Purification and characterization of a (1,4)-β-d-arabinoxylan arabinofuranohydrolase from Aspergillus awamori

Summary An enzyme able to split off arabinose sidechains from cereal arabinoxylans was isolated from a cell-free culture filtrate of Aspergillus awamori CMI 142717 containing milled oat straw as the carbon source. The enzyme was highly specific for arabinoxylans and, unlike other -l-arabinofuranosidases reported in the literature, did not show any activity towards p-nitrophenyl -l-arabinofuranoside, arabinans and arabinogalactans. This novel enzyme, which can be described as a (1,4)--d-arabinofuranohydrolase, had a molecular mass of 32 000 Da when determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and a specific activity of 22 units/mg on wheat arabinoxylan. Offprint requests to: A. G. J. Voragen     

Purification and Substrate Specificities of Two α-l-Arabinofuranosidases from Aspergillus awamori IFO 4033

α-l-Arabinofuranosidases I and II were purified from the culture filtrate of Aspergillus awamori IFO 4033 and had molecular weights of 81,000 and 62,000 and pIs of 3.3 and 3.6, respectively. Both enzymes had an optimum pH of 4.0 and an optimum temperature of 60°C and exhibited stability at pH values from 3 to 7 and at temperatures up to 60°C. The enzymes released arabinose from p-nitrophenyl-α-l-arabinofuranoside, O-α-l-arabinofuranosyl-(1→3)-O-β-d-xylopyranosyl-(1→4)-d-xylopyranose, and arabinose-containing polysaccharides but not from O-β-d-xylopyranosyl-(1→2)-O-α-l-arabinofuranosyl-(1→3)-O-β-d-xylopyranosyl-(1→4)-O-β-d-xylopyranosyl-(1→4)-d-xylopyranose. α-l-Arabinofuranosidase I also released arabinose from O-β-d-xylopy-ranosyl-(1→4)-[O-α-l-arabinofuranosyl-(1→3)]-O-β-d-xylopyranosyl-(1→4)-d-xylopyranose. However, α-l-arabinofuranosidase II did not readily catalyze this hydrolysis reaction. α-l-Arabinofuranosidase I hydrolyzed all linkages that can occur between two α-l-arabinofuranosyl residues in the following order: (1→5) linkage > (1→3) linkage > (1→2) linkage. α-l-Arabinofuranosidase II hydrolyzed the linkages in the following order: (1→5) linkage > (1→2) linkage > (1→3) linkage. α-l-Arabinofuranosidase I preferentially hydrolyzed the (1→5) linkage of branched arabinotrisaccharide. On the other hand, α-l-arabinofuranosidase II preferentially hydrolyzed the (1→3) linkage in the same substrate. α-l-Arabinofuranosidase I released arabinose from the nonreducing terminus of arabinan, whereas α-l-arabinofuranosidase II preferentially hydrolyzed the arabinosyl side chain linkage of arabinan.Recently, it has been proven that l-arabinose selectively inhibits intestinal sucrase in a noncompetitive manner and reduces the glycemic response after sucrose ingestion in animals (33). Based on this observation, l-arabinose can be used as a physiologically functional sugar that inhibits sucrose digestion. Effective l-arabinose production is therefore important in the food industry. l-Arabinosyl residues are widely distributed in hemicelluloses, such as arabinan, arabinoxylan, gum arabic, and arabinogalactan, and the α-l-arabinofuranosidases (α-l-AFases) (EC 3.2.1.55) have proven to be essential tools for enzymatic degradation of hemicelluloses and structural studies of these compounds.α-l-AFases have been classified into two families of glycanases (families 51 and 54) on the basis of amino acid sequence similarities (11). The two families of α-l-AFases also differ in substrate specificity for arabinose-containing polysaccharides. Beldman et al. summarized the α-l-AFase classification based on substrate specificities (3). One group contains the Arafur A (family 51) enzymes, which exhibit very little or no activity with arabinose-containing polysaccharides. The other group contains the Arafur B (family 54) enzymes, which cleave arabinosyl side chains from polymers. However, this classification is too broad to define the substrate specificities of α-l-AFases. There have been many studies of the α-l-AFases (3, 12), especially the α-l-AFases of Aspergillus species (2–8, 12–15, 17, 22, 23, 28–32, 36–39, 41–43, 46). However, there have been only a few studies of the precise specificities of these α-l-AFases. In previous work, we elucidated the substrate specificities of α-l-AFases from Aspergillus niger 5-16 (17) and Bacillus subtilis 3-6 (16, 18), which should be classified in the Arafur A group and exhibit activity with arabinoxylooligosaccharides, synthetic methyl 2-O-, 3-O-, and 5-O-arabinofuranosyl-α-l-arabinofuranosides (arabinofuranobiosides) (20), and methyl 3,5-di-O-α-l-arabinofuranosyl-α-l-arabinofuranoside (arabinofuranotrioside) (19).In the present work, we purified two α-l-AFases from a culture filtrate of Aspergillus awamori IFO 4033 and determined the substrate specificities of these α-l-AFases by using arabinose-containing polysaccharides and the core oligosaccharides of arabinoxylan and arabinan.     

