Publications

a novel strategy for the isolation of defined pyrg mutants and the development of a site-specific integration system for aspergillus awamori.	a homologous gene transfer system for aspergillus awamori for site-specific integration is described, based on two components. first, a defined a. awamori pyrg mutant strain constructed by a selection strategy for gene-replacement in fungi. second, a vector with a homologous pyrg selection marker containing a defined mutation at a site different from that of the mutations in the pyrg gene of the defined mutant strain. defined mutation in the a. awamori pyrg gene, isolated from a genomic library ...	1995	7553938
rice aspartic proteinase, oryzasin, expressed during seed ripening and germination, has a gene organization distinct from those of animal and microbial aspartic proteinases.	the gene organization and nucleotide sequence of an aspartic proteinase (ap) of plant origin were first disclosed by cdna and genomic dna cloning of a rice ap (oryzasin). the deduced amino acid sequence of oryzasin 1 was significantly similar to those of other aps (34-85%), with highest similarity (85%) to barley ap (hvap). oryzasin 1, as well as hvap, is distinct from animal and microbial aps in that the plant aps contain a unique 104-amino-acid insertion in the c-terminal region. the oryzasin ...	1995	7556174
molecular cloning of the cdna and gene for an elastinolytic aspartic proteinase from aspergillus fumigatus and evidence of its secretion by the fungus during invasion of the host lung.	hydrolysis of structural proteins in the lung by extracellular proteinases secreted by aspergillus fumigatus is thought to play a significant role in invasive aspergillosis. this fungus was found previously to secrete an elastinolytic serine proteinase and a metalloproteinase. we report that a. fumigatus also secretes an aspartic proteinase (aspergillopepsin f) that can catalyze hydrolysis of the major structural proteins of basement membrane, elastin, collagen, and laminin. the ph optimum for t ...	1995	7558282
extraction of charged fusion proteins in reversed micelles: comparison between different surfactant systems.	behavior of a series of fusion proteins of varying charge in reversed micellar extraction was studied. the proteins consisted of the enzyme glucoamylase from aspergillus awamori joined to short peptides containing from 0-10 additional aspartate residues. the fusions were partitioned into two different cationic surfactant systems, one based on the surfactant trioctylmethylammonium chloride (tomac) and the other on cetyltrimethylammonium bromide (ctab). these two systems differed chiefly in micell ...	1995	7619396
construction and heterologous expression of a synthetic copy of the cutinase cdna from fusarium solani pisi.	a copy of the cutinase cdna from fusarium solani pisi was constructed starting from synthetic oligonucleotides. for this construction three separate cassettes were made, which were subsequently assembled to form the cutinase gene. heterologous expression of the synthetic cutinase gene and the subsequent secretion of the recombinant enzyme was achieved in saccharomyces cerevisiae and aspergillus awamori.	1995	7632392
refined structure for the complex of d-gluco-dihydroacarbose with glucoamylase from aspergillus awamori var. x100 to 2.2 a resolution: dual conformations for extended inhibitors bound to the active site of glucoamylase.	the crystal structure at ph 4 of the complex of glucoamylase ii(471) from aspergillus awamori var. x100 with the pseudotetrasaccharide d-gluco-dihydroacarbose has been refined to an r-factor of 0.125 against data to 2.2 a resolution. the first two residues of the inhibitor bind at a position nearly identical to those of the closely related inhibitor acarbose in its complex with glucoamylase at ph 6. however, the electron density bifurcates beyond the second residue of the d-gluco-dihydroacarbose ...	1995	7821430
domain e of bacillus macerans cyclodextrin glucanotransferase: an independent starch-binding domain.	the starch-binding domains of glucoamylase i (sbd of ga-i) from aspergillus awamori and of cyclodextrin glucanotransferase (domain e of cgtase) from bacillus macerans were fused to the c-terminus of beta-galactosidase (beta-gal) the majority of the fusion proteins produced in escherichia coli were found as inclusion bodies. active fusion proteins were purified by partial solubilization of the inclusion bodies with 2 m urea followed by affinity chromatography. adsorption isotherms of purified fus ...	1995	18623437
increased thermostability of asn182 --> ala mutant aspergillus awamori glucoamylase.	asn182 --> ala aspergillus awamori glucoamylase expressed in saccharomyces cerevisiae had a first-order thermodeactivation coefficient 40% that of wild-type glucoamylase at ph 4.5 between 60 degrees and 65 degrees c, caused by the elimination of an asn-gly sequence subject to deamidation and eventual chain breakage. above 70 degrees c, and at phs 3.5 and 5.5, thermodeactivation coefficients of wild-type and mutant enzymes were roughly equal, because the fastest deactivation mechanism was no long ...	1994	18613315
the mechanism of binding of glucoamylase i from aspergillus awamori var. kawachi to cyclodextrins and raw starch.	glucoamylase i (gai) bound to β-cyclodextrin (β-cd) with the binding constants of kd = 17.7 μm and n = 1.69. binding resulted in formation of an inclusion complex, as indicated by the fact that organic guest compounds for β-cd inhibited the binding of gai to β-cd. titration of gai (a(1)-r(615)) with β-cd or amylose a showed critical spectral perturbations at 286 nm that reflected the aromatic side chains of tyr and trp, but no spectral changes were observed in gai' (a(1)-v(469)) and gp-i (a(470) ...	1994	27315704
heterologous expression of the cytotoxin restriction in aspergillus nidulans and aspergillus niger.	the cdna clone of restrictocin was placed under the control of the glucoamylase promoter from aspergillus awamori and was transformed into aspergillus nidulans and aspergillus niger. site-specific changes were introduced into cdna constructs and these were transformed into a. nidulans. the secretion signal sequence was deleted from one form of the gene and three mutations introduced single amino acid substitutions into the protein. culture conditions were optimized for maximum expression levels ...	1994	7827506
effect of amino acid deletions in the o-glycosylated region of aspergillus awamori glucoamylase.	aspergillus awamori glucoamylase (ga) contains globular catalytic and starch-binding domains (residues 1-471 and 509-616, respectively). a heavily o-glycosylated sequence comprises two parts. the first (residues 441-471) in the crystal structure wraps around an alpha/alpha-barrel formed by residues 1-440. the second (residues 472-508) is an extended, semi-rigid linker between the two domains. to investigate the functional role of this linker, we made internal deletions to remove residues 466-512 ...	1994	7831281
