共查询到20条相似文献,搜索用时 46 毫秒
1.
Sode Koji Sugiura Hiroto Tsugawa Wakako Watazu Yoshihumi Hamafuji Tetsuro 《Applied biochemistry and biotechnology》2000,84(1-9):947-954
A novel enzymatic organic synthesis was reported, utilizing glucose-3-dehydrogenase (G3DH) and its regeneration via electrochemical
methods. We combined the water-soluble G3DH prepared from a marine bacterium, Halomonas sp. α-15, and electron mediator with the electrode system in order to regenerate the enzyme. Using this system, the conversion
of 1,5-anhydro-d-glucitol (1,5AG), a diabetes marker in human blood, was investigated. The final yield of the product, 3-keto anhydroglucitol
(3-ketoAG), which was identified by 13C nuclear magnetic resonance, was 82% based on the initial amount of 1,5AG. The electrochemical yield of the reaction proceeded
almost stoichiometrically. The electrochemical conversion rate of 1,5AG was 1.24 mmol/(L·h), and the electrochemical yield
of 1,5AG consumption was 80%, whereas that for 3-ketoAG was 60%. 相似文献
2.
《Analytical letters》2012,45(3):431-440
Abstract Continuous glucose monitoring (CGM) is expected to become an ideal way to monitor glycemic levels in diabetic patients. On the other hand, biofuel cells can be used as an alternative energy source in future implantable devices, such as implantable glucose sensors in the artificial pancreas. Glucose dehydrogenase from Acinetobacter calcoaceticus, which harbors pyrroloquinoline quinone as the prosthetic group (PQQGDH), is one of the enzymes most attractive as a glucose sensor constituent and as the anode enzyme in biofuel cells, due to its high catalytic activity and insensitivity to oxygen. However, the application of PQQGDH for these purposes is inherently limited because an electron mediator is required for the electron transfer to the electrode. We have recently reported on the development of an engineered enzyme, quinohemoprotein glucose dehydrogenase (QH‐GDH), in which the cytochrome c domain of the quinohemoprotein ethanol dehydrogenase (QH‐EDH) was fused with PQQGDH, to enable electron transfer to the electrode in the absence of an artificial mediator. In this study, we constructed a direct electron‐transfer‐type CGM system employing QH‐GDH. This CGM system showed sufficient current response and high operational stability. Furthermore, we successfully constructed a compartmentless biofuel cell employing QH‐GDH. 相似文献
3.
Tetianec L Bratkovskaja I Kulys J Casaite V Meskys R 《Applied biochemistry and biotechnology》2011,163(3):404-414
The kinetic parameters of carbohydrate oxidation catalyzed by Acinetobacter calcoaceticus pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase (GDH) and Escherichia coli PQQ-dependent aldose sugar dehydrogenase (ASDH) were determined using various electron acceptors. The radical cations of
organic compounds and 2,6-dichlorophenolindophenol are the most reactive with both enzymes in presence of glucose. The reactivity
of dioxygen with ASDH is low; the bimolecular constant k
ox = 660 M−1 s−1, while GDH reactivity with dioxygen is even less. The radical cation of 3-(10H-phenoxazin-10-yl)propionic acid was used as electron acceptor for reduced enzyme in the study of dehydrogenases carbohydrates
specificity. Mono- and disaccharide reactivity with GDH is higher than the reactivity of oligosaccharides. For ASDH, the reactivity
increased with the carbohydrate monomer number increase. The specificity of quinoproteins was compared with specificity of
flavoprotein Microdochium nivale carbohydrate oxidase due to potential enzymes application for lactose oxidation. 相似文献
4.
We have used the initial-rate approach to characterize changes in the glucose consumption kinetics of baculovirus-infected
Spodoptera frugiperda clone 9 (Sf9) cells with the progression of the infection process. The specific glucose consumption rate (q
G) of cultured baculovirus-infected Sf9 cells was measured at 4, 8, 12, 16, and 24 h postinfection (h.p.i.) in media containing
4–35 mM glucose. Higher medium glucose concentrations resulted in higher final extracellular virus and recombinant β-galactosidase
yields. q
G was related to the extracellular glucose concentration by means of a Michaelis-Menten relationship. The apparent Michaelis-Menten
constant (K
m) for glucose consumption was found not to change significantly during the progression of the infection process, and remained
between 6.2 and 7.2 mM. However, the maximal specific glucose consumption rate (q
Gmax) was found to rapidly increase after infection, peaking at 16 h.p.i. at a value four times that for uninfected Sf9 cells.