Culture Conditions for the Production of Alkaline Protease from n-Paraffins by a Kabicidin Resistant Mutant No. 5–128B

A novel process for the microbial production of alkaline protease on an industrial scale was successfully established by using a kabicidin resistant mutant, No. 5–128B, derived from Fusarium sp. S–19–5. The most suitable carbon source for producing alkaline protease was n-paraffins (C₁₀~C₁₄) and the effective nitrogen source was dried-yeast cells containing no nucleic acid, the optimum concentrations being 12.5% (w/v) and 7.0% (w/v), respectively. The optimal temperature and initial pH for protease production were 24°C and 6.0, respectively. Under the optimal conditions using a shaker flask mutant No. 5–128B produced 41000 PU/ml of alkaline protease, which corresponded to about 10 times the amount produced by the parent strain. The relation between the high ability to produce alkaline protease and the resistance to kabicidin, a polyene antibiotic, is discussed.     

Production of β-Mannanase and β-Mannosidase from Aspergillus awamori K4 and Their Properties

β-Mannanase and β-mannosidase from Aspergillus awamori K4 was produced by solid culture with coffee waste and wheat bran. The optimum composition for enzyme production was 40% coffee waste–60% wheat bran. Two enzymes were partially purified. Optimum pH was about 5 for both enzymes, and optimum temperature was around 80°C for β-mannanase and 60–70°C for β-mannosidase. These enzymes produced some oligosaccharides from glucomannan and galactomannan by their hydrolyzing and transferring activities. β-Mannanase hydrolyzed konjak and locust bean gum 39.1% and 15.8%, respectively. Oligosaccharides of various molecular size were released from glucomannan of konjak, but on the addition of cellulase, mannobiose was released selectively. In locust bean gum, tetra-, tri-, and disaccharides (mannobiose) were mainly released by K4 β-mannanase. Tetra- and trisaccharides were heterooligosaccharides consisting of galactose and mannose residues. K4 β-mannosidase had a transglycosylation action, transferring mannose residue to alcohols and sugars like fructose. Received: 24 April 2000/Accepted: 20 October 2000     

Isolation of a Raw Starch-Binding Fragment from Barley α-Amylase

Barley α-amylase was purified by ammonium sulfate fraction, ion-exchange, ultrafiltration, and gel filtration to homogeneity. The purified enzyme was partially digested with trypsin, and the reaction mixture was applied to a cyclohepta-amylose epoxy Sepharose 6B column. Bound fragments were eluted by free cyclohepta-amylose, lyophilized, and separated on Tricine gels. Four fragments were shown to interact with β-cyclodextrin. The fragment that could be identified on the gel with the lowest molecular weight (11 kDa) was electroblotted onto PVDF membrane for sequencing. The N-terminal sequence of this fragment was determined with the N-terminal amino acid corresponding to Ala283 in the whole protein. The trypsin cleavage was at Lys282/Ala283 and the C-terminal cleavage occurred at Lys354/Ile355 to give a fragment size of 11 kDa as estimated by SDS-PAGE. The fragment would be located at the C-terminal region, forming a majority of the antiparallel β-sheets in domain C and the α₇-and α₈-helices of the (α/β)₈ domain.     

