共查询到20条相似文献,搜索用时 62 毫秒
1.
Byoung Chul Park Sukhoon Koh Changsoo Chang Se Won Suh Dae-Sil Lee Si Myung Byun 《Applied biochemistry and biotechnology》1997,62(1):15-27
The gene encoding xylose isomerase (xylA) was cloned fromThermus flavus AT62 and the DNA sequence was determined. ThexylA gene encodes the enzyme xylose isomerase (XI orxylA) consisting of 387 amino acids (calculated Mr of 44,941). Also, there was a partial xylulose kinase gene that was 4 bp overlapped
in the end of XI gene. The XI gene was stably expressed inE. coli under the control oftac promoter. XI produced inE. coli was simply purified by heat treatment at 90°C for 10 min and column chromatography of DEAE-Sephacel. The Mr of the purified
enzyme was estimated to be 45 kDa on SDS-polyacrylamide gel electrophoresis. However, Mr of the cloned XI was 185 kDa on native
condition, indicating that the XI consists of homomeric tetramer. The enzyme has an optimum temperature at 90°C. Thermostability
tests revealed that half life at 85°C was 2 mo and 2 h at 95°C. The optimum pH is around 7.0, close to where by-product formation
is minimal. The isomerization yield of the cloned XI was about 55% from glucose, indicating that the yield is higher than
those of reported enzymes. The Km values for various sugar substrates were calculated as 106 mM for glucose. Divalent cations
such as Mn2+, Co2+, and Mg2+ are required for the enzyme activity and 100 mM EDTA completely inhibited the enzyme activity. 相似文献
2.
A novel β-galactosidase of 120 kDa (BgaBM) from Bacillus megaterium 2-37-4-1 was purified, and its gene (bgaBM) was analyzed and expressed. It displayed wide acceptor specificity for transglycosylation with a series of acceptors, including
pentose, hexose, hydroxyl, and alkyl alcohol using o-nitrophenyl-β-d-galactoside (ONPG) as a donor. BgaBM preferentially hydrolyzed ONPG in all tested substrates and showed maximum activity
at pH 7.5–8.0 and 55 °C. It was stable at pH 6.0–9.0 below 40 °C. The K
m and V
max values for ONPG and lactose were 9.5 mM, 16.6 mM/min and 12.6 mM, 54.4 mM/min, respectively. The nucleotide sequence of the
bgaBM gene consists of an ORF of 3,105 bp corresponding to 118 kDa protein, which indicates that BgaBM is a modular enzyme in the
glycosyl hydrolase family 2, including conserved sugar-binding domain, acid–base catalyst, and immunoglobulin-like beta-sandwich
domain. The possible acid/base and nucleophile sites of BgaBM were estimated to be E481 and E547, respectively. Furthermore,
expression of the bgaBM gene in Escherichia coli and purification of the recombinant enzyme were performed. The recombinant enzyme showed similar biochemical characteristics
to natural enzyme. 相似文献
3.
Bacillus cellulyticus K-12 Avicelase (Avicelase I; EC 3.2.1.4) gene (ace A) has been cloned in Escherichia coli by using the vector pT7T3U19 and HindIII-HindIII libraries of the chromosomal inserts. The libraries were screened for the expression of avicelase by monitoring the immunoreaction
of the antiavicelase (immunoscreening). Positive clones (Ac-3, Ac-5, and Ac-7) contained the identical 3.5-kb HindIII fragment as determined by restriction mapping and Southern hybridization, and expressed avicelase efficiently and constitutively
using its own promoter in the heterologous host. From the immunoblotting analysis, a polypeptide that showed a carboxymethylcellulase
(CMCase) activity with an M
r
, of 64,000 was detected. The recombinant endo 1,4-β-
d
-glucanase I was purified to homogeneity from an intracellular fraction of E. coli by DEAE-Toyopearl M650, Phenyl Toyoperal M650, and TSK gel HW50S chromatography. The enzyme had a monomeric structure, its
relative molecular mass being 65 kDa by gel filtration and 64 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
The pI was 5.3 and the optimal pH was 4.6, and the enzyme was stable at pH 4.0–10.5. The enzyme had a temperature optimum of 50°C
and was stable at 55°C for 48 h, and retained approx 20% of its activity after 30 min at 70°C. It showed high activity toward
carboxymethylcellulose (CMC) as well as p-nitrophenyl-β-d-cellobioside, 4-methylumbelliferyl cellobioside, Avicel, filter paper, and some cellooligosaccharides. K
m
values for CMC and Avicel were 7.6 and 85.2 mg/mL, respectively, whereas V
max values were 201 and 9.2 μmol · min−1 · mg−1, respectively. Cellotetraose (G4) was preferentially cleaved into cellobiose (G2) and cellopentaose (G5) was cleaved into
G2 + cellotriose (G3), whereas cellohexaose (G6) was cleaved into G4 + G2 and, to a lesser extent, into G3 + G3. G3 was not
cleaved at all. G2 was the main product of Avicel hydrolysis. G2 inhibited whereas Mg++ stimulated the activity of CMCase and Avicelase. Hydrolysis of CMC took place with a rapid decrease in viscosity but a slow
liberation of reducing sugars. Based on these results, it appeared that the cellulase should be regarded as endo type, although
it hydrolyzed Avicel. 相似文献
4.
