共查询到20条相似文献,搜索用时 46 毫秒
1.
Kamila Rzeznicka Sebastian Schätzle Dominique Böttcher Joachim Klein Uwe T. Bornscheuer 《Applied microbiology and biotechnology》2010,85(5):1417-1425
The nitrile hydratase (NHase, EC 4.2.1.84) genes (α and β subunit) and the corresponding activator gene from Rhodococcus equi TG328-2 were cloned and sequenced. This Fe-type NHase consists of 209 amino acids (α subunit, Mr 23 kDa) and 218 amino acids (β subunit, Mr 24 kDa) and the NHase activator of 413 amino acids (Mr 46 kDa). Various combinations of promoter, NHase and activator genes were constructed to produce active NHase enzyme recombinantly
in E. coli. The maximum enzyme activity (844 U/mg crude cell extract towards methacrylonitrile) was achieved when the NHase activator
gene was separately co-expressed with the NHase subunit genes in E. coli BL21 (DE3). The overproduced enzyme was purified with 61% yield after French press, His-tag affinity chromatography, ultrafiltration
and lyophilization and showed typical Fe-type NHase characteristics: besides aromatic and heterocyclic nitriles, aliphatic
ones were hydrated preferentially. The purified enzyme had a specific activity of 6,290 U/mg towards methacrylonitrile. Enantioselectivity
was observed for aromatic compounds only with E values ranging 5–17. The enzyme displayed a broad pH optimum from 6 to 8.5, was most active at 30°C and showed the highest
stability at 4°C in thermal inactivation studies between 4°C and 50°C. 相似文献
2.
Two chimeric genes, XynA-Bs-Glu-1 and XynA-Bs-Glu-2, encoding Aspergillus sulphureus β-xylanase (XynA, 26 kDa) and Bacillus subtilis β-1,3-1,4-glucanase (Bs-Glu, 30 kDa), were constructed via in-fusion by different linkers and expressed successfully in Pichia pastoris. The fusion protein (50 kDa) exhibited both β-xylanase and β-1,3-1,4-glucanase activities. Compared with parental enzymes, the moiety activities were decreased in fermentation supernatants. Parental XynA and Bs-Glu were superior to corresponding moieties in each fusion enzymes because of lower Kn higher kcat. Despite some variations, common optima were generally 50°C and pH 3.4 for the XynA moiety and parent, and 40°C and pH 6.4 for the Bs-Glu counterparts. Thus, the fusion enzyme XynA-Bs-Glu-1 and XynA-Bs-Glu-2 were bifunctional. 相似文献
3.
For the first time, a β-glucosidase gene from the edible straw mushroom, Volvariella volvacea V1-1, has been over-expressed in E. coli. The gene product was purified by chromatography showing a single band on SDS-PAGE. The recombinant enzyme had a molecular
mass of 380 kDa with subunits of 97 kDa. The maximum activity was at pH 6.4 and 50 °C over a 5 min assay. The purified enzyme
was stable from pH 5.6–8.0, had a half life of 1 h at 45 °C. The β-glucosidase had a Km of 0.2 mM for p-nitrophenyl-β-D-glucopyranoside. 相似文献
4.
Yoshihiro Hakamada Kenzo Koike Tadashi Yoshimatsu Hajime Mori Tohru Kobayashi Susumu Ito 《Extremophiles : life under extreme conditions》1997,1(3):151-156
Thermostable alkaline cellulase (endo-1,4-β-glucanase, EC 3.2.1.4) activity was detected in the culture medium of a strictly
alkaliphilic strain of Bacillus, designated KSM-S237. This novel enzyme was purified to homogeneity by a two-step column-chromatographic procedure with high
yield. The N-terminal amino acid sequence of the purified enzyme was Glu-Gly-Asn-Thr-Arg-Glu-Asp-Asn-Phe-Lys-His-Leu-Leu-Gly-Asn-Asp-Asn-Val-Lys-Arg.
The enzyme had a molecular mass of approximately 86 kDa and an isoelectric point of pH 3.8. The enzyme had a pH optimum of
8.6–9.0 and displayed maximum activity at 45°C. The alkaline enzyme was stable up to 50°C and more than 30% of the original
activity was detectable after heating at 100°C and at pH 9.0 for 10 min. The enzyme hydrolyzed carboxymethylcellulose, lichenan
(β-1,3;1,4-linkage), and p-nitrophenyl derivatives of cellotriose and cellotetraose. Crystalline forms of cellulose (Avicel and filter paper), H3PO4-swollen cellulose, NaOH-swollen cellulose, curdlan (β-1,3-linkage), laminarin (β-1,3;1,6-linkage), and xylan were barely
hydrolyzed at all.
