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1.
S Chakraborti R K Sani U C Banerjee R C Sobti 《Journal of industrial microbiology & biotechnology》2000,24(1):58-63
An extracellular β-galactosidase which catalyzed the production of galacto-oligosaccharide from lactose was harvested from
the late stationary-phase of Bacillus sp MTCC 3088. The enzyme was purified 36.2-fold by ZnCl2 precipitation, ion exchange, hydrophobic interaction and gel filtration chromatography with an overall recovery of 12.7%.
The molecular mass of the purified enzyme was estimated to be about 484 kDa by gel filtration on a Sephadex G-200 packed column
and the molecular masses of the subunits were estimated to be 115, 86.5, 72.5, 45.7 and 41.2 kDa by sodium dodecyl sulfate-polyacrylamide
gel electrophoresis. The isoelectric point of the native enzyme, determined by polyacrylamide gel electrofocusing, was 6.2.
The optimum pH and temperature were 8 and 60°C, respectively. The Michaelis–Menten constants determined with respect to o-NO2-phenyl-β-D-galactopyranoside and lactose were 6.34 and 6.18 mM, respectively. The enzyme activity was strongly inhibited (68%) by galactose,
the end product of lactose hydrolysis reaction. The β-galactosidase was specific for β-D anomeric linkages. Enzyme activity was significantly inhibited by metal ions (Hg2+, Cu2+ and Ag+) in the 1–2.5 mM range. Mg2+ was a good activator. Catalytic activity was not affected by the chelating agent EDTA. Journal of Industrial Microbiology & Biotechnology (2000) 24, 58–63.
Received 09 February 1999/ Accepted in revised form 24 September 1999 相似文献
2.
Some properties of purified endo-l,4-β-D-xylanase (1,4-β-D-xylan xylanohydrolase, EC 3.2.1.8) from the ligniperdous fungusTrametes hirsuta were investigated. The enzyme was stable between pH 4.0 and 8.0 with optimum activity at pH 5.0–5.5. The temperature optimum
was 50 °C and the enzyme was stable for up to 30 min at 45 °C; however, it was denatured at higher temperatures. TheK
m for 4-O-methylgluourono-D-xylan was 6.36. 10−3 equivalents ofD-xylose per litre, the activation energy was 28 kJ mol−1. The molecular weight determined by means of gel chromatography was 22000–24000. The enzyme was activated by Ca2+ and inhibited by Ag+ and Hg2+. On the basis of the effect of 2-hy-droxy-5 nitrobenzyl bromide, N-bromosuccimmide and N-aeetyhmidazole it may be assumed
that trytophan and possibly tyrosine residues influence the enzyme catalysis. 相似文献
3.
Jin Zhou Ju Chu Yong-Hong Wang Si-Liang Zhang Ying-Ping Zhuang Zhong-Yi Yuan 《World journal of microbiology & biotechnology》2008,24(6):789-796
An intracellular S-adenosylmethionine synthetase (SAM-s) was purified from the fermentation broth of Pichia pastoris GS115 by a sequence chromatography column. It was purified to apparent homogeneity by (NH4)2SO4 fractionation (30–60%), anion exchange, hydrophobic interaction, anion exchange and gel filtration chromatography. HPLC showed
the purity of purified SAM-s was 91.2%. The enzyme was purified up to 49.5-fold with a final yield of 20.3%. The molecular
weight of the homogeneous enzyme was 43.6 KDa, as determined by electro-spray ionization mass spectrometry (ESI-MS). Its isoelectric
point was approximately 4.7, indicating an acidic character. The optimum pH and temperature for the enzyme reaction were 8.5
and 35 °C, respectively. The enzyme was stable at pH 7.0–9.0 and was easy to inactivate in acid solution (pH ≤ 5.0). The temperature
stability was up to 45 °C. Metal ions, such as, Mn2+ and K+ at the concentration of 5 mM had a slight activation effect on the enzyme activity and the Mg2+ activated the enzyme significantly. The enzyme activity was strongly inhibited by heavy metal ions (Cu2+ and Ag2+) and EDTA. The purified enzyme from the transformed Pichia pastoris synthesized S-adenosylmethionine (SAM) from ATP and l-methionine in vitro with a K
m of 120 and 330 μM and V
max of 8.1 and 23.2 μmol/mg/min for l-methionine and ATP, respectively. 相似文献
4.