The kinetic analysis of glucose consumption rates in baculovirus-infected Sf9 cells presented here will aid in the optimal
design and operation of bioreactor systems for the large-scale production of recombinant products from the baculovirus/insect
cell system. 相似文献
5.
Four myrosinase (β-thioglucosidase EC. 3.2.3.1) and seven disaccharase (β-fructofuranosidase, EC. 3.2.1.26) isoenzymes were isolated from turnip leaves. The most active enzymes were isolated in pure
form. Myrosinase and disaccharase mol wt was 62.0 × 103 and 69.5 × 103 dalton, respectively, on the basis of gel filtration on Sephadex G-200.
Myrosinase pH profile showed high activity between pH 5 and 7 with the optimum at pH 5.5. The purified enzyme was heat-stable
for 60 min at 30°C with only loss of 24% of activity. Its activity is strongly inhibited (100%) by Pb2+, Ba2+, Cu2+ and Ca2+ ions, and activated (70%) by EDTA at 0.04M. The pure enzyme failed to hydrolyze amylose, glycogen, lactose, maltose, and
sucrose. TheK
m andV
max values of myrosinase using sinigrin as specific substrate was 0.045 mM and 2.5 U, respectively.
The maximal activity of disaccharase enzyme was obtained at pH 4–5 and 35–37°C. The enzyme was heat-stable at 30°C for 30
min with only 10% loss of its activity. Its activity is strongly activated (70–240%) by Ca2+, Ba2+, Cu2+, and EDTA at 0.01M. The enzyme activity is specific to the disaccharide sucrose and failed to hydrolyze other disaccharides (maltose and lactose).
TheK
m andV
max of disaccharase were 0.123 mM and 3.33 U, respectively. 相似文献
6.
Urease from pigeonpea (Cajanus cajan L.) was covalently linked to crab shell chitosan beads using glutaraldehyde. The optimum immobilization (64% activity) was
observed at 4°C, with a protein concentration of 0.24 mg/bead and 3% glutaraldehyde. The immobilized enzyme stored in 0.05
M Trisacetate buffer, pH 7.3, at 4°C had a t
1/2 of 110 d. There was practically no leaching of enzyme (<3%) from the immobilized beads in 30 d. The immobilized urease was
used 10 times at an interval of 24 h between each use with 80% residual activity at the end of the period. The chitosan-immobilized
urease showed a significantly higher Michaelis constant (8.3 mM) compared to that of the soluble urease (3.0 mM). Its apparent optimum pH also shifted from 7.3 to 8.5. Immobilized urease showed an optimal temperature of 77°C, compared
with 47°C for the soluble urease. Time-dependent kinetics of the thermal denaturation of immobilized urease was studied and
found to be monophasic in nature compared to biphasic in nature for soluble enzyme. This immobilized urease was used to analyze
blood urea of some of the clinical samples from the clinical pathology laboratories. The results compared favorably with those
obtained by the various chemical/biochemical methods employed in the clinical pathology laboratories. A column packed with
immobilized urease beads was also prepared in a syringe for the regular and continuous monitoring of serum urea concentrations. 相似文献
7.
《Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy》1994,50(6):1179-1193
A new spectroscopic procedure for the measurement of glucose concentrations is described which is based on substrate induced quenching (SIQ) of an indicator fluorescence. The method exploits a novel photo reaction between thionine and NADH, the latter being generated due to the reduction of NAD+ in an enzymic reaction between glucose dehydrogenase (GDH) and glucose. The observed SIQ data was analysed using an empirical relation. A quenching constant of 1.8×103 (±100) M−1 is obtained for the substrate induced quenching of thionine by glucose. The reported method, which was investigated over the range 0–1000 μM, offers a glucose detection limit of 2.2 μM. Various applications of the proposed scheme are discussed, including its use to construct a fibre optic biosensor for glucose. 相似文献
8.
An extracellular exoinulinase was purified from the crude extract of Aspergillus fumigatus by ammonium sulfate precipitation, followed by successive chromatographies on DEAE-Sephacel, Sephacryl S-200, concanavalin
A-linked amino-activated silica, and Sepharose 6B columns. The enzyme was purified 25-fold, and the specific activity of the
purified enzyme was 171 IU/mg of protein. Gel filtration chromatography revealed a molecular weight of about 200 kDa, and
native polyacrylamide gel electrophoresis (PAGE) showed an electrophoretic mobility corresponding to a molecular weight of
about 176.5 kDa. Sodium dodecyl sulfate-PAGE analysis revealed three closely moving bands of about 66, 62.7, and 59.4 kDa,
thus indicating the heterotrimeric nature of this enzyme. The purified enzyme appeared as a single band on isoelectric focusing,
with a pI of about 8.8. The enzyme activity was maximum at pH 5.5 and was stable over a pH range of 4.0–9.5, and the optimum temperature
for enzyme activity was 60°C. The purified enzyme retained 35.9 and 25.8% activities after 4 h at 50 and 55°C, respectively.