isolation and characterization of a 1,4-beta-endoxylanase gene of a. awamori.	an enzyme with a particular 1,4-beta-xylanase activity was identified and purified from wheat-bran culture medium of an aspergillus awamori strain. with oligonucleotides based on the n-terminal amino-acid sequence of the enzyme, the exla gene of a. awamori, encoding 1,4-beta-xylanase a, has been cloned. based on the deduced amino-acid sequence, 1,4-beta-xylanase a is produced as a 211 amino-acid-residue-long precursor, which is converted post-translationally into a 184-aa residue-long mature pro ...	1994	7859305
structural similarities in glucoamylase by hydrophobic cluster analysis.	the model of the catalytic domain of aspergillus awamori var. x100 glucoamylase was related to 14 other glucoamylase protein sequences belonging to five subfamilies. structural features of the different sequences were revealed by multisequence alignment following hydrophobic cluster analysis. the alignment agreed with the hydrophobic microdomains, normally conserved throughout evolution, evaluated from the 3-d model. saccharomyces and clostridium glucoamylases lack the alpha-helix exterior to th ...	1994	7937705
site-directed mutagenesis of the catalytic base glutamic acid 400 in glucoamylase from aspergillus niger and of tyrosine 48 and glutamine 401, both hydrogen-bonded to the gamma-carboxylate group of glutamic acid 400.	replacement of the catalytic base glu400 by glutamine in glucoamylase from aspergillus niger affects both substrates ground-state binding and transition-state stabilization. compared to those of the wild-type enzyme, km values for maltose and maltoheptaose are 12- and 3-fold higher for the glu400-->gln mutant, with kcat values 35- and 60-fold lower, respectively, for the same substrates. this unusually high residual activity for a glycosylase mutant at a putative catalytic group is tentatively e ...	1994	7947792
circular dichroism studies of acid proteinases from aspergillus niger and aspergillus awamori.	acid proteinases produced by strains of aspergillus niger and aspergillus awamori were isolated by means of ethanol precipitation, gel filtration and anion-exchange high resolution chromatography. in each case, the purified proteinase showed a single band in polyacrylamide gel electrophoresis. their molecular weights were almost identical (approx. 45,000). however, the proteinase from aspergillus awamori contained 16% of neutral hexoses while the other enzyme (aspergillus niger) showed negligibl ...	1994	7981663
analysis of the raw starch-binding domain by mutation of a glucoamylase from aspergillus awamori var. kawachi expressed in saccharomyces cerevisiae.	carboxy-terminal deletions were introduced into the raw starch-binding domain (a-515 to r-615) encoded by the gene for glucoamylase i (gai) from aspergillus awamori var. kawachi. genes coding for proteins designated ga596 (a-1 to e-596), ga570 (a-1 to a-570), and ga559 (a-1 to n-559) were constructed and resulted in truncated proteins. all of the mutant genes were expressed heterologously in saccharomyces cerevisiae. ga596 adsorbed to raw starch and digested it. ga570 and ga559 did not adsorb to ...	1994	7993082
nmr spectroscopy of exchangeable protons of glucoamylase and of complexes with inhibitors in the 9-15-ppm range.	1h-nmr spectra have been recorded for glucoamylases i and ii from aspergillus awamori var. x100 and from a. niger in the 9-15-ppm region. at least 17 distinct peaks, many of them arising from single protons, are observed. these are designated a-q, a being the furthest downfield. at least 9 of these are lost rapidly by exchange when the enzyme is placed in d2o. peaks a, b, e and h undergo distinct shifts with ph change in the ph region 3-7. several others undergo smaller shifts. small differences ...	1994	8033904
substitution of asparagine residues in aspergillus awamori glucoamylase by site-directed mutagenesis to eliminate n-glycosylation and inactivation by deamidation.	aspergillus awamori glucoamylase is a secreted glycoprotein containing n-linked carbohydrate recognition sites at asn-171, asn-182 and asn-395. site-directed mutagenesis was performed at asn-182 and asn-395 to determine whether these residues were n-glycosylated by saccharomyces cerevisiae, to investigate the function of any glycans linked to them, and to determine the effect of their deamidation on glucoamylase thermostability. asn-171 and asn-395, but not asn-182, were n-glycosylated. deletion ...	1994	8037681
refined crystal structures of glucoamylase from aspergillus awamori var. x100.	the refined crystal structures of a proteolytic fragment of glucoamylase from aspergillus awamori var. x100 have been determined at ph 6 and 4 to a resolution of 2.2 a and 2.4 a, respectively. the models include the equivalent of residues 1 to 471 of glucoamylase from aspergillus niger and a complete interpretation of the solvent structure. the r-factors of the ph 6 and 4 structures are 0.14 and 0.12, respectively, with root-mean-square deviations of 0.014 a and 0.012 a from expected bondlengths ...	1994	8176747
structure-function relationships in the catalytic and starch binding domains of glucoamylase.	sixteen primary sequences from five sub-families of fungal, yeast and bacterial glucoamylases were related to structural information from the model of the catalytic domain of aspergillus awamori var. x100 glucoamylase obtained by protein crystallography. this domain is composed of thirteen alpha-helices, with five conserved regions defining the active site. interactions between methyl alpha-maltoside and active site residues were modelled, and the importance of these residues on the catalytic ac ...	1994	8177888
structures of small oligomers liberated from barley arabinoxylans by endoxylanase from aspergillus awamori.	arabinoxylans consisting of more than 90% of arabinose and xylose were extracted from water-insoluble cell wall material of dehusked barley grain, and degraded with a purified endo-(1-->4)-beta-d-xylanase from aspergillus awamori. twelve of the oligosaccharide fragments released were isolated by a combination of size-exclusion and anion-exchange chromatography, and their structures were determined by 1h nmr spectroscopy. the oligosaccharides identified consisted of (1-->4)-linked beta-d-xylopyra ...	1994	8180986
refined structure for the complex of acarbose with glucoamylase from aspergillus awamori var. x100 to 2.4-a resolution.	the three-dimensional structure of the pseudotetrasaccharide acarbose complexed with glucoamylase ii(471) from aspergillus awamori var. x100 has been determined to 2.4-a resolution. the model includes residues corresponding to 1-471 of glucoamylase i from aspergillus niger, a single molecule of bound acarbose, and 535 sites for water molecules. the crystallographic r factor from refinement is 0.124, and the root-mean-squared deviation in bond distances is 0.013 a. electron density for a single m ...	1994	8195212