Asiya Nazir Rohit Soni H. S. Saini R. K. Manhas B. S. Chadha 《World journal of microbiology & biotechnology》2009,25(7):1189-1197
An endoglucanase (1, 4-β-d glucan glucanohydrolase, EC 3.2.1.4) which was catalytically more active and exhibited higher affinity towards barley β-glucan,
xyloglucan and lichenin as compared to carboxymethylcellulose (CMC) was purified from Aspergillus terreus strain AN1 following ion-exchange and hydrophobic interaction chromatography and gel filtration. The purified enzyme (40-fold) that
apparently lacked a cellulose-binding domain showed a specific activity of 60 μmol mg−1 protein−1 against CMC. The purified enzyme had a molecular weight of 78 and 80 KDa as indicated by sodium dodecyl sulphate–polyacrylamide
gel electrophoresis and gel filtration, respectively, and a pI of 3.5. The enzyme was optimally active at temperature 60°C
and pH 4.0, and was stable over a broad range of pH (3.0–5.0) at 50°C. The endoglucanase activity was positively modulated
in the presence of Cu2+, Mg2+, Ca2+, Na+, DTT and mercaptoethanol. Endoglucanase exhibited maximal turn over number (K
cat) and catalytic efficiency (K
cat/km) of 19.11 × 105 min−1 and 29.7 × 105 mM−1 min−1 against barley β-glucan as substrate, respectively. Hydrolysis of CMC and barley β-glucan liberated cellobiose, cellotriose,
cellotetraose and detectable amount of glucose. The hydrolysis of xyloglucan, however, apparently yielded positional isomers
of cellobiose, cellotriose and cellotetraose as well as larger oligosaccharides. 相似文献
5.
Tony Marcio da Silva Alexandre Maller André Ricardo de Lima Damásio Michele Michelin Richard John Ward Izaura Yoshico Hirata João Atilio Jorge Héctor Francisco Terenzi Maria Lourdes T. M. de Polizeli 《Journal of industrial microbiology & biotechnology》2009,36(12):1439-1446
A glucoamylase from Aspergillus niveus was produced by submerged fermentation in Khanna medium, initial pH 6.5 for 72 h, at 40°C. The enzyme was purified by DEAE-Fractogel
and Concanavalin A-Sepharose chromatography. The enzyme showed 11% carbohydrate content, an isoelectric point of 3.8 and a
molecular mass of 77 and 76 kDa estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or Bio-Sil-Sec-400
gel filtration, respectively. The pH optimum was 5.0–5.5, and the enzyme remained stable for at least 2 h in the pH range
of 4.0–9.5. The temperature optimum was 65°C and retained 100% activity after 240 min at 60°C. The glucoamylase remained completely
active in the presence of 10% methanol and acetone. After 120 min hydrolysis of starch, glucose was the unique product formed,
confirming that the enzyme was a glucoamylase (1,4-alpha-d-glucan glucohydrolase). The K
m was calculated as 0.32 mg ml−1. Circular dichroism spectroscopy estimated a secondary structure content of 33% α-helix, 17% β-sheet and 50% random structure,
which is similar to that observed in the crystal structures of glucoamylases from other Aspergillus species. The tryptic peptide sequence analysis showed similarity with glucoamylases from A. niger, A. kawachi,
A. ficcum, A. terreus, A. awamori and A. shirousami. We conclude that the reported properties, such as solvent, pH and temperature stabilities, make A. niveus glucoamylase a potentially attractive enzyme for biotechnological applications. 相似文献
6.