The inulin hydrolysis activity was completely abolished with 1 mM Hg++, whereas EDTA inhibited about 63% activity. As compared to sucrose, stachyose, and raffinose, the purified enzyme had lower
K
m
(0.25 mM) and higher V
max (333.3 IU/mg) values for inulin. 相似文献
9.
A new high polygalacturonase (PG)-producing Kluyveromyces marxianus strain was isolated from coffee wet-processing wastewater. PG production in this strain is not repressed in the presence
of 100g/L of glucose and, being growth-associated, reached its maximum accumulation in the culture medium at the beginning
of the stationary phase. Oxygen and galacturonic acid negatively regulated enzyme synthesis, and glucose as the carbon source
afforded better enzyme yields than lactose. The data reported here show that this strain exhibits the highest index of PG
production among the wild-type strains reported so far (18.8U/mL). PG was readily purified by ion-exchange chromatography
on SP-Sepharose FF. The activity corresponded to a single protein with an M
r of 41.7 kDa according to sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was stable in the pH range
of 3.0–5.0 and displayed an optimal temperature of 55°C; it showed a typical endo-splitting way of substrate hydrolysis and exhibited a fair degree of activity on pectin with a high degree of esterification. 相似文献
10.
Xin-Liang Li Lars G. Ljungdahl Eduardo A. Ximenes Huizhong Chen Carlos R. Felix Michael A. Cotta Bruce S. Dien 《Applied biochemistry and biotechnology》2004,113(1-3):233-250
A β-glucosidase (BglA, EC 3.2.1.21) gene from the polycentric anaerobic fungus Orpinomyces PC-2 was cloned and sequenced. The enzyme containing 657 amino acid residues was homologous to certain animal, plant, and
bacterial β-glucosidases but lacked significant similarity to those from aerobic fungi. Neither cellulose- nor protein-binding
domains were found in BglA. When expressed in Saccharomyces cerevisiae, the enzyme was secreted in two forms with masses of about 110 kDa and also found in two forms associated with the yeast
cells. K
m
and V
max
values of the secreted BglA were 0.762 mM and 8.20 μmol/(min·mg), respectively, with p-nitrophenyl-β-d-glucopyranoside (pNPG) as the substrate and 0.310 mM and 6.45 μmol/(min·mg), respectively, for the hydrolysis of cellobiose. Glucose competitively inhibited the hydrolysis of
pNPG with a K
i
of 3.6 mM. β-Glucosidase significantly enhanced the conversion of cellulosic materials into glucose by Trichoderma reesei cellulase preparations, demonstrating its potential for use in biofuel and feedstock chemical production.
Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard
of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may be
suitable. 相似文献
11.
Tomohiko Yamazaki Wakako Tsugawa Koji Sode 《Applied biochemistry and biotechnology》1999,77(1-3):325-335
We previously reported a novel glucose dehydrogenase (GDH) showing two peaks in the optimum temperature for the reaction at
around 45°C and at around 75°C. Each peak derived from hetero-oligomeric enzyme, constructed from two distinct peptides with
an α-subunit (MWs 67,000) and β-subunit (MWs 43,000), and a single peptide enzyme containing an α-subunit alone. The function
of the two subunits in the thermostable co-factor binding GDH was investigated. The results of spectroscopic analyses indicated
that the α-subunit contained an unknown co-factor showing specific fluorescence spectra like pyrroloquinoline quinone (PQQ),
and the β-subunit was cytochrome c. Moreover, the results of a urea denaturation and reconstitution experiment suggested that the dissociation of the hetero-oligomeric
complex to a single peptide was reversible. The kinetic parameter analyses for glucose and the electron mediator also suggested
that the β-subunit was responsible for electron transfer from the catalytic center of the α-subunit to the electron mediator. 相似文献
12.