protein engineering of the relative specificity of glucoamylase from aspergillus awamori based on sequence similarities between starch-degrading enzymes.	aspergillus glucoamylase catalyzes hydrolysis of d-glucose from non-reducing ends of starch with an approximately 300-fold (kcat/km) preference for the alpha-1,4- over the alpha-1,6-glucosidic linkage determined using the substrates maltose and isomaltose. it is postulated that as most amylolytic enzymes act on either the alpha-1,4- or alpha-1,6-linkages, sequence comparison between active-site regions should enable the correlation of the substrate bond specificity with particular residues at ke ...	1994	7716159
purification and characterization of an extracellular aspartic proteinase from aspergillus fumigatus.	an aspartic proteinase (pep) from the culture supernatant of a clinical isolate of aspergillus fumigatus was purified to virtual homogeneity at a yield of 24%. the procedure involved affinity chromatography on pepstatin agarose, the interaction requiring a chaotropic salt (sodium trifluoroacetate) for complete elution of the enzyme. among 11 amino acids of the n-terminal region, nine were identical with the corresponding sequence of the aspartic proteinase aspergillopepsin a from aspergillus nig ...	1994	7738725
thermosensitive mutants of aspergillus awamori glucoamylase by random mutagenesis: inactivation kinetics and structural interpretation.	seven thermosensitive glucoamylase mutants generated by random mutagenesis and expressed in saccharomyces cerevisiae were sequenced and their inactivation kinetics were determined. wild-type glucoamylase expressed in s. cerevisiae was more glycosylated and more stable than the native aspergillus niger enzyme. all mutants had lower free energies of inactivation than wild-type glucoamylase. in the ala39-->val, ala302-->val and leu410-->phe mutants, small hydrophobic residues were replaced by large ...	1994	7809026
strain improvement of chymosin-producing strains of aspergillus niger var. awamori using parasexual recombination.	parasexual recombination was used to obtain improved chymosin-producing strains and to perform genetic analysis on existing strains. chlorate resistance was used to select for a variety of spontaneous nitrate assimilation pathway mutations in strains previously improved for chymosin production using classical strain improvement methods including mutation and screening, and selection for 2-deoxyglucose resistance (dgr). diploids of these improved strains were generated via parasexual recombinatio ...	1994	7764791
enhanced recovery and purification of aspergillus glucoamylase from saccharomyces cerevisiae by the addition of poly(aspartic acid) tails.	poly(aspartic acid) tails of different lengths were fused to the glucoamylase (ga) of aspergillus awamori by genetic engineering techniques. tails consisting of 5, 7, and 10 aspartate residues were fused to the n-terminus of the full-length mature ga (aa 1-616) downstream from the intact leader peptide to produce fusion proteins designated gand5, gand7, and gand10, respectively. three fusion proteins with c-terminal tails were also constructed, designated gacd0, gacd5, and gacd10 (0, 5, and 10 a ...	1993	7763957
use of aspergillus overproducing mutants, cured for integrated plasmid, to overproduce heterologous proteins.	aspergillus niger var. awamori was previously transformed with a vector designed to express a fused glucoamylase-prochymosin gene and bearing the neurospora crassa pyr4 gene as a selectable marker. mutant strains that overproduced the glucoamylase-prochymosin fusion protein were derived from one of the transformants. despite the fact that the expression vector was integrated into the genome of these strains it was possible to obtain strains from which the vector sequences had been removed. this ...	1993	7764120
the cloning and sequencing of the genes encoding phytase (phy) and ph 2.5-optimum acid phosphatase (aph) from aspergillus niger var. awamori.	the genes encoding phytase (ec 3.1.3.8) and ph 2.5-optimum acid phosphatase (ec 3.1.3.2) have been cloned and sequenced from aspergillus niger var. awamori. the translated nucleotide sequences yielded polypeptides of 467 and 479 amino acids (aa) for phytase and acid phosphatase, respectively. the genes were isolated using oligodeoxyribonucleotide probes based on the aa sequences of the purified proteins. recombinant a. niger var. awamori strains carrying additional copies of the gene sequences d ...	1993	8224894
mode of action of the xylan-degrading enzymes from aspergillus awamori on alkali-extractable cereal arabinoxylans.	alkali-extractable cereal arabinoxylan and oligosaccharides of known structure derived from it by enzymic hydrolysis were treated with endo-(1-->4)-beta-d-xylanases i and iii from aspergillus awamori cmi 142717 and the digests subjected to analysis by high performance anion-exchange chromatography. clear differences in the mode of action of the two endo-(1-->4)-beta-d-xylanases were observed. when counting from the reducing end, at least one unsubstituted xylopyranosyl residue adjacent to singly ...	1993	8275505
characterisation by 1h nmr spectroscopy of oligosaccharides derived from alkali-extractable wheat-flour arabinoxylan by digestion with endo-(1-->4)-beta-d-xylanase iii from aspergillus awamori.	alkali-extractable wheat-flour arabinoxylan, treated with endo-(1-->4)-beta-d-xylanase iii from aspergillus awamori cmi 142717, was fractionated by bio-gel p-2 size exclusion chromatography at 60 degrees c. column fractions, corresponding to oligosaccharides with degrees of polymerisation from 5 to 10, were collected, and subfractionated by high performance anion-exchange chromatography on carbopac pa-1. the structures of the oligosaccharides thus obtained were elucidated by 1h nmr spectroscopy, ...	1993	8275506
cassette mutagenesis of aspergillus awamori glucoamylase near its general acid residue to probe its catalytic and ph properties.	nine single amino acid mutations in the active site of aspergillus awamori glucoamylase were made by cassette mutagenesis to alter the ph dependence of the enzyme and to determine possible functions of the mutated residues. the glu179-->asp mutation expressed in yeast led to a very large decrease in kcat but to no change in km, verifying this residue's catalytic function. asp176-->glu and glu180-->asp mutations affected km more than kcat, implying that asp176 and glu180 are involved in substrate ...	1993	8309943
a new promoter-probe vector for saccharomyces cerevisiae using fungal glucoamylase cdna as the reporter gene.	a system is described for the selection of dna sequences showing promoter activity in the yeast saccharomyces cerevisiae using a heterologous reporter enzyme which is efficiently secreted by the yeast host. a multicopy shuttle plasmid of the yep-type was constructed so as to carry multiple unique cloning sites at the 5' end of the aspergillus awamori glucoamylase cdna. glucoamylase can only be expressed upon insertion at one of these unique cloning sites of a dna fragment from any source, provid ...	1993	8346676
