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1.
C. J. Baxter N. Magan B. Lane H. G. Wildman 《Applied microbiology and biotechnology》1998,49(3):328-332
A Phoma sp., known to produce the pharmaceutically active metabolites squalestatin 1 (S1) and squalestatin 2 (S2), was cultured on
malt-extract/agar (MEA) over a range of water activities (a
w, 0.995–0.90) and temperatures (10–35 °C) to investigate the influence on growth and metabolite production. Use of the ionic
solute NaCl to adjust a
w resulted in significantly lower (P < 0.01) squalestatin yields than when the Phoma sp. was grown on MEA amended with the non-ionic solute glycerol. Water activity and temperature and their interactions were
highly significant factors (P < 0.001) affecting growth of the Phoma sp., with optimum conditions of 0.998–0.980 a
w and 25 °C. Squalestatin production was similarly influenced by a
w, temperature, time and their interactions (P < 0.001). S1 and S2 production occurred over a narrower a
w and temperature range than growth, with a slightly lower optimum a
w range of 0.995–0.980 a
w. The optimum temperature for squalestatin production varied from 20 °C (S1) to 25 °C (S2) and yields of S2 were up to 1000
times lower than those of S1. The ratio of S1 and S2 produced by the Phoma sp. was influenced by a
w and temperature, with highest values at 0.99–0.98 a
w, and at 15 °C. Incubation times of 28 days gave highest yields of both S1 and S2. Up to 2000-fold increases in squalestatin
yields were measured at optimum environmental conditions, compared to the unmodified MEA. This indicates the need to consider
such factors in screening systems used to detect biologically active lead compounds produced by fungi.
Received: 2 June 1997 / Received last revision: 6 November 1997 / Accepted: 7 November 1997 相似文献
2.
H. Okada T. Sekiya K. Yokoyama H. Tohda H. Kumagai Y. Morikawa 《Applied microbiology and biotechnology》1998,49(3):301-308
A cbh2 cDNA encoding Trichoderma reesei QM9414 cellobiohydrolase II, located on the expression vector whose copy number is controlled by the level of gentamicin,
was successfully expressed under the control of a human cytomegalovirus promoter in the fission yeast, Schizosaccharomyces pombe. The 24-amino-acid leader peptide of the cbh2 gene was recognized by the yeast, enabling the efficient secretion of the heterologous cellobiohydrolase. The transformed
S. pombe strain produced over 115 μg cellobiohydrolase proteins/ml rich medium supplemented with malt extract and 100 μg/ml gentamicin.
The molecular masses of the recombinant cellobiohydrolases, secreted as two molecular species, were estimated to be 70 kDa
and 72 kDa by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE). Deglycosylation treatments revealed that
the recombinant enzymes were overglycosylated and scarcely susceptible to α-mannosidase. The recombinant enzymes showed no
carboxymethylcellulase activity, but showed similar characteristics to those of a native enzyme purified from T. reesei in their optimum pH and temperature, pH and temperature stabilities, and V
max values toward phosphoric-acid-swollen cellulose as substrate, except that their K
m values were about fourfold higher than that of the native enzyme.
Received: 4 August 1997 / Received revision: 13 October 1997 / Accepted: 31 October 1997 相似文献
3.
A number of nutritional factors influencing growth and glucose oxidase (EC 1.1.3.4) production by a newly isolated strain
of Penicillium pinophilum were investigated. The most important factors for glucose oxidase production were the use of sucrose as the carbon source,
and growth of the fungus at non-optimal pH 6.5. The enzyme was purified to apparent homogeneity with a yield of 74%, including
an efficient extraction step of the mycelium mass at pH 3.0, cation-exchange chromatography and gel filtration. The relative
molecular mass (M
r) of native glucose oxidase was determined to be 154 700 ± 4970, and 77 700 for the denatured subunit. Electron-microscopic
examinations revealed a sandwich-shaped dimeric molecule with subunit dimensions of 5.0 × 8.0 nm. Glucose oxidase is a glycoprotein
that contains tightly bound FAD with an estimated stoichiometry of 1.76 mol/mol enzyme. The enzyme is specific for d-glucose, for which a K
m value of 6.2 mM was determined. The pH optimum was determined in the range pH 4.0–6.0. Glucose oxidase showed high stability
on storage in sodium citrate (pH 5.0) and in potassium phosphate (pH 6.0), each 100 mM. The half-life of the activity was
considerably more than 305 days at 4 °C and 30 °C, and 213 days at 40 °C. The enzyme was unstable at temperatures above 40 °C
in the range pH 2.0–4.0 and at a pH above 7.0.