Chin-An Hsu Roch-Chui Yu Cheng-Chun Chou 《World journal of microbiology & biotechnology》2006,22(4):355-361
Summary β-galactosidase from Bifidobacterium longum CCRC 15708 was first extracted by ultrasonication then purified by Q Fast-Flow chromatography and gel chromatography on a
Superose 6 HR column. These steps resulted in a purification of 15.7-fold, a yield of 29.3%, and a specific activity of 168.6 U mg−1 protein. The molecular weight was 357 kDa as determined from Native-PAGE. Using o-nitrophenyl-β-d-galactopyranoside (ONPG) as a substrate, the pH and temperature optima of the purified β-galactosidase were 7.0 and 50 °C,
respectively. The enzyme was stable at a temperature up to 40 °C and at pH values of 6.5–7.0. K
m and V
max for this purified enzyme were noted to be 0.85 mM and 70.67 U/mg, respectively. Na+ and K+ stimulated the enzyme up to 10-fold, while Fe3+, Fe2+, Co2+, Cu2+, Ca2+, Zn2+, Mn2+ and Mg2+ inhibited the activity of β-galactosidase. Furthermore, although glucose, galactose, maltose, or raffinose exerted little
or no effect on the β-galactosidase activity, lactose and fructose inhibited the enzyme activity. The effect of lactose on
the enzyme activity for ONPG is probably a case of competitive inhibition.
A relatively high specific activity of β-galactosidase from B. longum CCRC 15708 could be obtained by Q Fast-Flow chromatography and gel chromatography on a Superose 6 HR column. In some aspects,
particularly the activation by monovalent cations, the properties of β-galactosidase of B. longum CCRC 15708 are different from those obtained from other sources.
Data collected in the present study are of value and indispensable when β-galactosidase from B. longum CCRC 15708 is employed in practical application. 相似文献
7.
Uchima CA Tokuda G Watanabe H Kitamoto K Arioka M 《Applied microbiology and biotechnology》2011,89(6):1761-1771
Neotermes koshunensis is a lower termite that secretes endogenous β-glucosidase in the salivary glands. This β-glucosidase (G1NkBG) was successfully
expressed in Aspergillus oryzae. G1NkBG was purified to homogeneity from the culture supernatant through ammonium sulfate precipitation and anion exchange,
hydrophobic, and gel filtration chromatographies with a 48-fold increase in purity. The molecular mass of the native enzyme
appeared as a single band at 60 kDa after gel filtration analysis, indicating that G1NkBG is a monomeric protein. Maximum
activity was observed at 50 °C with an optimum pH at 5.0. G1NkBG retained 80% of its maximum activity at temperatures up to
45 °C and lost its activity at temperatures above 55 °C. The enzyme was stable from pH 5.0 to 9.0. G1NkBG was most active
towards laminaribiose and p-nitrophenyl-β-d-fucopyranoside. Cellobiose, as well as cello-oligosaccharides, was also well hydrolyzed. The enzyme activity was slightly
stimulated by Mn2+ and glycerol. The K
m and V
max values were 0.77 mM and 16 U/mg, respectively, against p-nitrophenyl-β-d-glucopyranoside. An unusual finding was that G1NkBG was stimulated by 1.3-fold when glucose was present in the reaction mixture
at a concentration of 200 mM. These characteristics, particularly the stimulation of enzyme activity by glucose, make G1NkBG
of great interest for biotechnological applications, especially for bioethanol production. 相似文献
8.
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
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.
