzyme from plant origin compared with those from fungi, we studied the possibility of CPEM-β-G inhibiting the activities of β-glucosidases extracted from the cut flowers on the basis of their kinetic data.
Microbulbifer sp. strain JAMB-A3, isolated from the sediment in Sagami Bay, Japan, at a depth of 1174 m, was found to produce a novel β-agarase. The agarase gene was cloned and sequenced. It encodes a protein of 602 amino acids with a calculated molecular mass of 65,017 Da. The deduced amino acid sequence showed similarity to those of known β-agarases in glycoside hydrolase family 16, with 34-55% identity. Tandem sequences similar to a carbohydrate binding-like module were found in the C-terminal region of the enzyme. The recombinant agarase was hyper-produced extracellularly using Bacillus subtilis as the host, and the homogeneously purified enzyme had a high specific activity of 528 U/mg at pH 7.0 and 50°C. The optimal temperature and pH for activity were 54°C and around 7, respectively. The recombinant enzyme was thermostable with a half-life of 8.7 h at 50°C. It was very stable during incubation with EDTA, Na+, K+, Mg2+, Ca2+ ions, and surfactants at high concentrations. N-Bromosuccinimide abolished the enzymatic activity, and agarose oligosaccharides protected the enzyme from inactivation by this chemical, suggesting that a tryptophan(s) residue is involved in the catalysis of the enzyme. The pattern of agarose hydrolysis showed that the enzyme is an endo-type β-agarase, and the final main product is neoagarotetraose.
A fungus, strain YM-80, isolated from a soil sample produced β-glucosidase ( β-D-glucoside glucohydrolase EC 22.214.171.124) in solid culture. The fungus was identified as Aspergillus pulverulentus from its taxonomical characteristics. The β-glucosidase was purified 18-fold from the culture extract by (NH4)2SO4 precipitation, DEAE-Toyopearl 650M, Butyl-Toyopearl 650M and Sephacryl S-200 chromatography. The purified enzyme appeared as a single band on SDS-PAGE. The molecular weight of the purified enzyme was estimated to be 118,000 by SDS-PAGE and 240,000 by gel filtration. The enzyme was an acidic protein with a pI of 4.5 and the specific activity was 308 U/mg protein. The optimum temperature and pH for the enzyme activity were 60°C and 4.0, respectively. This enzyme was stable in the pH range 3.0-7.0 and also up to 50°C. The activity of the enzyme was completely inhibited by Hg2+ and SDS. The purified enzyme was active with a broad range of β-linked glucosides but not α-linked saccharides. Added during tofu processing, the enzyme was useful for the preparation of aglycon isoflavone-enriched tofu by hydrolysis of the glucoside form. However, isoflavones with 6"-O-acetyl and 6"-O-malonyl glucosides were not converted into the aglycon form.
Amylose content and molecular structures of starch from a mutant of wheat (Triticum aestivum L.) that lacks a starch granule bound protein, SGP-1, have been compared with those from normal wheat (cv. Chinese Spring). Actual amylose contents, calculated by taking iodine affinity of amylopectin into consideration, for the mutant (SGP-1 null) and normal wheat starches were 34 and 26%, respectively. The amylopectins from SGP-1 null and normal starches had number-average degree of polymerization (DPn) of 4200 and 4700, respectively. Three molecular species of amylopectin with different size were observed by fluorescent labeling/GPC. Number-average chain length (CLn) of the amylopectins was close (∼21) but their CL distributions were significantly different. SGP-1 null amylopectin contained 9.5% by weight of long unit-chains having CL of >100 while such long chains were absent in amylopectin from the normal starch. Molar ratio of A chains increased slightly and that of B1 chains conversely decreased in the mutant, and the molar ratio of (A+B1)/(B2+B3) was 13.8 and 13.1 for the mutant and normal amylopectins, respectively. DPn of amylose from SGP-1 null (∼800) was lower than that for the normal (∼1000). DP distribution of the amyloses on molar and weight bases showed the ratio of smaller amyloses was higher for the mutant than the normal starch. Branched molecules of amylose consisted of 24 and 23% by mole and had average number of chains of branched molecules (NCB) 13.1 and 7.5, respectively. The above structural data on a molar basis also revealed that the mutation caused the increase in number of amylose molecules but had no effect on the number of amylopectin molecules.
Cyclomaltodextrin glucanotransferase (EC 126.96.36.199, abbreviated as CGTase) from Bacillus stearothermophilus produced a series of transfer products from a mixture of cyclomaltohexaose and D-fructose. After digestion with glucoamylase, the resultant product contained turanose and trehalulose in addition to D-glucose and D-fructose. These results showed that CGTase transferred the glucose residue to the C3- and C1-hydroxy groups of fructopyranose. The optimum conditions on the turanose production from α-CD and D-fructose using CGTase were examined. The reaction mixture containing 30% of α-CD and 30% of D-fructose and 300 U/g of α-CD of CGTase from B. stearothermophilus was incubated at 60°C and pH 5.5 for 24 h and then treated with glucoamylase. Turanose was produced in a yield of about 45%.