Received: 18 November 1996 / Received revision: 3 March 1997 / Accepted: 7 March 1997 相似文献
4.
Effect of various inhibitors on the (NH4
+ + Na+)-activated ATPase of an anaerobic alkaliphile, Ep01(a strain of Amphibacillus xylanus), was examined. Among the chemicals tested, the enzyme was drastically inactivated by p-chloromercuribenzoic acid and diethyl pyrocarbonate. The ATPase activity of the enzyme, which was inactivated by p-chloromercuribenzoic acid and diethyl pyrocarbonate, was remarkably restored by β-mercaptoethanol and hydroxylamine, respectively,
suggesting the involvement of cysteine and histidine residues in the enzyme activity. Analysis of the inhibition kinetics
by diethyl pyrocarbonate indicated that modification of a single histidine residue per ATPase molecule was sufficient to inactivate
the enzyme.
Received: 2 June 1997 / Accepted: 7 July 1997 相似文献
5.
Geranyldiphosphate:4-hydroxybenzoate 3-geranyltransferase is a regulatory enzyme in the biosynthesis of shikonin, a phytoalexin
and pharmaceutical produced by cell cultures of Lithospermum erythrorhizon Sieb. et Zucc.. In Linsmaier-Skoog medium, the activity of this enzyme could be enhanced more than 200-fold by addition of
methyl jasmonate, and this culture material was used for the solubilization and purification of the enzyme. Of various detergents
examined, digitonin was the most suitable for the solubilization of the enzyme. The solubilized enzyme was purified 800-fold
by chromatography over diethylaminoethyl (DEAE)-Sephacel, Heparin-Sepharose, Reactive Green 19-Agarose, and Cholic Acid-Agarose.
The purified enzyme required magnesium ions as cofactor and was highly specific for geranyldiphosphate (GPP) and 4-hydroxybenzoate
(4HB) as substrates. The K
m values for 4HB and GPP were calculated by the method of Lineweaver and Burk as 18.4 μM and 13.8 μM, respectively.
Received: 2 July 1997 / Accepted: 14 October 1997 相似文献
6.
No increases in exopolysaccharide (EPS) yields in Aureobasidium pullulans were observed when grown with reduced-shear impellers instead of standard Rushton turbines in the same vessel. However, yields
were dramatically reduced when the organism was grown in an airlift reactor. This fall in production could be counteracted
by improving fluid circulation through the placement of impellers within the draught tube, a strategy that resulted in the
highest EPS concentration (approx. 13 g l−1) of all the fermenter configurations tested.
Received: 24 June 1997 / Revised revision: 25 September 1997 / Accepted: 29 September 1997 相似文献
7.
Cephalosporin production by Streptomyces clavuligerus is known to be negatively regulated by carbon sources, e.g., glycerol and starch, and by phosphate at high concentrations.
Formation of lysine ɛ-aminotransferase (LAT) activity, the first enzyme of the biosynthetic pathway, was affected by a high
concentration of carbon source. Whereas 3% starch more than doubled LAT activity production as compared to 1% starch, 3% glycerol
repressed LAT activity formation by 20%–30%. LAT activity production was not affected by 100 mM K2HPO4. Our results thus show that the negative effects of 2% glycerol and 3% starch and 100 mM phosphate on cephalosporin production
are not due to an effect on production of LAT activity. However, repression of LAT activity by 3% glycerol would be expected
to play a negative role in antibiotic production.
Received: 13 June 1997 / Received revision: 20 August 1997 / Accepted: 25 August 1997 相似文献
8.
The biosynthesis of poly(hydroxyalkanoates) (PHA) by Pseudomonas resinovorans from triglyceride substrates was investigated. Each triglyceride, whether animal fat or vegetable oil, supported cellular
growth to relatively high average cell yields (3.3 ± 0.2 g/l). PHA yields ranged from 1.1 g/l to 2.1 g/l, representing approximately
45% of the bacterial cell dry weight. The repeat-unit composition of the polymers was determined by gas chromatography (GC)
and GC/mass spectrometry of the β-hydroxyalkanoate methyl esters from the hydrolyzed polymers. With the exception of PHA from
soybean oil (PHA-soy), each polyester was composed of β-hydroxyacyl moieties with chain lengths ranging from C4 to C14, with C8 and C10 being the predominant species. PHA-soy contained an additional fraction (2%) of C16 monomers. The alkyl side-chains of the PHA contained varying degrees of unsaturation. PHA from coconut oil was composed entirely
of saturated side-chains, whereas PHA-soy contained 4.2 mol% olefinic groups in its side-chains. The increase in the degree
of side-chain unsaturation caused decreased melting temperatures, enthalpies of fusion, and glass transition temperatures.