Chengwei Hua Qiaojuan Yan Zhengqiang Jiang Yinan Li Priti Katrolia 《Applied microbiology and biotechnology》2010,88(2):509-518
In this study, a novel β-1,3-1,4-glucanase gene (designated as PtLic16A) from Paecilomyces thermophila was cloned and sequenced. PtLic16A has an open reading frame of 945 bp, encoding 314 amino acids. The deduced amino acid sequence shares the highest identity
(61%) with the putative endo-1,3(4)-β-glucanase from Neosartorya fischeri NRRL 181. PtLic16A was cloned into a vector pPIC9K and was expressed successfully in Pichia pastoris as active extracellular β-1,3-1,4-glucanase. The recombinant β-1,3-1,4-glucanase (PtLic16A) was secreted predominantly into
the medium which comprised up to 85% of the total extracellular proteins and reached a protein concentration of 9.1 g l−1 with an activity of 55,300 U ml−1 in 5-l fermentor culture. The enzyme was then purified using two steps, ion exchange chromatography, and gel filtration chromatography.
The purified enzyme had a molecular mass of 38.5 kDa on SDS–PAGE. It was optimally active at pH 7.0 and a temperature of 70°C.
Furthermore, the enzyme exhibited strict specificity for β-1,3-1,4-d-glucans. This is the first report on the cloning and expression of a β-1,3-1,4-glucanase gene from Paecilomyces sp. 相似文献
11.
S M Kotwal M M Gote M I Khan J M Khire 《Journal of industrial microbiology & biotechnology》1999,23(1):661-667
The thermophilic fungus Humicola sp constitutively produces intracellular α-galactosidase (1.33 U mg−1 protein) within 48 h at 45°C in shaken flasks, when grown in a medium containing 7% wheat bran extract as a carbon source
and 0.5% yeast extract as a nitrogen source. The enzyme has been purified to homogeneity by ultrafiltration, ethanol precipitation,
DEAE cellulose and Sephacryl S-300 chromatography with a 124-fold increase in specific activity and 29.5% recovery. The molecular
weight of the enzyme is 371.5 kDa by gel filtration on Sephacryl S-300 and 87.1 kDa by SDS-polyacrylamide gel electrophoresis.
The enzyme has an optimum temperature of 65°C and an optimum pH of 5.0. Humicola α-galactosidase is a glycoprotein with 8.3% carbohydrate content and is acidic in nature with a pI of 4.0. The K
m
S for p-nitrophenyl-α-D-galactopyranoside, O-nitrophenyl-α-D-galactopyranoside, raffinose and stachyose are 0.279, 0.40, 1.45 and 1.42 mM respectively. The enzyme activity was strongly
inhibited by Ag+ and Hg2+. D-Galactose inhibited α-galactosidase competitively and the inhibition constant (K
i) for galactose was 11 mM.
Received 28 January 1999/ Accepted in revised form 07 April 1999 相似文献
12.
Canakci S Belduz AO Saha BC Yasar A Ayaz FA Yayli N 《Applied microbiology and biotechnology》2007,75(4):813-820
The gene encoding an α-l-arabinofuranosidase from Geobacillus caldoxylolyticus TK4, AbfATK4, was isolated, cloned, and sequenced. The deduced protein had a molecular mass of about 58 kDa, and analysis
of its amino acid sequence revealed significant homology and conservation of different catalytic residues with α-l-arabinofuranosidases belonging to family 51 of the glycoside hydrolases. A histidine tag was introduced at the N-terminal
end of AbfATK4, and the recombinant protein was expressed in Escherichia coli BL21, under control of isopropyl-β-D-thiogalactopyranoside-inducible T7 promoter. The enzyme was purified by nickel affinity
chromatography. The molecular mass of the native protein, as determined by gel filtration, was about 236 kDa, suggesting a
homotetrameric structure. AbfATK4 was active at a broad pH range (pH 5.0–10.0) and at a broad temperature range (40–85°C),
and it had an optimum pH of 6.0 and an optimum temperature of 75–80°C. The enzyme was more thermostable than previously described
arabinofuranosidases and did not lose any activity after 48 h incubation at 70°C. The protein exhibited a high level of activity
with p-nitrophenyl-α-l-arabinofuranoside, with apparent K
m and V
max values of 0.17 mM and 588.2 U/mg, respectively. AbfATK4 also exhibited a low level of activity with p-nitrophenyl-β-d-xylopyranoside, with apparent K
m and V
max values of 1.57 mM and 151.5 U/mg, respectively. AbfATK4 released l-arabinose only from arabinan and arabinooligosaccharides. No endoarabinanase activity was detected. These findings suggest
that AbfATK4 is an exo-acting enzyme. 相似文献
13.