Received: April 28, 1997 / Accepted: May 24, 1997 相似文献
5.
A C S Rizzatti J A Jorge H F Terenzi C G V Rechia M L T M Polizeli 《Journal of industrial microbiology & biotechnology》2001,26(3):156-160
A β-D-xylosidase was purified from cultures of a thermotolerant strain of Aspergillus phoenicis grown on xylan at 45°C. The enzyme was purified to homogeneity by chromatography on DEAE-cellulose and Sephadex G-100. The
purified enzyme was a monomer of molecular mass 132 kDa by gel filtration and SDS-PAGE. Treatment with endoglycosidase H resulted
in a protein with a molecular mass of 104 kDa. The enzyme was a glycoprotein with 43.5% carbohydrate content and exhibited
a pI of 3.7. Optima of temperature and pH were 75°C and 4.0–4.5, respectively. The activity was stable at 60°C and had a K
m of 2.36 mM for p-nitrophenyl-β-D-xylopiranoside. The enzyme did not exhibit xylanase, cellulase, galactosidase or arabinosidase activities. The purified enzyme
was active against natural substrates, such as xylobiose and xylotriose. Journal of Industrial Microbiology & Biotechnology (2001) 26, 156–160.
Received 23 June 2000/ Accepted in revised form 29 September 2000 相似文献
6.
The expression of the Arabidopsis heat-shock protein (HSP) 18.2 promoter β-d-glucuronidase (GUS) chimera gene was studied in various organs of the transgenic Nicotiana plumbaginifolia during the recovery phase at normal temperatures (20–22 °C) following heat-shock (HS) treatment. The optimum HS temperature
for GUS activity in the anthers, petals and capsules was 42 °C, but in immature seeds and the placentas of capsules it was
36 °C and 39 °C, respectively. No apparent GUS activity was observed in any organs except for dry seeds after HS at 45 °C,
although the activity in dry seeds was apparent even after HS at 48 °C. After HS at 42 °C, GUS activity in the flower tissues
was the highest before anthesis and declined thereafter.
Received: 13 January 1998 / Revision received: 25 January 1999 / Accepted: 3 March 1999 相似文献
7.
Purification and partial characterization οf a new β-xylosidase from
Humicola grisea var. thermoidea
T. Iembo M.O. Azevedo C. Bloch Jr. E.X.F. Filho 《World journal of microbiology & biotechnology》2006,22(5):475-479
Summary The thermophilic fungus Humicola grisea var. thermoidea produces a mycelium-associated β-xylosidase activity when grown in liquid-state cultures on media containing oat spelt xylan
as the carbon source. The β-xylosidase was purified to apparent homogeneity by gel filtration and anion exchange chromatography.
Its molecular weight was 37 and 50 kDa, as determined by MALDI/TOF mass spectrometry and SDS-PAGE, respectively. The purified
enzyme exhibited maximum activity at 55 °C and pH 6.5. It was also active at pH 8.8, retaining 60% of its activity after 6 h
of incubation at 50 °C. β-xylosidase was strongly inactivated by NBS and slightly activated by DTT and β-mercaptoethanol.
The enzyme was highly specific for PNPX as the substrate. The purified β-xylosidase showed K
m and V
max values of 1.37 mM and 12.98 IU ml−1, respectively. 相似文献
8.
Sonia Ben Hadj Kalifa Ferid Limam M. Issam Smaali Thierry Maugard M. Nejib Marzouki 《World journal of microbiology & biotechnology》2007,23(10):1363-1370
The β-glucosidase enzyme β-glu2 isolated from Sclerotinia sclerotiorum was purified and used as tracer in enzyme linked immunosorbent assay. A novel purification procedure of the protein was developed
that consists of ammonium sulphate precipitation, gel filtration on Sephacryl S-200-HR column, ion exchange chromatography
on DEAE-Toyopearl and polybuffer exchanger PBE 94 TM chromatofocusing. The pI value was 4.45. K
m
and V
max values for the enzyme towards p-nitrophenyl-β-D-glucopyranoside were respectively 0.45 mM and 0.2 U/mL. Thermal stability showed that β-glu2 has a half-life
of 85 min at 55 °C and of 25 min at 65 °C. β-glu2 was conjugated to goat anti-rabbit antibodies with glutaraldehyde as cross
linking agent according to the one-step method. Conjugates were purified by HPLC gel filtration on TSK 2000. Enzymatic and
immunological activities of the β-glucosidase conjugate component were tested by the ELISA method. 相似文献
9.
