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1.
β-Glucosidase from almonds (EC 3.2.1.21) was covalently immobilized by a two-step technique. In the first step, double bonds
were introduced into the β-glucosidase by derivatization with itaconic anhydride. In separate studies with α-N-protected l-amino acids, it was established that itaconic anhydride acylated mainly primary amino groups of lysines and, to a much lesser
extent hydroxyl groups of tyrosines and sulfhydryl groups of cysteines. The acylated β-glucosidase showed no loss of activity
and the K
m decreased from 3.6 mM to 2.6 mM when p-nitrophenyl β-d-glucopyranoside was used as the substrate. In the second step, the derivatized β-glucosidase was co-polymerized radically
with N,N′-methylenebisacrylamide in buffer solution. The resulting acrylamide immobilizate possessed a much better storage stability
at 30–56 °C when compared to β-glucosidase immobilized on Eupergit C. However, the specific activity was higher with the Eupergit
immobilizate. Free and acrylamide-immobilized β-glucosidase were used for glucosylation of chloramphenicol by transglucosylation
in 20% (v/v) acetonitrile at 37 °C. The acrylamide immobilizate demonstrated a great enhancement of stability and approximately
50% more chloramphenicol β-glucoside was obtained after 5 h.
Received: 22 September 1997 / Accepted: 28 October 1997 相似文献
2.
M. J. Kujau C. Hoischen D. Riesenberg J. Gumpert 《Applied microbiology and biotechnology》1998,49(1):51-58
The paper describes the synthesis of the phosphorylcholine-binding miniantibody McPC603scFvDhl x in cell-wall-less L-form
strains of Escherichia coli and Proteus mirabilis. Cells of these strains were transformed with the plasmid pACK02scKan, carrying the miniantibody (miniAb) coding sequence
under the control of the lac promoter. L-form transformants of both species were able to synthesize the functional miniAb as an extracellular soluble
product. The highest quantities were obtained by P. mirabilis L-form strains after induction with 5 mM isopropyl β-d-thiogalactopyranoside (IPTG). Yields of 45–75 mg/l total antibody protein and of 10–18 mg/l functional miniAb were estimated
in the growth medium of shaking cultures 40–80 h after induction with IPTG. About 10% of the active miniAb remained cell-bound.
The yields of functional miniAb could be optimized by lowering the growth temperature from 37 °C to 26–32 °C and by supplementation
of the medium with 80 mM sodium fumarate. A comparison of the specific activities revealed that the P. mirabilis L-form strains have a similar synthesis capacity (2–4 mg functional miniAb/g cell dry weight) to that of the producer strain
E. coli RV308. The results show that the processes of correct folding and assembling of the miniAb molecules are possible without
the periplasmic compartment.
Received: 14 April 1997 / Received revision: 17 July 1997 / Accepted: 25 August 1997 相似文献
3.
N. Sriubolmas W. Panbangred S. Sriurairatana V. Meevootisom 《Applied microbiology and biotechnology》1997,47(4):373-378
Various concentrations of isopropyl β-d-thiogalactopyranoside (IPTG) were used to induce production of the enzyme penicillin G acylase by recom binant Escherichia coli harboring plasmid pQEA11. The plasmid pQEA11 carries a wild-type pga gene, which is under the control of the tac promoter and lacI q. At low IPTG concentrations (0.025 – 0.1 mM), enzyme activity increased with increasing IPTG concentrations. At higher IPTG
concentrations (0.2 and 0.5 mM), enzyme activity declined progressively. Examination of induced recombinant E. coli cells by transmission electron microscopy showed the presence of only periplasmic inclusion bodies at low IPTG concentrations
(up to 0.1 mM) and both periplasmic and cytoplasmic inclusion bodies at high IPTG concentrations (0.2 mM and 0.5 mM). Results
from sodium dodecyl sulfate/polyacrylamide gel electrophoresis and immunoblots of whole-cell proteins, membrane proteins and
inclusion body proteins in these cells indicated that cytoplasmic inclusion bodies constituted an accumulation of preproenzyme
(i.e., precursor polypeptide containing a signal peptide) and that periplasmic inclusion bodies constituted an accumulation
of proenzyme (i.e., precursor polypeptide lacking a signal peptide).