The molar masses of the polymers were relatively constant and ranged from 6.5 × 104 to 10.1 × 104 g/mol.
Received: 2 September 1997 / Received revision: 21 November 1997 / Accepted: 2 January 1998 相似文献
9.
Metabolism of EDTA and its metal chelates by whole cells and cell-free extracts of strain BNC1 总被引:5,自引:0,他引:5
The influence of metal ions on the metabolism of ethylenediaminetetraacetate (EDTA) by whole cells and cell-free extracts
of strain BNC1 was investigated. Metal-EDTA chelates with thermodynamic stability constants below 1012 were readily mineralized by whole cells with maximum specific turnover rates of 15 (MnEDTA) to 20 (Ca-, Mg-, and BaEDTA)
μmol g protein−1 min−1. With the exception of ZnEDTA, chelates with stability constants greater than 1012 were not oxidized at a significant rate. However, it was shown for Fe(III)EDTA that even strong complexes can be degraded
after pretreatment by addition of calcium and magnesium salts in the pH range 9–11. The range of EDTA chelates converted by
cell-free extracts of strain BNC1 did not depend on their thermodynamic stabilities. The EDTA chelates of Ba2+, Co2+, Mg2+, Mn2+, and Zn2+ were oxidized whereas Ca-, Cd-, Cu-, Fe-, Pb-, and SnEDTA were not. The first catabolic enzyme appears to be an EDTA monooxygenase
since it requires O2, NADH, and FMN for its activity and yields glyoxylate and ethylenediaminetriacetate as products. The latter is further degraded
via N,N′-ethylenediaminediacetate. The maximum specific turnover rate with MgEDTA, the favoured EDTA species, was 50–130 μmol g protein−1 min−1, and the K
m value was 120 μmol/l (K
s for whole cells = 8 μmol/l). Whole cells as well as cell-free extracts of strain BNC1 also converted several structural analogues
of EDTA.
Received: 4 July 1997 / Received revision: 25 September 1997 / Accepted: 29 September 1997 相似文献
10.
Production of high yields of arachidonic acid in a fed-batch system by Mortierella alpina ATCC 32222
Of six strains of Mortierella tested, Mortierella alpina ATCC 32222 produced the highest yields of arachidonic acid. Supplementation of soy flour (1% w/v) and vegetable oils (1%
v/v) significantly increased the biomass, lipid content and arachidonic acid level. Replacement of NaNO3 with corn steep liquor (1% w/v) also improved arachidonic acid production. A fed-batch culture system at 25 °C, producing
a high biomass (52.4 g/l) and arachidonic acid content (9.1 g/l) in 8␣days, was developed. A fed-batch system at low temperature
(15 °C) gave even higher arachidonic acid levels (11.1 g/l) in 11 days.
Received: 28 October 1996 / Received revision: 3 March 1997 / Accepted: 7 March 1997 相似文献
11.
O. Pines S. Shemesh E. Battat I. Goldberg 《Applied microbiology and biotechnology》1997,48(2):248-255
Saccharomyces cerevisiae accumulates l-malic acid through a cytosolic pathway starting from pyruvic acid and involving the enzymes pyruvate carboxylase and malate
dehydrogenase. In the present study, the role of malate dehydrogenase in the cytosolic pathway was studied. Overexpression
of cytosolic malate dehydrogenase (MDH2) under either the strong inducible GAL10 or the constitutive PGK promoter causes a
6- to 16-fold increase in cytosolic MDH activity in growth and production media and up to 3.7-fold increase in l-malic acid accumulation in the production medium. The high apparent K
m of MDH2 for l-malic acid (11.8 mM) indicates a low affinity of the enzyme for this acid, which is consistent with the cytosolic function
of the enzyme and differs from the previously published K
m of the mitochondrial enzyme (MDH1, 0.28 mM). Under conditions of MDH2 overexpression, pyruvate carboxylase appears to be
a limiting factor, thus providing a system for further metabolic engineering of l-malic acid production. The overexpression of MDH2 activity also causes an elevation in the accumulation of fumaric acid and
citric acid. Accumulation of fumaric acid is presumably caused by high intracellular l-malic acid concentrations and the activity of the cytosolic fumarase. The accumulation of citric acid may suggest the intriguing
possibility that cytosolic l-malic acid is a direct precursor of citric acid in yeast.