Monochoria vaginalis var. angustifolia，a new variety of the Pontederiaceae from Thailand

A new variety of Monochoria C. Presl from Thailand, M. vaginalis （N. L. Burman）Kunth var.angustifolia G. X. Wang, is described. This variety can be distinguished from the typical one, M. vaginalis var. vaginalis, by having mature leaves narrowly lanceolat     

Preparation of baicalein from baicalin using a baicalin-β-D-glucuronidase from Aspergillus niger b.48 strain

Baicalin-β-d-glucuronidase was produced from a culture of Aspergillus niger b.48 strain using Scutellaria root extract as an enzyme inducer, purified and characterized. The enzyme’s molecular weight was approximately 45 kDa; its optimal operating temperature and pH were 50 °C and 5.0, respectively. The enzyme specifically hydrolysed 7-O-β-d-glucuronide of baicalin into baicalein, weakly hydrolysed β-d-glucuronide of p-nitrophenyl-β-d-glucuronide and p-phenolphthalein-β-d-glucuronide, but did not hydrolyse β-d-glucuronide of glycyrrhizin. The Michaelis constant (Km) was 21.74 mM; Vmax was 11.63 mM/h. Common metallic ions almost did not effect enzyme activity; greater than 10 mM/L Cu²⁺ and greater 50 mM/L Fe³⁺ ion strongly inhibited enzyme activity. The use of pure enzyme in baicalin conversion to baicalein was costly, the crude baicalin-β-d-glucuronidase from A. niger b.48 strain was used in the preparation of baicalein from baicalin to keep costs low. The optimum conditions for baicalein production from crude enzyme reaction were 1% baicalin reacting for 20 h–24 h at pH 5.0 and 50 °C. Here, 10.7 g baicalein was obtained from 20 g baicalin using the crude enzyme, and the molar yield was 88.4 %. Therefore, active baicalein was successfully produced at low cost from baicalin using a non-transgenic crude enzyme from A. niger b.48.     

Functional properties of a chitinase promoter from cabbage （Brassica oleracea var.capitata）

The 5‘-region of the chitinase gene cabch29,derived from Brassica oleracea var.capitata,has been sequenced and analyzed for cis-acting elements important in controlling gene expression in transgenic tobacco plants.Different 5‘-deletion fragments were linked to reporter gene β-glucuronidase (GUS) as translational fusions,and the expression of these chimeric genes was analyzed in vegetative organs and tissues.Sequences up to-651 showed some basal GUS activity with nearly equal levels in wounded and intact tissues.The addition of further upstream sequences(-651 to-1284) enhanced expression level,and the expression driven by this fragment was inducible by a factor of two to three-fold by wounding.Histochemical analysis of different tissue from transgenic plants that contain cabch29 promoter-gus fusion gene demonstrated woundinducible and tissue-specific cabch29 promoter activity in plants containing the 1308 base pair fragment.The location of GUS activity appears to be cell-specific,being highest in vascular cells and epidermal cells of stem,leaf and roots.Meanwhile,the temporal and spatial expression of cabch29-GUS fusion gene has been investigated.Among the different vegetative organs,a high level of GUS activity was observed in stem and a moderate one in roots;whereas,wounding stress led to a high level of GUS in stem and moderate one in leaf.     

High-level production and characterization of a novel β-1,3-1,4-glucanase from Aspergillus awamori and its potential application in the brewing industry

A novel β-1,3-1,4-glucanase gene (AaBglu12A) from Aspergillus awamori was extracellularly expressed in Pichia pastoris. AaBglu12A showed amino acid identity of 96 % with a glycoside hydrolase family 12 cellulase from A. kawachii and 48 % with a β-1,3-1,4-glucanase from Magnaporthe oryzae. The highest β-1,3-1,4-glucanase activity of 159,500 ± 500 U/mL with protein concentration of 31.7 ± 0.3 g/L was achieved in a 5-L fermentor. AaBglu12A was purified until homogeneous with recovery yield of 92 %. Its maximal activity was found at 55 °C and pH 5.0. The enzyme was stable up to 60 °C and within the pH range of 2.0-9.0. It also demonstrated strict substrate specificity towards oat- and barley-glucans as well as lichenan. The K_m values for oat-, barley-glucans, and lichenan were 2.82, 3.51, and 2.53 mg/mL, respectively. The V_max values for oat-, barley-glucans, and lichenan were 12,068, 10,790, and 7236 μmol/min·mg, respectively. AaBglu12A hydrolyzed oat- and barley-β-glucans to produce tetra- and tri-saccharides. However, lichenan was hydrolyzed to yield trisaccharides as the main end product. The addition of AaBglu12A to the mashing process substantially decreased filtration time by 34.5 % and viscosity by 9.6 %. Therefore, the high-level production of AaBglu12A might be a promising strategy for the brewing industry owing to its favorable properties.     