effect of modification of carbohydrate component on properties of glucoamylase.	in this study, we investigated enzymatic deglycosylation of glucoamylase from aspergillus awamori x 100/d27, a glycoprotein which has two n-linked and about forty short mannose-bearing o-linked sugars per molecule. o-linked sugars were modified by treatment with alpha-mannosidase and n-linked sugars were removed using endo-beta-n-acetylglucosaminidase f. analysis of conformational changes following deglycosylation suggests that o-linked sugars essentially contribute to the stabilization of gluco ...	1993	8420800
functional roles of the invariant aspartic acid 55, tyrosine 306, and aspartic acid 309 in glucoamylase from aspergillus awamori studied by mutagenesis.	three mutants, asp55-gly, tyr306-->phe, and asp309-->asn, of aspergillus awamori glucoamylase (identical to aspergillus niger glucoamylase) were constructed to elucidate the roles of two conserved regions within fungal glucoamylases. kinetic studies indicate that both of these regions are closely associated with activity. the asp55-->gly mutation decreases the kcat approximately 200 times toward maltose and isomaltose, while km values remain similar to the wild-type. this localizes asp55 to subs ...	1993	8424940
refined structure for the complex of 1-deoxynojirimycin with glucoamylase from aspergillus awamori var. x100 to 2.4-a resolution.	the three-dimensional structure of the complex of 1-deoxynojirimycin with glucoamylase ii-(471) from aspergillus awamori var. x100 has been determined to 2.4-a resolution. the model includes residues corresponding to residues 1-471 of glucoamylase i from aspergillus niger, two molecules of bound 1-deoxynojirimycin and 605 sites for water molecules. the crystallographic r factor from refinement is 0.119, and the root-mean-squared deviation in bond distances is 0.012 a. the inhibitor complex confi ...	1993	8431441
functional roles and subsite locations of leu177, trp178 and asn182 of aspergillus awamori glucoamylase determined by site-directed mutagenesis.	fungal glucoamylases contain four conserved regions. one region from the aspergillus niger enzyme contains three key carboxylic acid residues, the general acid catalytic group, glu179, along with asp176 and glu180. three site-directed mutations, leu177-->his, trp178-->arg and asn182-->ala, were constructed near these acidic groups to reveal the function of other conserved residues in this region. leu177 and trp178 are strictly conserved among fungal glucoamylases, while an amide, predominantly a ...	1993	8433972
molecular analysis and overexpression of the gene encoding endothiapepsin, an aspartic protease from cryphonectria parasitica.	the gene, epn-1, encoding endothiapepsin (epn), an aspartic protease (aspp) synthesized and secreted by the ascomycete fungus responsible for chestnut blight, cryphonectria (endothia) parasitica, was identified and characterized. inspection of the nucleotide and deduced amino acid (aa) sequences revealed perfect agreement with the experimentally derived 330-aa sequence of mature epn [barkholt, eur. j. biochem. 167 (1987) 327-338] and an additional 89 aa of putative preprosequence. of the nine fu ...	1993	8462868
microencapsulation selection for isolation of yeast mutants with increased secretion of aspergillus awamori glucoamylase.	we have developed a microencapsulation selection method which allows the rapid and quantitative screening of >10(6) yeast cells for enhanced secretion of aspergillus awamori glucoamylase. the method provides a 400-fold single-pass enrichment for high-secreting mutants, and can be straightforwardly adapted for application to growth-based selection schemes with other microorganisms and enzymes.	1993	18613019
[dermatomycoses in workers in enterprises of the microbiological industry].	mycologic examination of 54 patients with clinical manifestations of dermatomycosis, engaged in glucose oxidase and catalase production has found a producer fungus, penicillium vitale in 31 (38%); 3 out of 37 people contacting with producers of glucamylase and pectofetidine appeared to be infected by aspergillus awamori (1.1%) and aspergillus foetidus (1.1%), respectively. the producers were isolated from the skin of 40 (16.44%) out of 243 workers having no clinical signs of dermatomycosis, main ...	1992	1427292
crystal structure of glucoamylase from aspergillus awamori var. x100 to 2.2-a resolution.	the crystal structure of a catalytically active fragment of glucoamylase-i from aspergillus awamori var. x100 has been determined to a resolution of 2.2 a. twelve of its 13 alpha-helices are arranged into an "alpha/alpha-barrel." an inner core of six mutually parallel alpha-helices are connected to each other through a peripheral set of six alpha-helices. the peripheral helices are parallel to each other, but approximately antiparallel to the inner core of alpha-helices. the putative active site ...	1992	1527049
crystallization and preliminary x-ray study of minor glucoamylase from aspergillus awamori variant x-100/d27.	crystals of the reduced form of glucoamylase were obtained from polyethylene glycol 6000 solution by the hanging-drop method. the protein was treated with alpha-mannosidase to partly remove the sugar component. the crystals belong to the space group p2(1)2(1)2(1) with cell dimensions a = 116.7 a, b = 104.3 a, c = 48.5 a and diffract beyond 2.5 a resolution.	1992	1619656
specific inhibition by cyclodextrins of raw starch digestion by fungal glucoamylase.	alpha-, beta-, and gamma-cyclodextrins (cds) completely inhibited raw starch digestion by glucoamylase i (ga i, mw 90,000) from aspergillus awamori var. kawachi, and inhibited by 85% the raw starch adsorption of ga i at the cd concentrations of 1-5 mm. cds at 1-5 mm did not inhibit gelatinized starch hydrolysis by ga i, but at the concentration of 50 mm, they inhibited such hydrolysis slightly. ga i was specifically adsorbed onto cd-sepharose 6b, but glucoamylase i' (ga i', mw 73,000), which doe ...	1992	1368209
promotive and inhibitory effects of raw starch adsorbable fragments from pancreatic alpha-amylase on enzymatic digestions of raw starch.	the enzymatically inactive but raw-starch-adsorbable peptide fragments designated as gp-pan p and gp-pan i were obtained from a tryptic digest of heat-inactivated hog pancreatic alpha-amylase. these two glycopeptide fragments were purified with sephadex g-75, deae-sephadex a-50, and hplc and were found to be homogeneous on disc gel electrophoresis. gp-pan p and i had molecular weights of 20,000 and 30,000 with sds-page, carbohydrate contents of 10% and 7%, n-terminal amino acids gly-trp and ala- ...	1991	1368657