Roland K. Robins Ganapathi R. Revankar Darrell E. O'Brien Robert H. Springer Thomas Novinson Anthony Albert Keitaro Senga Jon P. Miller David G. Streeter 《Journal of heterocyclic chemistry》1985,22(3):601-634
A number of new hypoxanthine analogs have been prepared as substrate inhibitors of xanthine oxidase. Most noteworthy inhibitory new hypoxanthine analogs are 3-(m-tolyl)pyrazolo[1,5-a]pyrimidin-7-one ( 47 ), ID50 0.06 μM and 3-phenylpyrazolo[1,5-a]pyrimidin-7-one ( 46 ), ID50 0.40 μM. 5-(p-Chlorophenyl)pyrazolo[1,5-a]pyrimidin-7-one ( 63 ) and the corresponding 5-nitrophenyl derivative 64 exhibited an ID50 of 0.21 and 0.23 μM, respectively. 7-Phenylpyrazolo[1,5-a]-s-triazin-4-one ( 40 ) is shown to exhibit an ID50 of 0.047 μM. The structure-activity relationships of these new phenyl substituted hypoxanthine analogs are discussed and compared with the xanthine analogs 3-m-tolyl- and 3-phenyl-7-hydroxypyrazolo[1,5-a]pyrimidin-5-ones ( 90 ) and ( 91 ), previously reported from our laboratory to have ID50 of 0.025 and 0.038 μM, respectively. The presence of the phenyl and substitutedphenyl groups contribute directly to the substrate binding of these potent inhibitors. This work presents an updated study of structure-activity relationships and binding to xanthine oxidase. In view of the recent elucidation of the pterin cofactor and the proposed binding of this factor to the molybdenum ion in xanthine oxidase, a detailed mechanism of xanthine oxidase oxidation of hypoxanthine and xanthine is proposed. Three types of substrate binding are viewed for xanthine oxidase. The binding of xanthine to xanthine oxidase is termed Type I binding. The binding of hypoxanthine is termed Type II binding and the specific binding of alloxanthine is assigned as Type III binding. These three types of substrate binding are analyzed relative to the most potent compounds known to inhibit xanthine oxidase and these inhibitors have been classified as to the type of inhibitor binding most likely to be associated with specific enzyme inhibition. The structural requirements for each type of binding can be clearly seen to correlate with the inhibitory activity observed. The chemical syntheses of the new 3-phenyl- and 3-substituted phenylpyrazolo[1,5-a]pyrimidines with various substituents are reported. The syntheses of various 8-phenyl-2-substituted pyrazolo-[1,5-a]-s-triazines, certain s-triazolo[1,5-a]-s-triazines and s-triazolo[1,5-a]pyrimidine derivatives prepared in connection with the present study are also described. 相似文献
13.
Rupesh KR Padma KR Saravanan P Jayachandran S 《Applied biochemistry and biotechnology》2007,141(2-3):161-174
Background: Seeds ofLathyrus sativus, a legume plant, contain 3-oxalyl and 2,3-dioxalyl DAP (O-DAP), neurotoxins which when consumed causes Neurolathyrism or
Osteolathyrism, in humans, affecting nervous system and bone formation respectively. Some microorganisms viz virulent and
non-virulentSalmonella typhimurium, Salmonella typhi and Pseudomonad have been shown to detoxifyL-α,β-diaminopropionate (DAP), the immediate precursor of O-DAP.
Result: The gene coding for diaminopropionate ammonia lyase (DAPAL) which detoxifies DAP was cloned from nonvirulentS. typhimurium PU011 intoEscherichia coli DH5α and the nucleotides sequenced (1212 bp). Whereas the specific enzyme activity of DAPAL obtained from recombinantE. coli PU018 was 0.346 U/mg, the specific activity of the enzyme from nonvirulentS. typhimurium PU011 was 0.351 U/mg. The DAPAL corresponding to 43 kDa protein was found both in nonvirulentS. typhimurium PU011 andE. coli PU018. The Km value was found to be 0.740 mM and 0.680 mM forS. typhimurium PU011 and 0.741 mM and 0.683 mM forE. coli PU018 when grown in minimal medium (MM+DAP) andL. sativus seed extracts respectively, indicating that both of them were capable of utilizing the neurotoxins present inL. sativus seeds. The biomass, enzyme production and the effect of pH and temperature on DAPAL enzyme activity from both non-virulentS. typhimurium PU011 andE. coli PU018 were found to be similar.
Conclusion: The recombinantE. coli PU018 as well as non-virulentS. typhimurium PU011 are as good as pathogenicS. typhimurium in detoxifying DAP, the immediate precursor of O-DAP present inL. sativus seeds. 相似文献
14.