Aspergillus Gracilis Bainier var. Sartoryi (Biourge) Batista,Lima e Vital n. var. — Sua importáncia filogenética

Summary We deal with a new variety ofAspergillus which we callAspergillus gracilis Bainiervar. sartoryi (Biourge) Batista, Lima and Vital.This variety appears to be very important under the phylogenetic point of view, apparently being an intermediate form betweenAspergillus andPenicillium.Its morphology resemblesA. gracilis but the conidia characteristics are distinct; we took the epithetsartoryi fromA. sartoryi Biourge which was not described by the respective author.We suggest, too, the transfer of the seriesA. restrictus, where we put our new variety, from the groupA. glaucus, to become independant, as a connective series in the phylogenetic development betweenAspergillus andPenicillium, since the type of the series and the other elements that it embraces does not produce sclerotia or cleistothecia and the conidial apparatus offers curious similarity toPenicillium. Besides, some fungi of the series grow well in certain culture media, in complete discordance with the members of the groupA. glaucus.

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Publiçacão no 4

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Chefe do Instituto de Micologia;

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Diretor do Instituto de Antibióticos e Prof. de Microbiologia da Escola de Química;

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Assistente micologista do Instituto de Micologia — (Todos da Universidade do Recife).

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Os autores declinam o seu agradecimento ao Dr.Heraldo da Silva maia, Assistente micologista eD. Marilene Maranhão Moreira, Auxiliar-Técnico, do Instituto de Micologia, pela colaboração que lhes prestaram, durante a realização do presente trabalho.     

Growth and β-Glucan 10C3K Production by a Mutant of Alcaligenes faecalis var. myxogenes in Defined Medium

A defined medium was developed in which Alcaligenes faecalis var. myxogenes 10C3 mutant K produced a large quantity of β-glucan 10C3K. The medium contained 4% glucose together with 0.1% citrate, succinate or fumarate as the carbon source, 0.15% (NH₄)₂HPO₄ as the nitrogen source and mineral salts. When NaNH₄HPO₄, KNO₃ or urea was used at a concentration of 0.03% nitrogen as the sole nitrogen source, salts of organic acid were not needed in addition to glucose.

In culture medium containing phosphate buffer (M/15, pH 6.5~8.0) large amounts of polysaccharide were formed and its yield from the 4% glucose added was about 50%. Thus, it was shown that polysaccharide production is enhanced greatly if a suitable pH for polysaccharide production is maintained during incubation.     

Properties of a Trypsin Inhibitor from Job’s-tears (Coix lacryma-jobi L. var. Ma-yuen Stapf)

A heat stable trypsin inhibitor was found in the bran of soft-shelled job’s-tears (Coix lacryma-jobi L. var. Ma-yuen Stapf) seeds. This inhibitor seemed to be a simple protein, and the molecular weight was about 12,000. Similar to other heat stable trypsin inhibitors, this inhibitor also contained many cysteine or cystine residues in the molecule. This inhibitor inhibited bovine trypsin at the molar ratio of 1 to 2, showing that it was double-headed. Its activity was stable against the change of pH at the range of 3 to 11 and high temperature of 100°C under certain conditions. However, the degree of heat stability of the inhibitory activity depended highly upon the kind of the solution in which this inhibitor was dissolved.     

Enzymatic Properties of β-Xylosidase from Malbranchea pulchella var. sulfurea No. 48

Some enzymatic properties of Malbranchea β-xylosidase were investigated. The β- xylosidase activity was inhibited by Hg²⁺, Zn²⁺, Cu²⁺, N-bromosuccinimide, p-chloromercuribenzoate and sodium laurylsulfate, while this activity was activated by Ca²⁺. The enzyme released xylose as the end product even from 10% xylobiose solution without forming any xylooligosaccharides. The enzyme well acted on aryl-β-d-xylosides, but showed no activity on alkyl-β-d-xylosides, and it was practically free from glucosidase activity. The Km and V_max values of this enzyme for xylobiose were calculated to be 2.86 × 10^?8 m and 34.5 μmoles/mg/min, respectively, and these values determined for phenyl-β-d-xyloside were 3.01 × 10^?8 m and 16.2 μmoles/mg/min, respectively.     