The high-molar mass from of β-glucosidase fromAspergillus niger strain NIAB280 was purified to homogeneity with a 46-fold increase in purification by a combination of ammonium sulfate precipitation,
hydrophobic interaction, ion-exchange and gel-filtration chromatography. The native and subunit molar mass was 330 and 110
kDa, respectively. The pH and temperature optima were 4.6–5.3 and 70°C, respectively. TheK
m andk
cat for 4-nitrophenyl β-d-glucopyranoside at 40°C and pH 5 were 1.11 mmol/L and 4000/min, respectively. The enzyme was activated by low and inhibited
by high concentrations of NaCl. Ammonium sulfate inhibited the enzyme. Thermolysin periodically inhibited and activated the
enzyme during the course of reaction and after 150 min of proteinase treatment only 10% activity was lost with concomitant
degradation of the enzyme into ten low-molar-mass active bands. When subjected to 0–9 mol/L transverse urea-gradient-PAGE
for 105 min at 12°C, the nonpurified β-glucosidase showed two major bands which denatured at 4 and 8 mol/L urea, respectively,
with half-lives of 73 min. 相似文献
10.
Xylaria regalis, a wood-grown ascomycete isolated in Taiwan, produces β-glucosidase (EC 3.2.1.21) extracellularly. The β-glucosidase was
purified to homogeneity by ammonium sulfate precipitation, ion-exchange, and gel filtration chromatography. The molecular
mass of the purified enzyme was estimated to be 85 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. With
p-nitrophenyl β-D-glucopyranoside (PNPG) as the substrate at pH 5.0 and 50°C, the K
m
was 1.72 mM and V
max
was 326 μmol/min/mg. Optimal activity with PNPG as the substrate was at pH 5.0 and 50°C. The enzyme was stable at pH 5.0
at temperatures up to 50°C. The purified β-glucosidase was active against PNPG, cellobiose, sophorose, and gentiobiose, but
did not hydrolyze lactose, sucrose, Avicel, and o-nitrophenyl β-D-galactopyranoside. The activity of β-glucosidase was stimulated by Ca2+, Mg2+, Mn2+, Cd2+ and β-mercaptoethanol, and inhibited by Ag+, Hg2+, SDS, and p-chloromercuribenzoate (PCMB).
Received: 30 March 1996 / Accepted: 3 May 1996 相似文献
11.
Beatriz Galán Concepción García Mendoza Myriam Calonje Monique Novaes-Ledieu 《Current microbiology》1999,38(3):190-193
Agaricus bisporus H 25 produced extracellular endo-1,3-β-glucanase when grown in a static culture at 25°C in a minimal synthetic medium supplemented
with A. bisporus cell walls plus fructose. Endo-1,3-β-glucanase was purified 17.85-fold from 20-day-old culture filtrates by precipitation
at 80% ammonium sulfate saturation, Sephadex G-75 gel filtration, and preparative PAGE followed by electroelution. The purified
enzyme yielded a single band in both native and SDS-polyacrylamide gels with a molecular mass of 32 kDa (SDS-PAGE) and 33.7
kDa (MALDI-MS), showing an isoelectric point of 3.7. The enzyme was active against β-1,3- linkages and, to a lesser extent,
against β-1,6-, exhibiting an endohydrolytic mode of action and a glycoprotein nature. Significant activities of the endo-glucanase
against laminarin and pustulan were observed between pH 4 and 5.5, and between 40° and 50°C for laminarin, and between 30°
and 50°C for pustulan. The optimum pH and temperature were 4.5 and 45°C for both substrates.
Received: 17 June 1998 / Accepted: 24 September 1998 相似文献
12.