Feng Xu Elizabeth J. Golightly Palle Schneider Randy M. Berka Kimberly M. Brown James A. Johnstone Denise H. Baker Claus C. Fuglsang Stephen H. Brown Allan Svendsen Alan V. Klotz 《Applied biochemistry and biotechnology》2000,88(1-3):23-32
The Fusarium spp. (Dactylium dendroides) galactose oxidase was expressed in Aspergillus oryzae and Fusarium venenatum hosts. Under the control of an A. niger α-amylase or a Fusarium trypsin promoter, high level galactose oxidase expression was achieved. The recombinant oxidase expressed in the A. oryzae host was purified and characterized. The purified enzyme had a molecular weight of 66 k Da on sodium dodecyl sulfate-polymerase
gel electrophoresis (SDS-PAGE) and 0.4 mol copper atom per mole protein. The stoichiometry increased to 1.2 after a Cu saturation.
Based on a peroxidase-coupled assay, the enzyme preparation showed an activity of 440 turnover per second toward d-galactose (0.1 M) at pH7 and 20°C. The enzyme had an optimal temperature of 60°C at pH 6.0 and an activation free Gibbs energy of 33 kJ/mol.
A series of d-galactose derivatives was tested as the reducing substrate for the oxidase. The difference in activity was interpreted by
the stereospecificity of the oxidase toward the substituents in the pyranose substrate, particularly on the C5 and the cyclic
hemiacetal O sites. The recombinan toxidase could act on some galactose-containing polysaccharides, such as guar gum, but
was not able to oxidize several common redox compounds that lacked a primary alcohol functional group. 相似文献
5.
An extracellular xylanase produced by a Mexican Aspergillus strain was purified and characterized. Aspergillus sp. FP-470 was able to grow and produce extracellular xylanases on birchwood xylan, oat spelt xylan, wheat straw, and corncob,
with higher production observed on corncob. The strain also produced enzymes with cellulase, amylase, and pectinase activities
on this substrate. A 22-kDa endoxylanase was purified 30-fold. Optimum temperature and pH were 60°C and 5.5, respectively,
and isoelectric point was 9.0. The enzyme has good stability from pH 5.0 to 10.0 retaining >80% of its original activity within
this range. Half-lives of 150 min at 50°C and 6.5 min at 60°C were found. K
m
and activation energy values were 3.8 mg/mL and 26 kJ/mol, respectively, using birch wood xylan as substrate. The enzyme
showed a higher affinity for 4-O-methyl-d-glucuronoxylan with a K
m
of 1.9 mg/mL. The enzyme displayed no activity toward other polysaccharides, including cellulose. Baking trials were conducted
using the crude filtrate and purified enzyme. Addition of both preparations improved bread volume. However, addition of purified
endoxylanase caused a 30% increase in volume over the crude extract. 相似文献
6.
Zhang Y Ji C Zhang X Yang Z Peng J Qiu R Xie Y Mao Y 《Applied biochemistry and biotechnology》2008,151(1):81-92
A gene-encoding alkaline phosphatase (AP) from thermophilic Geobacillus thermodenitrificans T2, termed Gtd AP, was cloned and sequenced. The deduced Gtd AP protein comprises 424 amino acids and shares a low homology with other known AP (<35% identity), while it exhibits the
conservation of the active site and structure element of Escherichia coli AP. The Gtd AP protein, without a predicted signal peptide of 30 amino acids, was successfully overexpressed in E. coli and purified as a hexa-His-tagged fusion protein. The pH and temperature optima for purified enzyme are 9.0 and 65 °C, respectively.