commercial levels of chymosin production by aspergillus.	we have increased the production of bovine chymosin in aspergillus niger var. awamori to more than one gram per liter of secreted authentic enzyme by combining a mutagenesis protocol with a novel robotic screening program. analysis of the superior chymosin producing strains indicated that they have enhanced capabilities to secrete extracellular proteins.	1991	1368725
the development of aspergillus niger var. awamori as a host for the expression and secretion of heterologous gene products.		1991	1783197
characterization of a polygalacturonase gene of aspergillus niger rh5344.	we have cloned a gene encoding a polygalacturonase (pg) in the filamentous fungus aspergillus niger rh5344. the structural gene comprises 1141 bp coding for 362 amino acids and the open reading frame is disrupted by one intron of 52 bp. eukaryotic consensus sequences for transcription regulation are found only in deviated forms. the biological functionality of the isolated pg gene was established by retransformation in a. niger and aspergillus awamori. in addition, we have found that the pg prot ...	1991	1787790
xylanase production by aspergillus awamori. development of a medium and optimization of the fermentation parameters for the production of extracellular xylanase and beta-xylosidase while maintaining low protease production.	a growth medium was developed for maximal production in batch culture of extracellular xylanase and beta-xylosidase by aspergillus awamori cmi 142717 and a mutant (aantg 43) derived from the wild-type strain. the optimum ph for the production of xylanase and beta-xylosidase was 4.0. the best temperature of xylanase production was 30 degrees c; 35 degrees c was optimal for beta-xylosidase. protease production was never completely suppressed under any of the conditions tested. however, protease ti ...	1991	18600845
isolation of mutants of aspergillus awamori with enhanced production of extracellular xylanase and β-xylosidase.	plate screening tests were designed for the selection and isolation of mutant strains of the fungus aspergillus awamori cmi 142717 showing over-production and constitutive synthesis of xylanase and β-xylosidase. following mutation by n-methyl-n-nitro-n-nitrosoguanidine, nitrous acid and uv (254 nm), two generations of mutants were isolated and cultured in shake fiasks containing glucose, ball-milled oat straw or oat speit xylan as carbon source. growth of a number of selected mutants in shake fl ...	1991	24425022
xylan-hydrolysing enzymes from thermophilic and mesophilic fungi.	screening of 40 mesophilic and 13 thermophilic fungi indicated that enzyme activities capable of degrading oat spelt xylan extensively were produced by only a few of the mesophilic species investigated. the relatively low degree of hydrolysis effected by the enzymes from thermophilic organisms could be explained, in part, by their lack of β-xylosidase. several strains of aspergillus awamori and aspergillus phoenicis were notable in producing high xylanase and β-xylosidase and low protease activi ...	1991	24425133
catalytic mechanism of fungal glucoamylase as defined by mutagenesis of asp176, glu179 and glu180 in the enzyme from aspergillus awamori.	asp176, glu179 and glu180 of aspergillus awamori glucoamylase appeared by differential labeling to be in the active site. to test their functions, they were replaced by mutagenesis with asn, gln and gln respectively, and kinetic parameters and ph dependencies of all enzyme forms were determined. glu179----gln glucoamylase was not active on maltose or isomaltose, while the kcat for maltoheptaose hydrolysis decreased almost 2000-fold and the km was essentially unchanged from wild-type glucoamylase ...	1990	1970434
activity and thermal stability of genetically truncated forms of aspergillus glucoamylase.	glucoamylase (ga) from aspergillus awamori (ec 3.2.1.3) is a secreted starch hydrolase with a large catalytic domain (aa 1-440), a starch-binding domain (aa 513-616), and a highly o-glycosylated region of 72 aa of unknown function that links the catalytic and starch-binding domains. we have genetically engineered a series of truncated forms of ga to determine how much of the highly o-glycosylated region is necessary for the activity or stability of gaii, a fully active form of the enzyme that la ...	1990	2119327
molecular cloning and deletion of the gene encoding aspergillopepsin a from aspergillus awamori.	we have cloned genomic pepa sequences encoding the aspartic proteinase aspergillopepsin a (pepa) from aspergillus awamori using a synthetic oligodeoxyribonucleotide probe. nucleotide sequence data from the pepa gene revealed that it is composed of four exons of 320, 278, 249, and 338 bp. three introns which interrupt the coding sequence are 51, 52, and 59 bp in length. directly downstream from the putative start codon lies a sequence encoding 69 amino acids (aa) which are not present in mature p ...	1990	2182390
molecular cloning and deletion of the gene encoding aspergillopepsin a from aspergillus awamori.		1990	2269444
cloning, characterization, and expression of two alpha-amylase genes from aspergillus niger var. awamori.	using synthetic oligonucleotide probes, we cloned genomic dna sequences encoding an alpha-amylase gene from aspergillus niger var. awamori (a. awamori) on a 5.8 kb ecori fragment. hybridization experiments, using a portion of this cloned fragment to probe dna from a. awamori, suggested the presence of two alpha-amylase gene copies which were subsequently cloned as 7 kb (designated as amya) and 4 kb (amyb) hindiii fragments. dna sequence analysis of the amya and amyb genes revealed the following: ...	1990	2340591
complete genomic sequence encoding trpc from aspergillus niger var. awamori.		1990	2395660
site-directed mutagenesis at the active site trp120 of aspergillus awamori glucoamylase.	trp120 of aspergillus awamori glucoamylase has previously been shown by chemical modification to be essential for activity and tentatively to be located near subsite 4 of the active site. to further test its role, restriction sites were inserted in the cloned a.awamori gene around the trp120 coding region, and cassette mutagenesis was used to replace it with his, leu, phe and tyr. all four mutants displayed 2% or less of the maximal activity (kcat) of wild-type glucoamylase towards maltose and m ...	1989	2510150
ethanol production from starch by a coimmobilized mixed culture system of aspergillus awamori and saccharomyces cerevisiae.	the development of a coimmobilized mixed culture sys tem of aerobic and facultative anaerobic microorganisms in ca-alginate gel beads and the production of useful metabolites by the system were investigated. a coimmobilized mixed culture system of aspergillus awamori (obligate aerobe) and saccharomyces cerevisiae (facultative anaerobe) in ca-alginate gel beads was used as a model system, and ethanol production from starch by the system was used as a model production. mold asp. awamori is an amyl ...	1989	18587973