Hong SY Lee JS Cho KM Math RK Kim YH Hong SJ Cho YU Cho SJ Kim H Yun HD 《Biotechnology letters》2007,29(6):931-936
An artificial bifunctional enzyme, cellulase-β-glucosidase, was prepared by gene fusion from the hyperthermophilic bacterium
Thermotoga maritima MSB8. The fusion protein exhibited both cellulase (Cel5C) and β-glucosidase (BglB) activity when the bglB gene was fused to downstream of cel5C, but not when cel5C was fused to downstream of bglB. The specific activity of the bifunctional enzyme was 70% lower than that of cellulase or β-glucosidase. The fusion enzyme
was purified, and the MW was estimated as 114 kDa. The fusion enzyme displayed optimum cellulase activity at pH 8.0 and 70°C
over 30 min, and optimal β-glucosidase activity at pH 7.0 and 80°C over 30 min. 相似文献
13.
The recombinant β-carotene 15,15′-monooxygenase from chicken liver was purified as a single 60 kDa band by His-Trap HP and Resource Q chromatography.
It had a molecular mass of 240 kDa by gel filtration indicating the native form to be tetramer. The enzyme converted β-carotene under maximal conditions (pH 8.0 and 37°C) with a k
cat of 1.65 min−1 and a K
m of 26 μM and its conversion yield of β-carotene to retinal was 120% (mol mol−1). The enzyme displayed catalytic efficiency and conversion yield for β-carotene, β-cryptoxanthin, β-apo-8′-carotenal, β-apo-4′-carotenal, α-carotene and γ-carotene in decreasing order but not for zeaxanthin, lutein, β-apo-12′-carotenal and lycopene, suggesting that the presence of one unsubstituted β-ionone ring in a substrate with a molecular weight greater than C30 seems to be essential for enzyme activity. 相似文献
14.
Pyruvate:ferredoxin oxidoreductase was purified to electrophoretic homogeneity from an aerobic, thermophilic, obligately
chemolithoautotrophic, hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, by precipitation with ammonium sulfate and fractionation by DEAE-Sepharose CL-6B, polyacrylate-quaternary amine, hydroxyapatite,
and Superdex-200 chromatography. The native enzyme had a molecular mass of 135 kDa and was composed of four different subunits
with apparent molecular masses of 46, 31.5, 29, and 24.5 kDa, respectively, indicating that the enzyme has an αβγδ-structure.
The activity was detected with pyruvate, coenzyme A, and one of the following electron acceptors in substrate amounts: ferredoxin
isolated from H. thermophilus, FAD, FMN, triphenyltetrazolium chloride, or methyl viologen. NAD, NADP, and ferredoxins from Chlorella spp. and Clostridium pasteurianum were ineffective as the electron acceptor. The temperature optimum for pyruvate oxidation was approximately 80° C. The pH
optimum was 7.6–7.8. The apparent K
m values for pyruvate and coenzyme A at 70° C were 3.45 mM and 54 μM, respectively. The enzyme was extremely thermostable under
anoxic conditions; the time for a 50% loss of activity (t
50%) at 70° C was approximately 8 h.
Received: 9 September 1996 / Accepted: 27 December 1996 相似文献
15.
The invertase of Lactobacillus reuteri CRL 1100 is a glycoprotein composed by a single subunit with a molecular weight of 58 kDa. The enzyme was stable below 45°C
over a wide pH range (4.5–7.0) with maximum activity at pH 6.0 and 37°C. The invertase activity was significantly inhibited
by bivalent metal ions (Ca++, Cu++, Cd++, and Hg++), β-mercaptoethanol, and dithiothreitol and partially improved by ethylenediaminetetraacetic acid. The enzyme was purified
32 times over the crude extract by gel filtration and ion-exchange chromatography with a recovery of 17%. The K
m
and Vmax values for sucrose were 6.66 mM and 0.028 μmol/min, respectively. An invertase is purified and characterized for the first
time in Lactobacillus, and it proved to be a β-fructofuranosidase.
Received: 13 August 1999 / Accepted: 15 September 1999 相似文献
16.
Sumra Afzal Mahjabeen Saleem Riffat Yasmin Mamoona Naz Muhammad Imran 《Molecular biology reports》2010,37(4):1717-1723
Consistent with its precloning characterization from the cellulolytic Bacillus sp., β-1,4-endoglucanase purified from the recombinant E. coli exhibited maximum activity at 60°C and pH 7.0. It was highly specific for CMC hydrolysis, with stability up to 70°C and over
a pH range of 6.0–8.0. The K
m and V
max values for CMCase activity of the enzyme were 4.1 mg/ml and 25 μmole/ml min−1, respectively. The purified enzyme was a monomer of 65 kDa, as determined by SDS-PAGE. The presence of sucrose and IPTG in
fermentation media increased the endoglucanase activity of the recombinant enzyme to 5.2-folds as compared with that of the
actual one. 相似文献
17.