Received: 27 March 1996 / Received revision: 2 July 1996 / Accepted: 10 November 1996 相似文献
4.
Byun SG Kim MD Lee WH Lee KJ Han NS Seo JH 《Applied microbiology and biotechnology》2007,74(4):768-775
A recombinant Escherichia coli strain was developed to produce guanosine 5′-diphosphate (GDP)-l-fucose, donor of l-fucose, which is an essential substrate for the synthesis of fucosyloligosaccharides. GDP-d-mannose-4, 6-dehydratase (GMD) and GDP-4-keto-6-deoxymannose 3, 5-epimerase 4-reductase (WcaG), the two crucial enzymes for
the de novo GDP-l-fucose biosynthesis, were overexpressed in recombinant E. coli by constructing inducible overexpression vectors. Optimum expression conditions for GMD and WcaG in recombinant E. coli BL21(DE3) were 25°C and 0.1 mM isopropyl-β-d-thioglucopyranoside. Maximum GDP-l-fucose concentration of 38.9 ± 0.6 mg l−1 was obtained in a glucose-limited fed-batch cultivation, and it was enhanced further by co-expression of NADPH-regenerating
glucose-6-phosphate dehydrogenase encoded by the zwf gene to achieve 55.2 ± 0.5 mg l−1 GDP-l-fucose under the same cultivation condition. 相似文献
5.
A novel phosphorylase from Clostridium phytofermentans belonging to the glycoside hydrolase family (GH) 65 (Cphy1874) was characterized. The recombinant Cphy1874 protein produced
in Escherichia coli showed phosphorolytic activity on nigerose in the presence of inorganic phosphate, resulting in the release of d-glucose and β-d-glucose 1-phosphate (β-G1P) with the inversion of the anomeric configuration. Kinetic parameters of the phosphorolytic activity
on nigerose were k
cat = 67 s−1 and K
m = 1.7 mM. This enzyme did not phosphorolyze substrates for the typical GH65 enzymes such as trehalose, maltose, and trehalose
6-phosphate except for a weak phosphorolytic activity on kojibiose. It showed the highest reverse phosphorolytic activity
in the reverse reaction using d-glucose as the acceptor and β-G1P as the donor, and the product was mostly nigerose at the early stage of the reaction. The
enzyme also showed reverse phosphorolytic activity, in a decreasing order, on d-xylose, 1,5-anhydro-d-glucitol, d-galactose, and methyl-α-d-glucoside. All major products were α-1,3-glucosyl disaccharides, although the reaction with d-xylose and methyl-α-d-glucoside produced significant amounts of α-1,2-glucosides as by-products. We propose 3-α-d-glucosyl-d-glucose:phosphate β-d-glucosyltransferase as the systematic name and nigerose phosphorylase as the short name for this Cphy1874 protein. 相似文献
6.
T. Ohshiro M. Shinji Y. Morita Y. Takayama Y. Izumi 《Applied microbiology and biotechnology》1997,48(4):546-548
Microorganisms capable of cleaving the urethane bond of t-butoxycarbonyl (Boc) amino acids in a whole-cell reaction were screened among stock cultures, and Corynebacterium aquaticum IFO12154 was the most promising. The conversion of Boc-Ala to Ala was stimulated by CoSO4 in the medium and reaction mixture. The optimum whole-cell concentration was 25 mg lyophilized cells/ml. Boc-l-Met was the best substrate for this reaction, and other Boc-L-amino acids, as well as benzyloxycarbonyl-l-amino acids with hydrophobic residues, were also good substrates. Boc-d- and Z-d-amino acids were inert. When the reactions had proceeded for 24 h with each substrate at 10 mM, the molar conversion rates
from Boc-l-, dl- and d-Met were 100%, 50%, and 0% respectively. From 150 mM Boc-l-Met, 143 mM l-Met was formed at a molar yield of 95.3%.
Received: 3 September 1996 / Received last revision: 7 April 1997 / Accepted: 19 April 1997 相似文献
7.