oxygen diffusivity in gel beads containing viable cells.	this article proposes a simple steady-state method for measuring the effective diffusion coefficient of oxygen (d(e)) in gel beads entrapping viable cells. we applied this method to the measurement of d(e) in ca- and ba-alginate gel beads entrapping saccharomyces cerevisiae and pseudomonas ovalis. the diffusivity of oxygen through gel beads containing viable cells was measured within an accuracy of +/-7% and found not to be influenced by cell density (0-30 g/l gel), cell type, and cell viability ...	1989	18588184
l-lactic acid production from starch by coimmobilized mixed culture system of aspergillus awamori and streptococcus lactis.		1988	18581580
fermentation of corn starch to ethanol with genetically engineered yeast.	expression of the glucoamylase gene from aspergillus awamori by laboratory and distiller's strains of saccharomyces cerevisiae allowed them to ferment soluble starch. approximately 95% of the carbohydrates in the starch were utilized. glycerol production was significantly decreased when soluble starch was used instead of glucose. ethanol yield on soluble starch was higher than that on glucose. the rate of starch fermentation was directly related to the level of glucoamylase activity. strains wit ...	1988	18584739
role of camp in the mediation of glucose catabolite repression of glucoamylase synthesis in aspergillus awamori.	exogenous camp was shown to mimic glucose in its effect on the synthesis of glucoamylase in aspergillus awamori. camp (4 mm) when added to the starch medium blocked the derepression of glucoamylase by inhibiting its protein synthesis by about twofold. the repressive effect of camp on the glucoamylase synthesis was found to be due to twofold reduction in the glucoamylase specific mrna levels. this camp related repression of the glucoamylase mrna levels was most likely brought about at the level o ...	1988	2848632
translational control of alpha-amylase gene expression in aspergillus awamori.		1988	3265327
translational control of alpha-amylase gene expression in aspergillus awamori.	regulation of alpha-amylase gene expression in aspergillus awamori was studied by analyzing the enzyme activity levels, rate of protein synthesis, and alpha-amylase-specific mrna levels under various conditions of growth. alpha-amylase synthesis was sensitive to catabolite repression as glucose repressed its synthesis by about fourfold. the stimulation of alpha-amylase synthesis in the presence of its substrate starch was shown to be due to derepression rather than induction as the enzyme was sy ...	1987	3499155
differences between saccharomyces cerevisiae and bacillus subtilis in secretion of human lysozyme.	saccharomyces cerevisiae secreted human lysozyme in the medium as an active form when the signal peptides of chicken lysozyme and a chicken lysozyme-aspergillus awamori glucoamylase hybrid were used, whereas it did not synthesize any human lysozyme protein by using the signal peptide of a. awamori glucoamylase. the secreted lysozyme was easily purified and crystallized. on the other hand, bacillus subtilis secreted an inactive human lysozyme, which seemed to have incorrect disulfide bonds, with ...	1987	3109419
production of multiple forms of glucoamylase in aspergillus awamori.	the biosynthesis of glucoamylases in aspergillus awamori was studied by in vivo protein labelling and analysis of glucoamylase-specific mrnas. two types of glucoamylases with molecular weights of 100,000 and 82,000 were shown to be synthesized de novo. deglycosylation of the 100,000 molecular weight glucoamylase type resulted in the formation of another glucoamylase form with molecular weight of about 94,000. de novo synthesis of two types of glucoamylases was further confirmed by the existence ...	1987	3124870
ethanol production from starch by a coimmobilized mixed culture system of aspergillus awamori and zymomonas mobilis.	the production of ethanol from starch by a coimmobilized mixed culture system of aerobic and anaerobic microorganisms in ca-alginate gel beads was investigated. the mold aspergillus awamori was used as an aerobic amylolytic microorganism and an anaerobic bacterium, zymomonas mobilis, as an ethanol producer. by controlling the mixing ratio of the microorganisms in the inoculum size, a desirable coimmobilized mixed culture system, in which the aerobic mycelia grew on and near the oxygen-rich surfa ...	1986	18555292
expression, glycosylation, and secretion of an aspergillus glucoamylase by saccharomyces cerevisiae.	a strain of saccharomyces cerevisiae capable of simultaneous hydrolysis and fermentation of highly polymerized starch oligosaccharides was constructed. the aspergillus awamori glucoamylase enzyme, form gai, was expressed in saccharomyces cerevisiae by means of the promoter and termination regions from a yeast enolase gene. yeast transformed with plasmids containing an intron-free recombinant glucoamylase gene efficiently secreted glucoamylase into the medium, permitting growth of the transforman ...	1985	17811549
large-scale purification of fungal glucoamylases using anion-exchange resin chromatography.	anion-exchange chromatography on polystyrene resin is shown to be more effective than deae-cellulose for purification of glucoamylase from crude enzyme extracts of aspergillus awamori or from commercial preparations. the glucoamylase from a. awamori culture medium was purified to electrophoretic homogeneity with yields approaching 80%.	1984	6435476
molecular cloning and characterization of the glucoamylase gene of aspergillus awamori.	the filamentous ascomycete aspergillus awamori secretes large amounts of glucoamylase upon growth in medium containing starch, glucose, or a variety of hexose sugars and sugar polymers. we examined the mechanism of this carbon source-dependent regulation of glucoamylase accumulation and found a several hundredfold increase in glucoamylase mrna in cells grown on an inducing substrate, starch, relative to cells grown on a noninducing substrate, xylose. we postulate that induction of glucoamylase s ...	1984	6440004
[effect of modifications of a series of amino acid radicals on the enzymatic activity of glucoamylase from aspergillus awamori].	the effect of chemical modification of various amino acid residues on the enzymatic activity of glucoamylase from asp. awamori was studied. modification of the carboxyl groups by taurine in the presence of water-soluble carbodiimide results in complete inactivation of the enzyme. the inactivation process includes two steps, namely non-specific modification and modification of the active center carboxyls. the rate constants of inactivation at both steps were measured in the presence and absence o ...	1983	6414534
continuous production of high-glucose syrup by chitin-immobilized amylase.	a simple method of preparing a chitin-immobilized alpha-amylase and glucoamylase from the protease- and glycosidase-less mutant hf-15 of aspergillus awamori var. kawachi was developed and used for the production of high-glucose syrup. the glucoamylase was tightly bound onto chitin without the aid of a crosslinking agent because the enzyme contained a specific binding site for chitin. continuous production of high glucose concentrate from a highly concentrated alpha-amylasetreated gelatinized sta ...	1983	18551543