Received: 22 January 1997 / Received revision: 14 April 1997 / Accepted: 19 April 1997 相似文献
12.
S A Shaikh J M Khire M I Khan 《Journal of industrial microbiology & biotechnology》1997,19(4):239-245
A thermostable β-galactosidase was produced extracellularly by a thermophilic Rhizomucor sp, with maximum enzyme activity (0.21 U mg−1) after 4 days under submerged fermentation condition (SmF). Solid state fermentation (SSF) resulted in a nine-fold increase
in enzyme activity (2.04 U mg−1). The temperature range for production of the enzyme was 38–55°C with maximum activity at 45°C. The optimum pH and temperature
for the partially purified enzyme was 4.5 and 60°C, respectively. The enzyme retained its original activity on incubation
at 60°C up to 1 h. Divalent cations like Co2+, Mn2+, Fe2+ and Zn2+ had strong inhibitory effects on the enzyme activity. The K
m and V
max for p-nitrophenyl-β- D-galactopyranoside and o-nitrophenyl-β - D-galactopyranoside were 0.39 mM, 0.785 mM and 232.1 mmol min−1 mg−1 respectively. The K
m and V
max for the natural substrate lactose were 66.66 μM and 0.20 μ mol min−1 mg−1.
Received 10 March 1997/ Accepted in revised form 17 July 1997 相似文献
13.
Z. D. Knezevic S. S. Siler-Marinkovic L. V. Mojovic 《Applied microbiology and biotechnology》1998,49(3):267-271
Candida rugosa lipase has been used to investigate the hydrolysis of palm oil in a lecithin/isooctane reversed micellar system. The reaction
obeys Michaelis-Menten kinetics for the initial conditions. Kinetic parameters such as maximum rate and Michaelis constant
(K
m) were determined for lipase-catalyzed hydrolysis in n-hexane and isooctane. According to the K
m values, the enzyme affinity towards the substrate was increased in isooctane. The maximum degree of hydrolysis was generally
decreased as the initial substrate concentration was increased. This may suggest that the hydrolysis in lecithin reversed
micelles should be regarded as a one-substrate first-order reversible reaction. It is shown in this study that the proposed
one-substrate first-order kinetic model can serve for the precise prediction of the degree of hydrolysis for a known reaction
time or vice versa, when the initial substrate concentration is less than 0.325 mol/dm3. A disagreement with this model was found when the initial substrate concentration was higher than approximately 0.3 mol/dm3. This may be due to the effects of the products on lipase activity or even to the conversion of the reversed micellar system
to other systems.
Received: 16 May 1997 / Received revision: 22 October 1997 / Accepted: 24 October 1997 相似文献
14.
Glucose oxidase from Penicillium amagasakiense was purified to homogeneity by ion-exchange chromatography and deglycosylated with endoglycosidase H. On the basis of gas
chromatography and sodium dodecyl sulphate/polyacrylamide gel electrophoretic (SDS-PAGE) analyses, the protein-bound high-mannose-type
carbohydrate moiety corresponded to 13% of the molecular mass of glycosylated glucose oxidase. A total of six N-glycosylation sites per dimer were determined from the N-acetylglucosamine content. The enzymatically deglycosylated enzyme contained less than 5% of the original carbohydrate moiety.
A molecular mass of 130 kDa (gel filtration) and 133 kDa (native PAGE) was determined for the dimer and 67 kDa (SDS-PAGE)
for the monomer of the deglycosylated enzyme. The N-terminal sequence, which has not been published for glucose oxidase from
P. amagasakiense to date and which showed less than 50% homology to the N terminus of glucose oxidase from Aspergillus niger, and the amino acid composition were not altered by the deglycosylation. Deglycosylation also did not affect the kinetics
of glucose oxidation or the pH and temperature optima. It also did not increase the susceptibility of the enzyme to proteolytic
degradation. However, deglycosylated glucose oxidase exhibited decreased pH and thermal stability. The thermal stability of
both enzymes was shown to be dependent on the buffer concentration and was enhanced by certain additives, particularly 1 M
(NH4)2SO4, which stabilised glucose oxidase 100- to 300-fold at 50 °C and pH 7–8, and 2 M KF, which stabilised the enzyme up to 36-fold
at 60 °C and pH 6. In sodium acetate buffer, changes in pH (4–6) affected the affinity for glucose but had no effect on the
V
max of the reaction. In contrast, in TRIS buffer, pH 8, a 10-fold decrease in V
max and a 2-fold decrease in K
m were observed.