A novel species of alkaliphilic Bacillus (JAMB-602), isolated from a soil sample, was found to exoproduce an alkaline mannanase. The mannanase gene was cloned by the shotgun method and sequenced. Its open reading frame encodes a protein of 490 amino acids with a calculated molecular mass of 53,763 Da. The deduced amino acid sequence showed similarity to those of known mannanases in glycoside hydrolase family 5, with only 32-58% identity. The recombinant mature enzyme with a molecular mass of 50 kDa was hyper-exoproduced with Bacillus subtilis as the host, corresponding to a level of approximately 2 g/L. The homogeneously purified enzyme had a molecular mass of 50 kDa, an optimal temperature of 65°C, and an optimal pH of around 9. It was thermostable with a half-life of 2 h at 55°C. N-Bromosuccinimide abolished the enzymatic activity. The pattern of mannan hydrolysis showed that the enzyme is an endo-type mannanase.
A xylanase gene was cloned from the cDNA library of Aspergillus sojae. The nucleotide sequence suggested that the mature enzyme is preceded by a 22 amino acid signal sequence and the molecular mass was predicted to be 32,403 Da. The deduced amino acid sequence of the enzyme exhibits good identity (98%) with that of Aspergillus oryzae xynF3 (T. Kimura etal.: Biosci. Biotechnol. Biochem., 66, 285-292, 2002), which is a enzyme that belongs to family 10 of the glycosyl hydrolases. The mature xylanase gene was expressed in Escherichia coli as a cellulose-binding domain tag fusion protein. The recombinant protein expressed high enzyme activity and the temperature stability was increased remarkably more with CBIND™ 100 Resin in the reaction mixture than was the case without the resin.
Using potato cultivars grown in Hokkaido, the starch properties in potato tubers were characterized. The starch properties determined in this study were the contents of amylose and phosphorus, granule size distribution, and pasting properties using a rapid visco-analyzer. There were wide variations in starch parameters among the 96 potato starch samples used. A high phosphorus content of starch was closely associated with high peak viscosity and breakdown and low peak viscosity temperature. Although the amylose content also had an effect on pasting properties, the correlation coefficients between the phosphorus content and pasting properties were higher than those between the amylose content and pasting properties.
Inulin is a fructan in which linear chains of β-(2→1)-linked D-fructofuranose molecules are attached to sucrose at the reducing end. Inulinases have been encountered in higher plants and in microorganisms, including filamentous fungi, yeasts and bacteria. Exoinulinases exo- and endoinulinases from filamentous fungi have been purified and characterized as monomeric glycoproteins. The inulinase genes from Aspergillus and Penicillium spp. have been cloned and the deduced amino acid sequences included conserved sequences in the β-fructofuranosidase superfamily. A phylogenetic analysis showed that fungal exo- and endoinulinases have independently evolved the respective hydrolytic activities toward terminal and internal β-(2→1)-fructofuranosidic linkages in inulin. Aspergillus niger inulinases in an immobilized form were applied to the continuous production of fructose syrup or inulo-oligosaccharides from inulin. High concentrations of ethanol were produced from pure inulin or Jerusalem artichoke tubers by a simultaneous saccharification and fermentation process using A. niger and Saccharomyces cerevisiae.
The production and rheology of exomannan by the yeast Rhokotorula mucilaginosa YR-2 was studied. Cultures were incubated at 30°C in medium (sucrose: 100 g/L; ammonium sulfate: 20 g/L). Fermentation used a 10-liter fermentor aerated at 1 vvm, and 550 rpm. During culture, the pH was adjusted to 1.8 with ammonia solution. The maximum amounts of exopolysaccharide (7.8 g/L) and biomass (39.2 g/L) were obtained at 120 h. This mannan was much less viscous than other polysaccharides, and had excellent physical stability in terms of pH, freezing, and NaCl concentration.
The tandem-cluster structure of amylopectin must be a refined product that higher plants have evolved from the cluster-free random branched structure of glycogen. During the evolutionary process, plants have increased the number of isozymes of three classes of enzymes, namely soluble starch synthase (SS), starch branching enzyme (BE) and starch debranching enzymes (DBE), by giving each individual enzyme a distinct function in constructing the amylopectin cluster structure. On the basis of biochemical and molecular analyses of starch mutants and transgenic plants of rice, the present paper presents a model for amylopectin cluster biosynthesis in rice endosperm to understand how each enzyme is involved in this process. The author also proposes a notion called “cluster-world” where by the structure of amylopectin varies through changes in several distinct patterns of the cluster structure, and alteration of activities of key enzymes such as SSIIa, BEIIb and isoamylase1 results in the structural variation within the fixed pattern depending on which enzyme activity is altered.