production and characteristics of raw starch-digesting glucoamylase o from a protease-negative, glycosidase-negative aspergillus awamori var. kawachi mutant.	production of a raw starch-digesting glucoamylase o (ga o) by protease-negative, glycosidase-negative mutant strain hf-15 of aspergillus awamori var. kawachi was undertaken under submerged culture conditions. the purified ga o was electrophoretically homogeneous and similar to the parent glucoamylase i (ga i) in the hydrolysis curves toward gelatinized potato starch, raw starch, and glycogen and in its thermostability and ph stability, but it was different in molecular weight and carbohydrate co ...	1983	16346254
production and characteristics of raw starch-digesting glucoamylase o from a protease-negative, glycosidase-negative aspergillus awamori var. kawachi mutant.	[this corrects the article on p. 906 in vol. 45.].	1983	16346379
[medium proteins as a factor of regulation of the synthesis of secreted proteinases].	it was shown that medium proteins possess a double regulatory effect on the synthesis of proteinase secreted by aspergillus awamori 78-2. these proteins are nitrous nutrition that provides for the utilization of the proteolysis products in the course of their gradual formation, which excludes their inhibiting action on the enzyme synthesis. on the other hand, medium proteins possess the ability to induce secreted proteinase synthesis. it is assumed that the concept "enzyme synthesis inducer" can ...	1982	6756488
[active center of glycoamylase from aspergillus awamori].	the maltooligosaccharides--triose, tetrose, pentose and hexose have been obtained by fractionation of partially hydrolyzed cyclohexamylose. the values of free energies for the binding of the first six sites of glucose residue binding in the enzyme active center were calculated according to the hiromi model and were found to be equal to -0.6, -4.5, -1.68, -0.66, -0.25 and +-0.06 kcal/mole, respectively. the hiromi model was extrapolated to p-nitrophenyl-alpha-d-glucoside, p-nitrophenyl-alpha-d-ma ...	1982	6803848
[carboxyl groups in the active center of glucoamylase from aspergillus awamori].	the values for the ionization constants of the catalytic groups of the active site of glucoamylase from asp. awamori for the free enzyme and for the enzyme--substrate complex were calculated. the temperature dependence of the alkaline branch of the ph-dependence curve and the ph dependence in the presence of methanol were determined. the ionization enthalpy delta h = 1.5 +/- 0.3 kcal/mole, the ionization entropy delta s = 20.5 +/- 1.2 e. u. it was assumed that two carboxyl groups are involved in ...	1982	6816298
production and composition of an exocellular nigeran-protein complex isolated from cultures of aspergillus awamori.	aspergillus awamori nakazawa (qm873) hyphae maintained on a nitrogen-deficient medium produced an exocellular complex consisting of the wall alpha-glucan, nigeran (94%), water-soluble nigeran oligosacchrides (1 to 2%), protein (2 to 4%), and a small amount of beta-glycan (less than 1%). nigeran was not covalently linked to protein. the complex appeared in the growth medium only under those temporal or nutritional conditions where the hyphal wall content to nigeran reached at least 30% of the cel ...	1982	7061398
production by aspergillus awamori of homologous oligosaccharides, each with an uneven number of glucosyl units and a reducing terminal (alpha , 1 leads to 4) linkage.	homologous oligosaccharides with degrees of polymerization of 7 to 15 have been isolated from the nigeran-protein complex of aspergillus awamori nakazawa (qm873). each contains an uneven number of glucose units, differs from its nearest homolog by two residues, and contains the sequence glc(alpha , 1 leads to 3)glc- (alpha , 1 leads to 4)glc at its reducing terminus.	1982	7061404
mutants of aspergillus awamori unable to germinate.		1980	7466182
[immobilization of acid proteinase from aspergillus awamori on inorganic carriers with carboxylic groups].	to achieve acid proteinase immobilization on inorganic carriers with carboxylic groups, water soluble carbodiimides and n-carbethoxy-2-ethoxy-1,2-dihydrochinoline were used. the influence of different concentrations of the native enzyme and carbodiimides on the immobilization process was studied. the preparations of immobilized acid proteinase were obtained. its specific activity was 73% in relation to initial activity of enzyme. amount of binding protein was 31% and amount of binding activity w ...	1980	6992135
[carbohydrates of the medium and the nature of ontogeny in aspergillus awamori in relation to glucoamylase biosynthesis].	the biological rhythm of the transition from the processes of blocking and nuclear division to active rna biosynthesis and growth in submerged culture of aspergillus awamori producing glucoamylase was studied by the technique of quantitative cytochemistry, and was characterized by distinct daily oscillation in a medium with starch. the rhythm was disturbed when other carbon sources, viz. maltose, sucrose and particularly glucose, were used: the rhythm became longer and uneven, the rate of nuclea ...	1980	6774215
enzyme formation during solid-substrate fermentation in rotating vessels.	aspergillus awamori nrrl 4869 was cultured on the solid substrate, wheat bran, in a modified rollacell apparatus to produce alpha-galactosidase and invertase. the swivel cap on the elongated bottle permits the introduction of air while the bottle rotates. parameters of air flow rate (0.05-0.2 liter/kg/min), rpm (0.15-15 rpm), and weight of solids (150 and 300 g) were varied. at low air flow rates (0.05 liter/kg solids/min), alpha-galactosidase production was minimal independent of the rotation r ...	1980	6243500
electron microscopy of some rock phosphate dissolving bacteria and fungi.	bacteria pseudomonas striata, bacillus polymyxa, b. megaterium and b. pulvifaciens, and fungi aspergillus awamori, a. niger and penicillium digitatum dissolve tricalcium phosphate and, much less, mussorie and udaipur rock phosphate. the solubilizing power of fungi was higher than that of bacteria, the highest being with a. awamori and a. niger, and with p. striata. electron microscopy of the various cultures showed an electron-dense layer on the bacterial surface after negative staining. the siz ...	1979	527907
[preparation and properties of acid proteinase from aspergillus awamori].	from the culture liquid filtrate of aspergillus awamori 78-2 a preparation of acid proteinase was isolated and its properties were described. this producer was shown to have a peculiar capacity of synthesizing under submerged conditions extracellular acid proteinase as the sole proteolytic enzyme. the preparation of acid proteinase showed a comparatively high hydrolyzing capacity. under optimal conditions the enzyme degraded hemoglobin and albumin almost completely. the possibility of purifying ...	1979	42050