The enzyme retained a high activity at 45–60 °C, while it could be quickly inactivated by a heat treatment at 80 °C for 15 min,
exhibiting a half-life of 8 min at 70 °C. The K
m and V
max for pNPP were determined to be 31.5 μM and 430 μM/min at optimal conditions. A divalent cation is essential, with a combination
of Mg2+ and Co2+ or Zn2+ preferred. The enzyme was strongly inhibited by 10 mM ethylenediaminetetraacetic acid (EDTA) and vanadate but highly resistant
to urea and dithiothreitol. The properties of Gtd AP make it suitable for application in molecular cloning or amplification. 相似文献
7.
Chitinase was purified from the culture medium of Bacillus licheniformis SK-1 by colloidal chitin affinity adsorption followed by diethylamino ethanol-cellulose column chromatography. The purified
enzyme showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecular size and pI of chitinase 72 (Chi72) were 72 kDa and 4.62 (Chi72) kDa, respectively. The purified chitinase revealed two activity optima
at pH 6 and 8 when colloidal chitin was used as substrate. The enzyme exhibited activity in broad temperature range, from
40 to 70°C, with optimum at 55°C. It was stable for 2 h at temperatures below 60°C and stable over a broad pH range of 4.0–9.0
for 24 h. The apparent K
m and V
max of Chi72 for colloidal chitin were 0.23 mg ml−1 and 7.03 U/mg, respectively. The chitinase activity was high on colloidal chitin, regenerated chitin, partially N-acetylated chitin, and chitosan. N-bromosuccinamide completely inhibited the enzyme activity. This enzyme should be a good candidate for applications in the
recycling of chitin waste. 相似文献
8.
Chemical modification was evaluated to stabilize pig kidney d-amino acid oxidase (pkDAAO), which is required for analytical determination of d-amino acids. Optimization of modification conditions was performed to obtain high recovery yield and stability, and chemical
modification at 30°C for 12 h with a highly concentrated enzyme solution gave dextran-conjugated pkDAAO with a 70% yield of
activity. pkDAAO was stable at less than 55°C at pH 6.0, while the conjugated enzyme was stable even at 70°C. In addition,
the conjugated enzyme showed decreased K
m values for d-amino acids. Because of these outstanding charcteristics, this new material is expected to be available for use as a liquid
assay reagent. 相似文献
9.
Christakopoulos P Katapodis P Hatzinikolaou DG Kekos D Macris BJ 《Applied biochemistry and biotechnology》2000,87(2):127-133
An α-l-arabinofuranosidase from Fusarium oxysporum F3 was purified to homogeneity by a two-step ion exchange intercalated by a gel filtration chromatography. The enzyme had
a molecular mass of 66 kDa and was optimally active at pH 6.0 and 60°C. It hydrolyzed aryl α-l-arabinofuranosides and cleaved arabinosyl side chains from arabinoxylan and arabinan. There was a marked synergistic effect
between the α-l-arabinofuranosidase and an endo-(1 →4)-β-d-xylanase produced by F. oxysporum in the extensive hydrolysis of arabinoxylan. 相似文献
10.
Production of chitinolytic enzymes with Trichoderma longibrachiatum IMI 92027 in solid substrate fermentation 总被引:1,自引:0,他引:1
Kovacs K Szakacs G Pusztahelyi T Pandey A 《Applied biochemistry and biotechnology》2004,118(1-3):189-204
Thirty Trichoderma strains representing 15 species within the genus were screened for extracellular production of chitinolytic enzymes in solid
substrate fermentation. Trichoderma longibrachiatum IMI 92027 (ATCC 36838) gave the highest yield (5.0 IU/g of dry matter of substrate) after 3 d of fermentation on wheat bran-crude
chitin (9:1 mixture) medium. The optimal moisture content (66.7%), chitin content (20%), initial pH of the medium (2.0–5.0),
and time course (5 d) of solid substrate fermentation were determined for strain IMI 92027. Cellulase, xylanase, α-amylase,
and β-xylosidase activities were also detected. The pH and temperature optima of the chitinase complex of T. longibrachiatum IMI 92027 were 4.5 and 55°C, respectively. The enzyme totally lost its activity at 70°C in 5 min in the absence of the substrate
but retained about 15% of its initial activity even at 70°C after a 60-min incubation in the presence of solid substrate fermentation
solids. Purification of protein extract from the solid substrate fermentation material revealed high chitinolytic activities
between pI 5.9 and 4.8, where N-acetyl-β-d-hexosaminidase and chitinase peaks have been found in the same pI range. Two chitinases of 43.5 and 30 kDa were purified at acidic pI. 相似文献
11.