Received: 8 October 1996 / Received revision: 14 January 1997 / Accepted: 17 January 1997 相似文献
15.
M. V. Gonchar L. B. Kostryk A. A. Sibirny 《Applied microbiology and biotechnology》1997,48(4):454-458
Mutant strains of the methylotrophic yeast Hansenula polymorpha defective in catalase (cat) and in glucose repression of alcohol oxidase synthesis (gcr1) have been isolated following multiple UV mutagenesis steps. One representative gcr1 cat mutant C-105 grows during batch cultivation in a glucose/methanol medium. However, growth is preceded by a prolonged lag
period. C-105 and other gcr1 cat mutants do not grow on methanol medium without an alternative carbon source. A large collection of second-site suppressor
catalase-defective (scd) revertants were isolated with restored ability for methylotrophic growth (Mth+) in the absence of catalase activity. These Mth+
gcr1 cat scd strains utilize methanol as a sole source of carbon and energy, although biomass yields are reduced relative to the wild-type
strain. In contrast to the parental C-105 strain, H2O2 does not accumulate in the methanol medium of the revertants. We show that restoration of methylotrophic growth in the suppressor
strains is strongly correlated with increased levels of the alternative H2O2-destroying enzyme, cytochrome c peroxidase. Cytochrome c peroxidase from cell-free extracts of one of the scd revertants has been purified to homogeneity and crystallized.
Received: 9 December 1996 / Received revision: 5 May 1997 / Accepted: 25 May 1997 相似文献
16.
S. Halldórsdóttir E. T. Thórólfsdóttir R. Spilliaert M. Johansson S. H. Thorbjarnardóttir A. Palsdottir G. Ó. Hreggvidsson J. K. Kristjánsson O. Holst G. Eggertsson 《Applied microbiology and biotechnology》1998,49(3):277-284
A gene library from the thermophilic eubacterium Rhodothermus marinus, strain ITI 378, was constructed in pUC18 and transformed into Escherichia coli. Of 5400 transformants, 3 were active on carboxymethylcellulose. Three plasmids conferring cellulase activity were purified
and were all found to contain the same cellulase gene, celA. The open reading frame for the celA gene is 780 base pairs and encodes a protein of 260 amino acids with a calculated molecular mass of 28.8 kDa. The amino acid
sequence shows homology with cellulases in glycosyl hydrolase family 12. The celA gene was overexpressed in E. coli when the pET23, T7 phage RNA polymerase system was used. The enzyme showed activity on carboxymethylcellulose and lichenan,
but not on birch xylan or laminarin. The expressed enzyme had six terminal histidine residues and was purified by using a
nickel nitrilotriacetate column. The enzyme had a pH optimum of 6–7 and its highest measured initial activity at 100 °C. The
heat stability of the enzyme was increased by removal of the histidine residues. It then retained 75% of its activity after
8 h at 90 °C.
Received: 5 August 1997 / Received revision: 6 November 1997 / Accepted: 7 November 1997 相似文献
17.
K. P. Hebbar R. D. Lumsden S. M. Poch J. A. Lewis 《Applied microbiology and biotechnology》1997,48(6):714-719
Conditions for optimizing spore production, especially chlamydospores, by host-specific mycoherbicidal strains of Fusarium oxysporum causing vascular wilts in coca (Erythroxylum coca) and poppy (Papaver somniferum) were studied in 2.5-1 fermentors. The fermentor dissolved oxygen and pH had significant effects on the growth characteristics
of F. oxysporum strains. The effect of the fungal strain, however was not significant for most of the variables studied except for chlamydospore
formation. After 14 days of fermentation, the spore types produced were microconidia and chlamydospores, with very little
production of macroconidia. While the total viable counts were significantly higher under high than under low dissolved O2, the chlamydospore counts were significantly higher under low than under high dissolved O2. The percentage of chlamydospores obtained, as a proportion of total viable was significantly higher when the fermentor pH
was increased, than when it was not. Scaling-up the liquid fermentation to 20 l, yielded log10
c = 6.8 (where c = chlamydospores ml−1) after 14 days' fermentation, with biomass viable counts of log10
v∼8.0 (where v = viable counts g−1 air-dried biomass). A single-step liquid fermentation reported in this study increased chlamydospore yields and reduced the
time required for their production with techniques currently available from 5 weeks to less than 2 weeks.