Zafar MN Beden N Leech D Sygmund C Ludwig R Gorton L 《Analytical and bioanalytical chemistry》2012,402(6):2069-2077
In this study, different flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenases (FADGDHs) were characterized electrochemically
after “wiring” them with an osmium redox polymer [Os(4,4′-dimethyl-2,2′-bipyridine)2(PVI)10Cl]+ on graphite electrodes. One tested FADGDH was that recently discovered in Glomerella cingulata (GcGDH), another was the recombinant form expressed in Pichia pastoris (rGcGDH), and the third was a commercially available glycosylated enzyme from Aspergillus sp. (AspGDH). The performance of the Os-polymer “wired” GDHs on graphite electrodes was tested with glucose as the substrate. Optimal
operational conditions and analytical characteristics like sensitivity, linear ranges and current density of the different
FADGDHs were determined. The performance of all three types of FADGDHs was studied at physiological conditions (pH 7.4). The
current densities measured at a 20 mM glucose concentration were 494 ± 17, 370 ± 24, and 389 ± 19 μA cm−2 for GcGDH, rGcGDH, and AspGDH, respectively. The sensitivities towards glucose were 2.16, 1.90, and 1.42 μA mM−1 for GcGDH, rGcGDH, and AspGDH, respectively. Additionally, deglycosylated rGcGDH (dgrGcGDH) was investigated to see whether the reduced glycosylation would have an effect, e.g., a higher current density, which
was indeed found. GcGDH/Os-polymer modified electrodes were also used and investigated for their selectivity for a number of different sugars. 相似文献
15.
The direct bioelectrocatalysis was demonstrated for pyrroloquinoline quinone‐dependent glucose dehydrogenase (PQQ‐dependent GDH) covalently attached to single‐walled carbon nanotubes (SWNTs). The homogeneous ink‐like SWNT suspension was used for both creating the SWNT network on the microelectrode carbon surface and for enzyme immobilization. Functionalization of the SWNT surface by forming active ester groups was found to considerably enhance SWNT solubility in water with a range from 0.1 to 1.0 mg/mL. The PQQ‐dependent GDH immobilized on the surface of the SWNTs exhibited fast heterogeneous electron transfer with a rate constant of 3.6 s?1. Moreover, the immobilized PQQ‐dependent GDH retained its enzymatic activity for glucose oxidation. A fusion of PQQ‐dependent GDH with SWNTs has a great potential for the development of low‐cost and reagentless glucose sensors and biofuel cells. 相似文献
16.
Colm P. McMahon Sarah J. Killoran Robert D. ONeill 《Journal of Electroanalytical Chemistry》2005,580(2):193-202
The glucose sensitivity and oxygen dependence of a variety of implantable biosensors based on glucose oxidase (GOx), incorporating an electrosynthesized poly-o-phenylenediamine (PPD) permselective barrier on 125-μm diameter Pt disks (PtD) and cylinders (PtC, 1-mm length), were measured and compared. Full glucose calibrations and experimental monitoring of solution oxygen concentration allowed us to determine apparent Michaelis–Menten parameters for glucose and oxygen. In the linear region of glucose response, the most sensitive biosensor design studied was PtD/PPD/GOx (enzyme deposited over polymer) that was 20 times more sensitive than the more widely used PtC/GOx/PPD (enzyme immobilized before polymer deposition) configuration. The oxygen dependence, quantified as KM(O2), of both active and less active designs was surprisingly similar, a finding that could be rationalized in terms of an increase in KM(G) with increased enzyme loading. The PtD/PPD/GOx design will now enable us to explore glucose concentration dynamics in smaller and layered brain regions with good sensitivity and minimal interference from fluctuations in tissue pO2. 相似文献
17.
Helmut Schübel Joachim Stckigt Richard Feicht Helmut Simon 《Helvetica chimica acta》1986,69(3):538-547
A novel highly substrate-specific Rauwolfia enzyme, raucaffricine β-D -glucosidase, was isolated from cell-suspension cultures of R. serpentina. The enzyme has been purified ca. 1350-fold, its major characteristics such as Mr = 66600 ± 5%, pH optimum 5.1, temperature optimum 38°, and inhibition of its activity by glucose and fructose were investigated. Its limited distribution in different cell cultures and differentiated plants indicates that the enzyme is present in significant amounts exclusively in cultured Rauwolfia cells. 相似文献
18.