Uchida H Hojyo M Fujii Y Maeda Y Kajimura R Yamanaka H Sakurai A Sakakibara M Aisaka K 《Applied microbiology and biotechnology》2007,74(4):805-812
Formate oxidase was found in cell-free extracts of Debaryomyces vanrijiae MH201, a soil isolate. After purification by column chromatography, the preparation showed a protein band corresponding to
a molecular mass (MM) of 64 kDa on sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The MM, estimated by a gel filtration,
was 99 kDa. The preparation showed two and three bands on isoelectric focusing under denaturing and native conditions, respectively.
These results suggest that the preparation contained three isoforms, each of which might be composed of αα, αβ, and ββ subunits
with apparently similar MM. The preparation acted on formate with K
m and V
max values of 11.7 mM and 262 μmol min−1 mg−1, respectively, at pH 4.5 and 25°C, but showed no evidence of activity on the other compounds tested. The optimum pH and temperature
were pH 4.0 and 35°C, respectively. The preparation showed activities of 85% of the initial activity after storage at pH 6.0
and 4°C for 8 weeks. When 10 mM formaldehyde was reacted with 2.0 U ml−1 of the enzyme preparation at pH 5.5 and room temperature in the presence of 2.0 U ml−1 of a microbial aldehyde oxidase and 100 U ml−1 of catalase for 180 min, neither of formate nor formaldehyde was detected, suggesting that the reaction involved the quantitative
conversion of formaldehyde to carbon dioxide. 相似文献
14.
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
15.
Mohammad Shafiei Abed-Ali Ziaee Mohammad Ali Amoozegar 《Journal of industrial microbiology & biotechnology》2011,38(2):275-281
A halophilic α-amylase produced by Nesterenkonia sp. strain F was purified to homogeneity by 80% ethanol precipitation, Q-Sepharose anion exchange, and Sephacryl S-200 gel
filtration chromatography. The purified amylase exhibited specific activity of 357 unit/mg protein that corresponds to twofold
purification. The molecular mass of the amylase was determined to be 57 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) and gel filtration chromatography. The optimal pH and temperature for enzyme activity were 6.5 and 45°C, respectively.
The amylase was active over a wide range of salt concentrations (0–4 M) with maximum activity at 0.75–1 M NaCl. The α-amylase
activity was stimulated by Ca2+ and inhibited by ethylenediamine tetraacetic acid (EDTA), suggesting that this enzyme is a metalloenzyme. The purified enzyme
showed remarkable stability towards surfactants (Tween 20, Tween 80, and Triton X-100), and its activity was increased by
β-mercaptoethanol. The halophilic α-amylase was stable in the presence of various organic solvents such as benzene, chloroform,
toluene, and cyclohexane. These properties indicate wide potential applications of this α-amylase in starch-processing industries. 相似文献
16.
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
17.