The soluble acid invertase (β-D-fructofuranoside fructohydrolase, EC 3.2.1.26) from potato (Solanum tuberosum L. cv. Kennebec) tubers was located in the vacuoles. Although the functionality of this invertase in the vacuoles has been
assumed, the activity of the enzyme has never been shown within isolated vacuoles. Vacuoles were prepared by gentle osmotic
shock from free protoplasts obtained by enzymic digestion of tuber tissues. The mean volume of these vacuoles, (0.26 ± 0.05) × 10−2 μl, was estimated by optical microscopy. Sucrose, glucose and fructose concentrations were calculated to be 100 mM, 20 mM
and 40 mM, respectively, in the vacuoles. Sucrose hydrolysis and the increase in glucose and fructose concentrations within
the vacuoles were measured during vacuolar incubations. An almost identical pattern of sucrose hydrolysis by invertase was
found by an in-vitro assay reproducing the vacuolar conditions. In view of the determinations of internal vacuolar pH (5.2),
the possibility of spontaneous hydrolysis of sucrose was disregarded. Vacuoles were shown to be free from proteinaceous inhibitors,
confirming the extravacuolar location of these inhibitors. The vacuolar hydrolytic pattern of sucrose confirms the regulatory
role of the reaction products previously proposed for in-vitro assays.
Received: 21 July 1997 / Accepted: 31 August 1997 相似文献
8.
J. Q. Liu S. Ito T. Dairi N. Itoh S. Shimizu H. Yamada 《Applied microbiology and biotechnology》1998,49(6):702-708
Low-specificity l-threonine aldolase, catalyzing the reversible cleavage/condensation reaction between l-threonine/l-allo-threonine and glycine plus acetaldehyde, was purified to homogeneity from Pseudomonas sp. NCIMB 10558. The enzyme has an apparent molecular mass of approximately 145 kDa and consists of four identical subunits
with a molecular mass of 38 kDa. The enzyme, requiring pyridoxal- 5′-phosphate as a coenzyme, is strictly l-specific at the α position, whereas it can not distinguish between threo and erythro forms at the β position. Besides the reversible cleavage/condensation of threonine, the enzyme also catalyzes the reversible
interconversion between glycine plus various aldehydes and l-β-hydroxy-α-amino acids, including l-β-(3,4-dihydroxyphenyl)serine, l-β-(3,4-met‐hylenedioxyphenyl)serine and l-β-phenylserine, providing a new route for the industrial production of these important amino acids.
Received: 10 November 1997 / Received revision: 7 January 1998 / Accepted 30 January 1998 相似文献
9.
An DS Cui CH Sung BH Yang HC Kim SC Lee ST Im WT Kim SG 《Applied microbiology and biotechnology》2012,94(3):673-682
The gene encoding an α-l-arabinofuranosidase that could biotransform ginsenoside Rc {3-O-[β-d-glucopyranosyl-(1–2)-β-d-glucopyranosyl]-20-O-[α-l-arabinofuranosyl-(1–6)-β-d-glucopyranosyl]-20(S)-protopanaxadiol} to ginsenoside Rd {3-O-[β-d-glucopyranosyl-(1–2)-β-d-glucopyranosyl]-20-O-β-d-glucopyranosyl-20(S)-protopanaxadiol} was cloned from a soil bacterium, Rhodanobacter ginsenosidimutans strain Gsoil 3054T, and the recombinant enzyme was characterized. The enzyme (AbfA) hydrolyzed the arabinofuranosyl moiety from ginsenoside
Rc and was classified as a family 51 glycoside hydrolase based on amino acid sequence analysis. Recombinant AbfA expressed
in Escherichia coli hydrolyzed non-reducing arabinofuranoside moieties with apparent K
m values of 0.53 ± 0.07 and 0.30 ± 0.07 mM and V
max values of 27.1 ± 1.7 and 49.6 ± 4.1 μmol min−1 mg−1 of protein for p-nitrophenyl-α-l-arabinofuranoside and ginsenoside Rc, respectively. The enzyme exhibited preferential substrate specificity of the exo-type
mode of action towards polyarabinosides or oligoarabinosides. AbfA demonstrated substrate-specific activity for the bioconversion
of ginsenosides, as it hydrolyzed only arabinofuranoside moieties from ginsenoside Rc and its derivatives, and not other sugar
groups. These results are the first report of a glycoside hydrolase family 51 α-l-arabinofuranosidase that can transform ginsenoside Rc to Rd. 相似文献
10.