[properties of immobilized acid proteinase from aspergillus awamori].	ph-optimum, thermal stability and storage stability of immobilized acid proteinase from aspergillus awamori obtained by covalent binding on silochrome through glutaraldehyde were studied. acid proteinase immobilization was found to shift towards the acid range by a unit. thermal stability of the immobilized preparation was lower than that of the native enzyme.	1978	31618
[comparative study of glycosidase from cattle liver and exoglycanase from aspergillus awamori].	anomerities of products were estimated for glucosidases from cattle liver and aspergillus awamori. it was demonstrated that the enzyme from cattle liver is alpha-glucosidase and that from asp. awamori is exogluconase. it was demonstrated that alpha-glucosidase hydrolyzes the c1--o bond in the course of reaction. delta-lactone of gluconic acid is a competitive inhibitor for both enzymes. the secondary kinetic isotope effects for both enzymes were measured. the isotope effect for alpha-glucosidase ...	1978	369619
[optimization of immobilization conditions for acid proteinase from aspergillus awamori].	to optimize the immobilization conditions for acid proteinase from aspergillus awamori by covalent binding through glutaraldehyde, experiments were carried out using the box-wilson method. the optimization process was assessed on the basis of absolute activity a, coefficient of activity retention gamma and their product a gamma. the following conditions can be recommended: glutaraldehyde concentration 50--60 mg/g, enzyme concentration not less than 40 mg/g, time of glutaraldehyde treatment 2--2. ...	1978	674118
distribution and conformation of crystalline nigeran in hyphal walls of aspergillus niger and aspergillus awamori.	hyphal walls of aspergillus awamori containing increased amount of the alpha-glucan, nigeran, became correspondingly more opaque when viewed in the electron microscope as shadowed preparations. however, increased polymer deposition was not accompanied by any significant change in wall thickness. the nigeran of both a. awamori and aspergillus niger occurred in situ in a crystalline conformation identical to that of single crystals prepared with pure polysaccharide. furthermore, this polymer was t ...	1977	914782
utilization of agricultural wastes of aspergillus awamori for the production of glucoamylase.	the principal industrial use of glucoamylase is in the production of crystalline d-glucose. aspergillus awamori was used. variations in the specific type of carbohydrate source affected the yield of enzyme markedly. of all the substrates tried, maltose supported satisfactory enzyme yields. sucrose, lactose, fructose, and galactose gave abundant growth which seemed to be necessary but no sufficient condition for maximum yields. the specificity of the carbohydrate structure for gluco-amylase produ ...	1977	333823
[modification of the carboxyl and amine groups of the cellulolytic enzymes of aspergillus awamori].	the activity and stability of some enzymes of asp. awamori cellulolytic complex were studied as affected by chemical modification of carboxylic groups with n,n'-dicyclohexyl carbodiimide (dccd) and amine groups with glutaric aldehyde. the carboxylic groups are established to be necessary for manifestation of the activities of c1- and c2-cellulases, cx-exo- and cx-endoglucanases. their role is negligible in the action of beta-glucosidase. the activity of individual cellulases was studied as affec ...	1976	1021913
[role of intramolecular bonds in stability of certain enzymes of the cellulolytic complex].	stability of c1- and c2-cellulases, cx-exo- and cx-endoglucanases and beta-glucosidase of aspergillus awamori was studied as affected by monoatomic aliphatic alcohols --methanol, ethanol, propanol and isopropanol; bi- and triatomic alcohols - ethylene glycol and glycerol, urea as well as detergents of dodecyl sulphate and sodium nonilate. the mentioned enzymes are established to manifest the highest activity in 40-60% glycerol. it is also shown that their stability is changed differently under t ...	1976	1258159
chromatography of acid proteinases and chymotrypsin on a sorbent containing 2,4-dinitrophenyl residues.	a sorbent obtained by treatment of cyanogen bromide-activated sepharose 4b with mono-n-dnp-hexamethylenediamine has been shown to be effective in the affinity chromatography of pepsin, pepsinogen and acid proteinase from aspergillus awamori. it is considered that 2,4-dinitrophenyl residues of the sorbent interact specifically with the hydrophobic zone of the enzyme, which may belong to the substrate binding site. the chromatography of chymotrypsin on the same sorbent supports this assumption.	1975	1097457
[effect of the inoculum on beta-fructofuranosidase synthesis by aspergillus awamori].	the influence of the age and amount of the inoculum on the beta-fructofuranosidase synthesis by aspergillus awamori 16 was studied during submerged cultivation. the level of the synthesis increased significantly when the 36-hour mycelial inoculum was used.	1975	1208374
[the effect of iron concentration on pectin decomposition by aspergillus niger and aspergillus awamori].	the effect of cations fe2+ and fe3+ on the decomposition of apple pectin by the enzymic preparations of asp. niger and asp. awamori has been examined. fe ions have delayed the process of enzymic decomposition of the pectin molecule. in the presence of fe3+ far less amounts of monogalacturonic acid are formed. the presence of fe ions makes the pectin molecule more stable to the effect of pectolytic enzymes.	1975	1208377
[effect of amino acids on the hemicellulase synthesis by the fungus aspergillus awamori 16-4e].	the effect of different amino acids on the hemicellulase synthesis by the fungus aspergillus awamori 16-4e was investigated. the favorable influence of beta-alanine on the enzyme synthesis was demonstrated. quantitative changes of beta-alanine and glutamic acid in the cell of asp. awamori 16-4e were followed during the fungal development.	1975	1208421
[multiple forms of acid phosphatase of the mould aspergillus awamori str. 22].	by isoelectric focusing and deae-cellulose and sephadex g-150 chromatography it has been shown that extracellular acid phosphatase produced by aspergillus awamori str. 22 exists as a mixture of oligomers of molecular weights ranging from 30,000 to 140,000. with an increase of aggregation of molecules of acid phosphatase the isoelectric point increases from 4.6 to 5.4.	1975	1208425
[isolation and properties of acid-stable alpha-amylase of aspergillus awamori].		1974	4830970
[glucoamylase from aspergillus awamori].		1974	4848835
[lipase preparations from deep cultures of aspergillus awamori 259 and their characteristics].		1974	4463356