Mrudula Vasudevan Ushasree Paramasamy Gunasekaran Ashok Pandey 《Applied biochemistry and biotechnology》2012,167(5):981-990
Periplasmic phytase, appA from E. coli has been noticed as a superior feed and food additive owing to its high specific activity, acidic pH optimum and resistance to gastric proteases. E. coli phytase was expressed as a fusion protein with maltose-binding protein, affinity-purified to homogeneity and, subsequently, immobilized in one step using a cost-effective matrix prepared from starch agar bead. Immobilized enzyme revealed an activity optimum at pH 6, while that of free enzyme was observed at pH 4. Both the immobilized and free enzyme showed a temperature optimum at 60?°C. Cleavage of 87?kDa fusion protein using factor Xa released 45?kDa appA. Hydrolysis of soy milk using immobilized enzyme led to 10% increase in release of inorganic phosphate at 50?°C relative to free fusion protein. This study suggests the usability of MBP as an immobilizing linker to other food enzymes for economical use in industry. 相似文献
12.
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
13.
To express high-active soluble d-amino acid oxidase (DAAO), a constitutive plasmid that is regulated by a native hydantoinase promoter (PHase), was constructed. A d-amino acid oxidase gene (dao) was ligated with the PHase and cloned into pGEMKT to constitutively express protein of DAAO without the use of any inducer such as isopropyl β-d-1-thiogalactopyranoside which is poisonous to the cells and environment. The ribosome binding site region, host strain, and
fermentation conditions were optimized to increase the expression level. When cultivated in a 5-m3 fermenter, the enzyme activity of JM105/pGEMKT-R-DAAO grown at 37 °C was found to be 32 U/mL and increase 16-fold over cells
of BL21(DE3)/pET-DAAO grown at 28 °C. These results indicate the success of our approaches to overproducing DAAO in soluble
form in Escherichia coli. 相似文献
14.
Smaali M. Issam Gargouri Mohamed Legoy Marie Dominique Maugard Thierry Limam Farid Marzouki Nejib 《Applied biochemistry and biotechnology》2004,112(2):63-77
The filamentous fungus Sclerotinia sclerotiorum, grown on a xylose medium, was found to excrete one β-glucosidase (β-glu x). The enzyme was purified to apparent homogeneity
by ammonium sulfate precipitation, gel filtration, anion-exchange chromatography, and high-performance liquid chromatography
(HPLC) gel filtration chromatography. Its molecular mass was estimated to be 130 kDa by HPLC gel filtration and 60 kDa by
sodium dodecyl sulfate polyacrylamide gel electrophoresis, suggesting that β-glu x may be a homodimer. For p-nitrophenyl β-d-glucopyranoside hydrolysis, apparent K
m and V
max values were found to be 0.09 mM and 193 U/mg, respectively, while optimum temperature and pH were 55–60°C and pH 5.0, respectively. β-Glu x was strongly
inhibited by Fe2+ and activated about 35% by Ca2+. β-Glu x possesses strong transglucosylation activity in comparison with commercially available β-glucosidases. The production
rate of total glucooligosaccharides (GOSs) from 30% cellobiose at 50°C and pH 5.0 for 6 h with 0.6 U/mL of enzyme preparation
was 80 g/L. It reached 105 g/L under the same conditions when using cellobiose at 350 g/L (1.023 M). Finally, GOS structure was determined by mass spectrometry and 13C nuclear magnetic resonance spectroscopy. 相似文献
15.
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
16.
Purification and partial characterization of a lipase from Bacillus coagulans ZJU318 总被引:1,自引:0,他引:1
An extracellular lipase was purified from the fermentation broth of Bacillus coagulans ZJU318 by CM-Sepharose chromatography, followed by Sephacryl S-200 chromatography. The lipase was purified 14.7-fold with
18% recovery and a specific activity of 141.1 U/mg. The molecular weight of the homogeneous enzyme was (32 kDa), determined
by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The enzyme activity was maximum at pH 9.0 and was stable
over a pH range of 7.0–10.0, and the optimum temperature for the enzyme reaction was 45°C. Little activity loss (6.2%) was
observed after 1 h of incubation at 40°C. However, the stability of the lipase decreased sharply at 50 and 60°C. The enzyme
activity was strongly inhibited by Ag+ and Cu2+, whereas EDTA caused no inhibition. SDS, Brij 30, and Tween-80 inhibited lipase, whereas Triton X-100 did not significantly
inhibit lipase activity. 相似文献
17.