An operon, bglABC, that encodes two sugar permeases and a β-glucosidase was cloned from a cellulolytic actinomycete, Thermobifida fusca, into Escherichia coli and sequenced. The bglC gene encoding an intracellular β-glucosidase (β-d-glucoside glucohydrolase, EC 3.2.1.21) belonging to glycosyl hydrolase
family 1 was subcloned and expressed in E. coli. The purified enzyme (MW 53,407 Da; pI 4.69) hydrolyzed substrates containing both β 1 → 4 and β 1 → 2 glycosidic bonds,
and was most active against cellobiose (Vmax= 29, K
m
= 0.34 mm), cellotriose, cellotetraose, and sophorose. The enzyme also showed aryl-β-glucosidase activity on p-nitrophenyl-β-d-glucopyranoside and p-nitrophenyl-β-d-cellobioside. BglC had a pH optimum of 7.0 and a temperature optimum
of 50°C. The enzyme was stable at 60°C, but was rapidly inactivated at 65°C. BglC was inhibited by low concentrations of gluconolactone,
but was insensitive to end-product inhibition by glucose and was not affected by Ca or Mg ions or EDTA. Its properties are
well suited for use in a process to hydrolyze biomass cellulose to glucose.
Received: 21 August 2000 / Accepted: 4 October 2000 相似文献
18.
S Hayashi S Sako H Yokoi Y Takasaki K Imada 《Journal of industrial microbiology & biotechnology》1999,22(3):160-163
β-Glucosidase hydrolyzing cellobiose was extracted from Aureobasidium sp ATCC 20524 and purified to homogeneity. The molecular mass was estimated to be about 331 kDa. The enzyme contained 26.5%
(w/w) carbohydrate. The optimum pH and temperature for the enzyme reaction were pH 4 and 80°C, respectively. The enzyme was
stable at a wide range of pH, 2.2–9.8, after 3 h and at 75°C for 15 min. The kinetic parameters were determined. The enzyme
was relatively stable against typical organic enzyme inhibitors. The enzyme also hydrolyzed gentiobiose, p-nitrophenyl-β-glucoside and salicin.
Received 05 November 1998/ Accepted in revised form 14 February 1999 相似文献
19.
Hayashi S Ohno T Ito M Yokoi H 《Journal of industrial microbiology & biotechnology》2001,26(5):276-279
β-Xylosidase was extracted from Aureobasidium sp. ATCC 20524 and purified to homogeneity. The molecular mass was estimated at 411 kDa. The enzyme contained 15.3% (w/w)
carbohydrate. The optimum pH and temperature were pH 3.5 and 80°C, respectively. The enzyme was stable at pH 3.5–9 after 3
h and at 80°C after 15 min. The Michaelis constant (K
m) and maximum velocity (V
max) toward p-nitrophenyl-β-D-xyloside were 2.0 mmol l−1 and 0.94 mmol min−1 mg−1 protein, respectively. The enzyme was inhibited strongly by mercury, lead, and copper ions. Journal of Industrial Microbiology & Biotechnology (2001) 26, 276–279.
Received 02 August 2000/ Accepted in revised form 15 December 2000 相似文献
20.
Moreland D. Gibbs Rosalind A. Reeves Anwar Sunna Peter L. Bergquist 《Current microbiology》1999,39(6):351-357
A β-mannanase gene (manA) was isolated from the extremely thermophilic bacterium Dictyoglomus thermophilum Rt46B.1. ManA is a single-domain enzyme related to one group of β-mannanases (glycosyl hydrolase family 26). The manA gene was expressed in the heat-inducible vector pJLA602 and the expression product, ManA, purified to homogeneity. The recombinant
ManA is a monomeric enzyme with a molecular mass of 40 kDa and an optimal temperature and pH for activity of 80°C and 5.0.
In the absence of substrate, the enzyme showed no loss of activity at 80°C over 16 h, while at 90°C the enzyme had a half-life
of 5.4 min. Hydrolysis of the galactomannan locust bean gum (LBG) by purified ManA released mainly mannose, mannobiose, and
mannotriose, confirming that ManA is an endo-acting β-mannanase. Sequence comparisons with related β-mannanases has allowed
the design of consensus PCR primers for the identification and isolation of related genes.
Received: 7 June 1999 / Accepted: 6 July 1999 相似文献