D. Segura C. Santana R. Gosh L. Escalante S. Sanchez 《Applied microbiology and biotechnology》1997,48(5):615-620
In Streptomyces peucetius var. caesius, the production of anthracyclines was suppressed either by 330 mM d-glucose or 25 mM phosphate. In addition, the anthracycline doxorubicin and the glucose analogue 2-deoxyglucose inhibited
the growth of this microorganism at concentrations of 0.025 mM and 10 mM respectively. Spontaneous and induced mutants, resistant
to the action of these compounds, were isolated, tested and chosen by their ability to overproduce anthracyclines. Genetic
recombination between representative mutants was carried out by the protoplast fusion technique. Some recombinants carrying
resistance to doxorubicin, phosphate and 2-deoxyglucose produced more than 40-fold greater levels of anthracyclines than those
obtained with the parental strain. This improvement resulted in total antibiotic titres of more than 2 g/l culture medium
at 6 days of fermentation.
Received: 14 April 1997 / Received revision: 19 June 1997 / Accepted: 4 July 1997 相似文献
11.
Syntheses of l-dopa 1a glucoside 10a,b and dl-dopa 1b glycosides 10–18 with d-glucose 2, d-galactose 3, d-mannose 4, d-fructose 5, d-arabinose 6, lactose 7, d-sorbitol 8 and d-mannitol 9 were carried out using amyloglucosidase from Rhizopus mold, β-glucosidase isolated from sweet almond and immobilized β-glucosidase. Invariably, l-dopa and dl-dopa gave low to good yields of glycosides 10–18 at 12–49% range and only mono glycosylated products were detected through glycosylation/arylation at the third or fourth
OH positions of l-dopa 1a and dl-dopa 1b. Amyloglucosidase showed selectivity with d-mannose 4 to give 4-O-C1β and d-sorbitol 8 to give 4-O-C6-O-arylated product. β-Glucosidase exhibited selectivity with d-mannose 4 to give 4-O-C1β and lactose 7 to give 4-O-C1β product. Immobilized β-glucosidase did not show any selectivity. Antioxidant and angiotensin converting enzyme inhibition
(ACE) activities of the glycosides were evaluated glycosides, out of which l-3-hydroxy-4-O-(β-d-galactopyranosyl-(1′→4)β-d-glucopyranosyl) phenylalanine 16 at 0.9 ± 0.05 mM and dl-3-hydroxy-4-O-(β-d-glucopyranosyl) phenylalanine 11b,c at 0.98 ± 0.05 mM showed the best IC50 values for antioxidant activity and dl-3-hydroxy-4-O-(6-d-sorbitol)phenylalanine 17 at 0.56 ± 0.03 mM, l-dopa-d-glucoside 10a,b at 1.1 ± 0.06 mM and dl-3-hydroxy-4-O-(d-glucopyranosyl)phenylalanine 11a-d at 1.2 ± 0.06 mM exhibited the best IC50 values for ACE inhibition.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
12.
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
13.
Sieve-tube exudate protein (STEP) from Ricinus communis L. seedlings consists of a characteristic set of more than 100 different polypeptides, against which a complex antiserum
was raised. This antiserum cross-reacted with dominant protein species (molecular weights 10–30 kDa) present in the sieve-tube
exudate and, to a lesser extent, with proteins in tissue extracts of Ricinus and a wide range of other plant species. For further elucidation of the nature of individual STEPs in the sieve tubes the
anti-STEP serum was used to screen a cDNA expression library constructed from Ricinus cotyledon mRNA. Two clones that differed in the 3′ untranslated region encoded a protein of 11 kDa which showed striking
homology to bacterial and eucaryotic glutaredoxin sequences. Glutaredoxin activity was confirmed for the recombinant protein
after overexpression in Escherichia coli and characterised in detail in sieve-tube exudate. Michaelis Menten constants (K
m) for reduced glutathione and cysteine were 2 mM and 50 μM, respectively. Besides l-cysteine, dehydroascorbate and protein disulphides were also reduced by the activity present in the sieve-tube exudate. Glutathione,
which is the obligate donor of reduced thiols for glutaredoxin, was present in sieve-tube sap in millimolar concentrations
(up to 3 mM) with a ratio of total to oxidised glutathione of 3:1. It is suggested that glutaredoxin and glutathione in sieve
tubes prevent oxidative damage and may be involved in redox regulation of sieve-tube proteins.