Hirokawa Y Fujiwara S Suzuki M Akiyama T Sakamoto M Kobayashi S Tsuzuki M 《Planta》2008,227(3):589-599
The storage β-polyglucan and catabolic enzyme activities of the haptophyte Pleurochrysis haptonemofera were characterized. The storage β-polyglucan was prepared by the dimethylsulfoxide-extraction method. 13C- and 1H-NMR spectroscopy revealed that the polyglucan consists of β-(1→3)- and β-(1→6)-linked glucose polymers, with a β-(1→6)-
to β-(1→3)-linkage ratio of 1.5. Gel permeation chromatography showed that the molecular weight of the polyglucan is 1.1–8.4 × 104 Da, with a peak at 3.4 × 104 Da. The degree of polymerization, which was estimated from the amounts of total carbohydrate and reduced ends, was 203, corresponding
to 3.3 × 104 Da. A method for measurement of the β-polyglucan in a small amount of liquid culture involving a mixture of β-glucanases,
Westase, was established. The β-polyglucan was localized in the soluble fraction of cells. The amount of β-polyglucan per
cell increased at the stationary phase under continuous illumination and decreased in the dark, like those of storage α-polyglucans,
starch of green algae and glycogen of cyanobacteria. The activities of β-1,3- and β-1,6-glucanases involved in the degradation
of the storage β-polyglucan were assayed in vitro, both being optimal at pH 5.0. The β-1,3-glucanase activity, which was detected
on active staining after native polyacrylamide gel electrophoresis, was partially purified by ammonium sulfate precipitation
and anion exchange chromatography. 相似文献
18.
Sakamoto T Tanaka H Nishimura Y Ishimaru M Kasai N 《Applied microbiology and biotechnology》2011,90(5):1701-1710
A type II arabinogalactan-degrading enzyme, termed Exo-1,3-Gal, was purified to homogeneity from the culture filtrate of Sphingomonas sp. 24T. It has an apparent molecular mass of 48 kDa by SDS–PAGE. Exo-1,3-Gal was stable from pH 3 to 10 and at temperatures
up to 40 °C. The optimum pH and temperature for enzyme activity were pH 6 to 7 and 50 °C, respectively. Galactose was released
from β-1,3-d-galactan and β-1,3-d-galactooligosaccharides by the action of Exo-1,3-Gal, indicating that the enzyme was an exo-β-1,3-d-galactanase. Analysis of the reaction products of β-1,3-galactotriose by high-performance anion-exchange chromatography revealed
that the enzyme hydrolyzed the substrate in a non-processive mode. Exo-1,3-Gal bypassed the branching points of β-1,3-galactan
backbones in larch wood arabinogalactan (LWAG) to produce mainly galactose, β-1,6-galactobiose, and unidentified oligosaccharides
1 and 2 with the molar ratios of 7:19:62:12. Oligosaccharides 1 and 2 were enzymatically determined to be β-1,6-galactotriose
and β-1,6-galactotriose substituted with a single arabinofuranose residue, respectively. The ratio of side chains enzymatically
released from LWAG was in good agreement with the postulated structure of the polysaccharide previously determined by chemical
methods. 相似文献
19.
In vitro transgenic hairy root cultures provide a rapid system for physiological, biochemical studies and screening of plants
for their phytoremediation potential. The hairy root cultures of Brassica juncea L. showed 92% decolorization of Methyl orange within 4 days. Out of the different redox mediators that were used to achieve
enhanced decolorization, 2, 2′-Azinobis, 3-ethylbenzothiazoline-6-sulfonic acid (ABTS) was found to be the most efficient.
Laccase activity of 4.5 U mg−1 of protein was observed in hairy root cultures of Brassica juncea L., after the decolorization of Methyl orange. Intracellular laccase produced by B. juncea root cultures grown in MS basal medium was purified up to 2.0 fold with 6.62 U mg−1 specific activity using anion-exchange chromatography. Molecular weight of the purified laccase was estimated to be 148 kDa
by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified enzyme efficiently oxidized ABTS which was also
required for oxidation of the other tested substrates. The pH and temperature optimum for laccase activity were 4.0 and 40°C,
respectively. The purified enzyme was stable up to 50°C and was stable in the pH range of 4.0–6.0. Laccase activity was strongly
inhibited by sodium azide, EDTA, dithiothreitol and l-cysteine. The purified enzyme decolorized various textile dyes in the presence of ABTS as an efficient redox mediator. These
findings contribute to a better understanding of the enzymatic process involved in phytoremediation of textile dyes by using
hairy roots. 相似文献
20.
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