Stajic M Persky L Hadar Y Friesem D Duletic-Lausevic S Wasser SP Nevo E 《Applied biochemistry and biotechnology》2006,128(1):87-96
Copper (Cu2+) and manganese (Mn2+) ions influenced laccase (Lac) and peroxidase production in Pleurotus eryngii, Pleurotus ostreatus, and Pleurotus pulmonarius. In P. eryngii, the optimum Cu2+ concentration for Lac production was 1 mM and for peroxidases 10mM, and Mn2+ concentration of 5mM led to peaks of Lac and peroxidase activity. In P. ostreatus HAI 493, the highest level of Lac activity was at Cu2+ concentrations of 1 and 10 mM and Mn2+ concentration of 1mM, respectively. The absence of Cu2+ and Mn2+ caused the highest levels of peroxidase production. In P. ostreatus HAI 494, the highest level of Lac activity was at a Cu2+ concentration of 5 mM and at Mn2+ concentration of 1 mM, respectively. High levels of peroxidase activity were found in the medium without and with 1mM Cu2+, and at 1 and 5 mM Mn2+, respectively. In P. pulmonarius, the highest Lac activity was found in the presence of 5 mM Cu2+ and 5 mM Mn2+, respectively. The absence of Cu2+ and Mn2+ as well as their presence at a concentration of 1 mM led to the peaks of peroxidase activities. 相似文献
19.
Li Yang Jing Shi Cuijie Chen Shaoming Wang Liande Zhu Wenlin Xie Liping Guo 《Electrophoresis》2009,30(20):3527-3533
A new method for high‐sensitive determination of glutamate was developed and evaluated based on CE by using dual‐enzyme co‐immobilized capillary microreactor combined with substrate recycling. The capillary microreactor was prepared by covalently co‐immobilizing glutamate dehydrogenase (GDH) and glutamic pyruvic transaminase (GPT) on the inner surface of a capillary and was characterized by SEM, ultraviolet‐visible spectroscopy, and fluorescence spectroscopy. The GDH‐GPT co‐immobilized capillary microreactor showed great stability and reproducibility. The apparent Km for glutamate with GDH‐GPT coupled reaction was determined to be 0.61±0.06 mM but 2.56±0.24 mM when only GDH was immobilized. Glutamate determination was based on on‐column monitoring UV absorption at 340 nm of the reaction product reduced nicotinamide adenine dinucleotide, of which peak area was directly related to the glutamate concentration. The response of the present co‐immobilized GDH‐GPT assay for glutamate is greatly enhanced over single enzyme system, and a 15.7‐fold improvement in sensitivity was obtained. The detection limit of the proposed method is 0.15 μM glutamate (S/N=3). Selectivity for glutamate is good over most of the 20 amino acids. Finally, this method was successfully applied to determine the glutamate content in rat plasma and serum samples. 相似文献
20.
Rey MW Brown KM Golightly EJ Fuglsang CC Nielsen BR Hendriksen HV Butterworth A Xu F 《Applied biochemistry and biotechnology》2003,111(3):153-166
Thielavia terrestris is a soil-borne thermophilic fungus whose molecular/cellular biology is poorly understood. Only a few genes have been cloned
from the Thielavia genus. We detected an extracellular glucoamylase in culture filtrates of T. terrestris and cloned the corresponding glaA gene. The coding region contains five introns. Based on the amino acid sequence, the glucoamylase was 65% identical to Neurospora crassa glucoamylase. Sequence comparisons suggested that the enzyme belongs to the glycosyl hydrolase family 15. The T. terrestris glaA gene was expressed in Aspergillus oryzae under the control of an A. oryzae α-amylase promoter and an Aspergillus niger glucoamylase terminator. The 75-kDa recombinant glucoamylase showed a specific activity of 2.8 μmol/(min·mg) with maltose
as substrate. With maltotriose as a substrate, the enzyme had an optimum pH of 4.0 and an optimum temperature of 60°C. The
enzyme was stable at 60°C for 30 min. The K
m
and k
cat
of the enzyme for maltotriose were determined at various pHs and temperatures. At 20°C and pH 4.0, the enzyme had a K
m
of 0.33±0.07 mM and a k
cat
of (5.5±0.5)×103 min−1 for maltotriose. The temperature dependence of k
cat
/K
m
indicated an activation free energy of 2.8 kJ/mol across the range of 20–70°C. Overall, the enzyme derived from the thermophilic
fungus exhibited properties comparable with that of its homolog derived from mesophilic fungi. 相似文献