Received: 13 December 1996 / Accepted: 28 December 1996 相似文献
14.
P. L. Schuerman J. S. Liu H. Mou A. M. Dandekar 《Applied microbiology and biotechnology》1997,47(5):560-565
Escherichia coli strains that did not have the ability to use sucrose as a sole carbon source gained this ability after receiving a cloned
fragment of DNA from Agrobacterium tumefaciens. No invertase was detected in the sucrose-metabolizing E. coli, but evidence for the activity of certain enzymes, known to be produced by biotype 1 strains of Agrobacterium, were found. Evidence was found for the presence of d-glucoside 3-dehydrogenase (G3DH) and α-3-ketoglucosidase. The activity of enzyme extracts on 3-ketosucrose also indicated
that 3-ketoglucose reductase, or some enzyme that acts on 3-ketoglucose, was present in the Suc+
E. coli as well. The fragment was found to complement a G3DH mutant of A. tumefaciens and was also found to confer chemotaxis towards sucrose in E. coli.
Received: 13 September 1996 / Received revision: 15 January 1997 / Accepted: 24 January 1997 相似文献
15.
S. S. Sudge K. B. Bastawde D. V. Gokhale U. R. Kalkote T. Ravindranathan 《Applied microbiology and biotechnology》1998,49(5):594-599
About 1000 bacterial colonies isolated from sea water were screened for their ability to convert dl-5-phenylhydantoin to d(−)N-carbamoylphenylglycine as a criterion for the determination of hydantoinase activity. The strain M-1, out of 11 hydantoinase-producing
strains, exhibited the maximum ability to convert dl-5-phenylhydantoin to d(−)N-carbamoylphenylglycine. The strain M-1 appeared to be a halophilic Pseudomonas sp. according to morphological and physiological characteristics. Optimization of the growth parameters revealed that nutrient
broth with 2% NaCl was the preferred medium for both biomass and enzyme production. d-Hydantoinase of strain M-1 was not found to be inducible by the addition of uracil, dihydrouracil, β-alanine etc. The optimum
temperature for enzyme production was about 25 °C and the organism showed a broad pH optimum (pH 6.5–9.0) for both biomass
and hydantoinase production. The organism seems to have a strict requirement of NaCl for both growth and enzyme production.
The optimum pH and temperature of enzyme activity were 9–9.5 and 30 °C respectively. The biotransformation under the alkaline
conditions allowed the conversion of 80 g l−1
dl-5-phenylhydantoin to 82 g l−1
d(−)N-carbamoylphenylglycine within 24 h with a molar yield of 93%.
Received: 15 September 1997 / Received revision: 5 January 1998 / Accepted: 6 January 1998 相似文献
16.
The callose synthase (UDP-glucose: 1,3-β-d-glucan 3-β-d-glucosyl transferase; EC 2.4.1.34) enzyme (CalS) from pollen tubes of Nicotiana alata Link et Otto is responsible for developmentally regulated deposition of the cell wall polysaccharide callose. Membrane preparations
from N. alata pollen tubes grown in liquid culture were fractionated by density-gradient centrifugation. The CalS activity sedimented to
the denser regions of the gradient, approximately 1.18 g · ml−1, away from markers for Golgi, endoplasmic reticulum and mitochondria, and into fractions enriched in ATPase activity and
in membranes staining with phosphotungstic acid at low pH. This suggests that pollen-tube CalS is localised in the plasma
membrane. Callose synthase activity from membranes enriched by downward centrifugation was solubilised with digitonin, which
gave a 3- to 4-fold increase in enzyme activity, and the solubilised activity was then enriched a further 10-fold by product
entrapment. The complete procedure gave final CalS specific activities up to 1000-fold higher than those of pollen-tube homogenates.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that several polypeptides co-fractionated with CalS activity
through purification, with a polypeptide of 190 kDa being enriched in product-entrapment pellets.
Received: 24 September 1997 / Accepted: 12 November 1997 相似文献
17.