Received: 24 April 1997 / Received revision: 6 August 1997 / Accepted: 29 August 1997 相似文献
18.
Overexpression of a cytosolic hydroxymethylglutaryl-CoA reductase leads to squalene accumulation in yeast 总被引:4,自引:0,他引:4
The enzyme 3-hydroxy-3-methylglutaryl-coenzyme-A (HMG-CoA) reductase is known as the rate-limiting enzyme in early sterol
biosynthesis in eukaryotic cells. To eliminate this regulation in the yeast Saccharomyces cerevisiae, a truncated HMG1 gene, producing a form of the enzyme that lacks the membrane-binding region (i.e. amino acids 1–552), was constructed and
overexpressed in this yeast. The transformed strains accumulated large amounts of the sterol precursor squalene, while the
levels of ergosterol and a number of other sterol compounds were only slightly elevated. These findings suggest that HMG-CoA
reductase is not the only rate-limiting step in sterol synthesis and its overexpression cannot significantly influence this
pathway beyond the sterol precursor squalene.
Received: 9 June 1997 / Received revision: 1 September 1997 / Accepted: 19 September 1997 相似文献
19.
C. M. G. A. Fontes J. H. Clarke G. P. Hazlewood T. H. Fernandes H. J. Gilbert L. M. A. Ferreira 《Applied microbiology and biotechnology》1998,49(5):552-559
Cellulose-binding domains (CBD) play a pivotal role during plant cell wall hydrolysis by cellulases and xylanases from aerobic
soil bacteria. Recently we␣have reported the molecular characterisation of a single-domain endoglucanase from Cellvibrio mixtus, suggesting that some cellulases produced by this aerobic bacterium preferentially hydrolyse soluble cellulosic substrates.
Here we describe the complete nucleotide sequence of a second cellulase gene, celB, from the soil bacterium C. mixtus. It revealed an open reading frame of 1863 bp that encoded a polypeptide, defined as cellulase B (CelB), with a predicted
M
r of 66 039. CelB contained a glycosyl hydrolase family 5 catalytic domain at its N terminus followed by two repeated domains,
which exhibited sequence identity with type VI CBD previously found in xylanases. Full-length CelB bound to cellulose while
catalytically active truncated cellulase derivatives were unable to bind the polysaccharide, confirming that CelB is a modular
enzyme and that the type VI CBD homologues were functional. Analysis of the biochemical properties of CelB revealed that the
enzyme hydrolyses a range of cellulosic substrates, although it was unable to depolymerise Avicel. We propose that type VI
CBD, usually found in xylanases, provide an additional mechanism by which cellulases can accumulate on the surface of the
plant cell wall, although they do not potentiate cellulase activity directly. These results demonstrate that C. mixtus, in common with other aerobic bacteria, is able to produce cellulases that are directed to the hydrolysis of cellulose in
its natural environment, the plant cell wall.
Received: 6 October 1997 / Received revision: 22 December 1997 / Accepted: 2 January 1998 相似文献
20.
The extremely thermophilic archaeon Thermococcus hydrothermalis, isolated from a deep-sea hydrothermal vent in the East Pacific Rise at 21°N, produced an extracellular pullulanase. This
enzyme was purified 97-fold to homogeneity from cell-free culture supernatant. The purified pullulanase was composed of a
single polypeptide chain having an estimated molecular mass of 110 kDa (gel filtration) or 128 kDa (sodium dodecyl sulfate/polyacryl
amide gel electrophoresis). The enzyme showed optimum activity at pH 5.5 and 95 °C. The thermostability and the thermoactivity
were considerably increased in the presence of Ca2+. The enzyme was activated by 2-mercaptoethanol and dithiothreitol, whereas N-bromosuccinimide and α-cyclodextrin were inhibitors. This enzyme was able to hydrolyze, in addition to the α-1,6-glucosidic
linkages in pullulan, α-1,4-glucosidic linkages in amylose and soluble starch, and can therefore be classified as a type II
pullulanase or an amylopullulanase. The purified enzyme displayed Michaelis constant (K
m) values of 0.95 mg/ml for pullulan and 3.55 mg/ml for soluble starch without calcium and, in the presence of Ca2+, 0.25 mg/ml for pullulan and 1.45 mg/ml for soluble starch.
Received: 19 November 1997 / Received revision: 9 March 1998 / Accepted: 14 March 1998 相似文献