Fluorescence studies of ATP-diphosphohydrolase from Solanum tuberosum var. Desirée

Chemical modification of potato apyrase suggests that tryptophan residues are close to the nucleotide binding site. Kd values (+/- Ca2+) for the complexes of apyrase with the non-hydrolysable phosphonate adenine nucleotide analogues, adenosine 5'-(beta,gamma-methylene) triphosphate and adenosine 5'-(alpha,beta-methylene) diphosphate, were obtained from quenching of the intrinsic enzyme fluorescence. Other fluorescent nucleotide analogues (2'(3')-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate, 2'(3')-O-(2,4,6-trinitrophenyl) adenosine 5'-diphosphate. 1,N6-ethenoadenosine triphosphate and 1,N6-ethenoadenosine diphosphate) were hydrolysed by apyrase in the presence of Ca2+, indicating binding to the active site. The dissociation constants for the binding of these analogues were calculated from both the decrease of the protein (tryptophan) fluorescence and enhancement of the nucleotide fluorescence. Using the sensitised acceptor (nucleotide analogue) fluorescence method, energy transfer was observed between enzyme tryptophans and ethene-derivatives. These results support the view that tryptophan residues are present in the nucleotide-binding region of the protein, appropriately oriented to allow the energy transfer process to occur.     

Purification and biochemical characterization of a novel α-glucosidase from Aspergillus niveus

An extracellular α-glucosidase produced by Aspergillus niveus was purified using DEAE-Fractogel ion-exchange chromatography and Sephacryl S-200 gel filtration. The purified protein migrated as a single band in 5% PAGE and 10% SDS–PAGE. The enzyme presented 29% of glycosylation, an isoelectric point of 6.8 and a molecular weight of 56 and 52 kDa as estimated by SDS-PAGE and Bio-Sil-Sec-400 gel filtration column, respectively. The enzyme showed typical α-glucosidase activity, hydrolyzing p-nitrophenyl α-d-glucopyranoside and presented an optimum temperature and pH of 65°C and 6.0, respectively. In the absence of substrate the purified α-glucosidase was stable for 60 min at 60°C, presenting t ₅₀ of 90 min at 65°C. Hydrolysis of polysaccharide substrates by α-glucosidase decreased in the order of glycogen, amylose, starch and amylopectin. Among malto-oligosaccharides the enzyme preferentially hydrolyzed malto-oligosaccharide (G10), maltopentaose, maltotetraose, maltotriose and maltose. Isomaltose, trehalose and β-ciclodextrin were poor substrates, and sucrose and α-ciclodextrin were not hydrolyzed. After 2 h incubation, the products of starch hydrolysis measured by HPLC and thin layer chromatography showed only glucose. Mass spectrometry of tryptic peptides revealed peptide sequences similar to glucan 1,4-alpha-glucosidases from Aspergillus fumigatus, and Hypocrea jecorina. Analysis of the circular dichroism spectrum predicted an α-helical content of 31% and a β-sheet content of 16%, which is in agreement with values derived from analysis of the crystal structure of the H. jecorina enzyme.     

Kinetic characterization of a crude β-d-glucosidase from Aspergillus wentii Pt 2804

The kinetic characteristics of β-d-glucosidase (cellobiase, β-d-glucosidase glucohydrolase, EC 3.2.1.21) from the filtered broth of a well grown culture of Aspergillus wentii have been studied. Both cellobiose and 4-nitrophenyl-β-d-glucoside (4NPG) were used as substrates and values of K_m, V_max for both the substrates were determined. Activity was maximum over a pH range of 4.5–5.5 but declined sharply beyond 5.5 for both substrates. The optimum temperature was between 60 and 65°C. Half-life of the cellobiase was ～38.0 h at 60°C and ～6.3 h at 65°C. However, the enzyme was found to be quite stable at 50°C. The activation and deactivation energies for 4NPG hydrolysis were 33.2 and 111.3 kJ mol^?1 K^?1, and 43.6 and 63.7 kJ mol K^?1 for cellobiose hydrolysis. Product inhibition was found to be of the competitive type. Preliminary experiments showed that marked synergistic activity exists between Trichoderma reesei and A. wentii cellulases [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] for cellulose hydrolysis.