Xanthomonas campestris pv. campestris, the causal agent of black-rot disease of cruciferous plants, and an important industrial microbe, was able to express the
Escherichia coliβ-glucuronidase reporter gene (uidA) when fused to the E. coli lactose operon promoter on a wide-host-range plasmid vector. The gene fusion is expressed constitutively at high levels in
both complex and defined media using a wide range of carbon sources, and is not repressible by glucose or inducible by the
gratuitous lac inducer isopropyl β-d-thiogalactoside. An X. campestris campestris strain with a lesion in the clp (catabolite-repressor-like protein) locus, and containing the plac/uidA fusion, was tested for β-glucuronidase activity. We found that the expression of the plac/uidA fusion gene is dependent on the presence of catabolite-repressor-like protein, with an approximately 75% reduction of expression
in the clp -deficient mutant.
Received: 1 April 1996 / Received revision: 21 June 1996 / Accepted: 15 July 1996 相似文献
18.
J. M. Obón J. R. Maiquez M. Cánovas H.-P. Kleber J. L. Iborra 《Applied microbiology and biotechnology》1999,51(6):760-764
The use of a biological procedure for l-carnitine production as an alternative to chemical methods must be accompanied by an efficient and highly productive reaction
system. Continuous l-carnitine production from crotonobetaine was studied in a cell-recycle reactor with Escherichia coli O44 K74 as biocatalyst. This bioreactor, running under the optimum medium composition (25 mM fumarate, 5 g/l peptone), was
able to reach a high cell density (26 g dry weight/l) and therefore to obtain high productivity values (6.2 g l-carnitine l−1 h−1). This process showed its feasibility for industrial l-carnitine production. In addition, resting cells maintained in continuous operation, with crotonobetaine as the only medium
component, kept their biocatalytic capacity for 4 days, but the biotransformation capacity decreased progressively when this
particular method of cultivation was used.
Received: 10 December 1998 / Received revision: 19 February 1999 / Accepted: 20 February 1999 相似文献
19.
M. J. Artolozaga E. Kubátová J. Volc H. M. Kalisz 《Applied microbiology and biotechnology》1997,47(5):508-514
Pyranose 2-oxidase (P2O) was purified 43-fold to apparent homogeneity from the basidiomycete Phanerochaete chrysosporium using liquid chromatography on phenyl Sepharose, Mono Q (twice) and phenyl Superose. The native enzyme has a molecular mass
of about 250 kDa (based on native PAGE) and is composed of four identical subunits of 65 kDa. It contains three isoforms of
isoelectric point (pI) 5.0, 5.05 and 5.15 and does not appear to be a glycoprotein. P2O is optimally stable at pH 8.0 and
up to 60 °C. It is active over a broad pH range (5.0–9.0) with maximum activity at pH 8.0–8.5 and at 55 °C, and a broad substrate
specificity. d-Glucose is the preferred substrate, but 1-β-aurothioglucose, 6-deoxy-d-glucose, l-sorbose, d-xylose, 5-thioglucose, d-glucono-1,5-lactone, maltose and 2-deoxy-d-glucose are also oxidised at relatively high rates. A Ping Pong Bi Bi mechanism was demonstrated for the P2O reaction at
pH 8.0, with a catalytic constant (k
cat) of 111.0 s−1 and an affinity constant (K
m) of 1.43 mM for d-glucose and 83.2 μM for oxygen. Whereas the steady-state kinetics for glucose oxidation were unaffected by the medium at
pH ≥ 7.0, at low pH both pH and buffer composition affected the P2O kinetics with the k
cat/K
m value decreasing with decreasing pH. The greatest effect was observed in acetate buffer (0.1 M, pH 4.5), where the k
cat decreased to 60.9 s−1 and the K
m increased to 240 mM. The activity of P2O was completely inhibited by 10 mM HgCl2, AgNO3 and ZnCl2, and 50% by lead acetate, CuCl2 and MnCl2.
Received: 28 August 1996 / Received revision: 25 November 1996 / Accepted: 29 November 1996 相似文献
20.
Aspergillus tamarii produced extracellular xylanase and intracellular β-xylosidase inductively in washed glucose-grown mycelia incubated with
xylan and methyl β-d-xyloside, a synthetic glycoside. Methyl β-d-xyloside was a more effective inducer than xylan at the same concentration for both enzymes. Glucose and cycloheximide were
found to inhibit xylanase production by methyl β-d-xyloside. Methyl β-d-xyloside was hydrolyzed to xylose by mycelial extract in vitro.
Received: 23 May 1996 / Received revision: 5 September 1996 / Accepted: 13 October 1996 相似文献