Maalej I Belhaj I Masmoudi NF Belghith H 《Applied biochemistry and biotechnology》2009,158(1):200-212
A thermostable xylanase from a newly isolated thermophilic fungus Talaromyces thermophilus was purified and characterized. The enzyme was purified to homogeneity by ammonium sulfate precipitation, diethylaminoethyl
cellulose anion exchange chromatography, P-100 gel filtration, and Mono Q chromatography with a 23-fold increase in specific
activity and 17.5% recovery. The molecular weight of the xylanase was estimated to be 25kDa by sodium dodecyl sulfate–polyacrylamide
gel electrophoresis and gel filtration. The enzyme was highly active over a wide range of pH from 4.0 to 10.0. The relative
activities at pH5.0, 9.0, and 10.0 were about 80%, 85.0%, and 60% of that at pH7.5, respectively. The optimum temperature
of the purified enzyme was 75°C. The enzyme showed high thermal stability at 50°C (7days) and the half-life of the xylanase
at 100°C was 60min. The enzyme was free from cellulase activity. K
m and V
max values at 50°C of the purified enzyme for birchwood xylan were 22.51mg/ml and 1.235μmol min−1 mg−1, respectively. The enzyme was activated by Ag+, Co2+, and Cu2+; on the other hand, Hg2+, Ba2+, and Mn2+ inhibited the enzyme. The present study is among the first works to examine and describe a secreted, cellulase-free, and
highly thermostable xylanase from the T. thermophilus fungus whose application as a pre-bleaching aid is of apparent importance for pulp and paper industries. 相似文献
18.
The gene encoding a glycoside hydrolase family 39 xylosidase (BH1068) from the alkaliphile Bacillus halodurans strain C-125 was cloned with a C-terminal His-tag, and the recombinant gene product termed BH1068(His)6 was expressed in Escherichia coli. Of the artificial substrates tested, BH1068(His)6 hydrolyzed nitrophenyl derivatives of β-d-xylopyranose, α-l-arabinofuranose, and α-l-arabinopyranose. Deviation from Michaelis−Menten kinetics at higher substrate concentrations indicative of transglycosylation
was observed, and k
cat and K
m values were measured at both low and high substrate concentrations to illuminate the relative propensities to proceed along
this alternate reaction pathway. The pH maximum was 6.5, and under the conditions tested, maximal activity was at 47°C, and
thermal instability occurred above 45°C. BH1068(His)6 was inactive on arabinan, hydrolyzed xylooligosaccharides, and released only xylose from oat, wheat, rye, beech, and birch
arabinoxylan, and thus, can be classified as a xylosidase with respect to natural substrate specificity. The enzyme was not
inhibited by up to 200 mM xylose. The oligomerization state was tetrameric under the size-exclusion chromatography conditions
employed. 相似文献
19.
Purification and characterization of an alkaline protease prot 1 frombotrytis cinerea 总被引:1,自引:0,他引:1
Alkaline thiol protease named Prot 1 was isolated from a culture filtrate ofBotrytis cinerea. The enzyme was purified by ammonium sulfate fractionation, gel filtration, and ion-exchange chromatography. Thus, the enzyme
was purified to homogeneity with specific activity of 30-fold higher than that of the crude broth. The purified alkaline protease
has an apparent molecular mass of 43 kDa under denaturing conditions as estimated by sodium dodecyl sulfate-polyacrylamide
gel electrophoresis. The native molecular mass (45 kDa), determined by gel filtration, indicated that the alkaline protease
has a monomeric form. The purified protease was biochemically characterized. The enzyme is active at alkaline pH and has a
suitable and high thermostability. The optimal pH and temperature for activity were 9.0–10.0 and 60°C, respectively. This
protease was stable between pH 5.0 and 12.0. The enzyme retained 85% of its activity by treatment at 50°C over 120 min; it
maintained 50% of activity after 60 min of heating at 60°C. Furthermore, the protease retained almost complete activity after
4 wk storage at 25°C. The activity was significantly affected by thiol protease inhibitors, suggesting that the enzyme belongs
to the alkaline thiol protease family. With the aim on industrial applications, we focused on studying the stability of the
protease in several conditions. Prot 1 activity was not affected by ionic strength and different detergent additives, and,
thus, the protease shows remarkable properties as a biodetergent catalyst. 相似文献
20.
Manchumas Hengsakul Prousoontorn Supranee Pantatan 《Journal of inclusion phenomena and macrocyclic chemistry》2007,57(1-4):39-46
Cyclodextrin glycosyltransferase (CGTase) isolated and purified from Paenibacillus sp. A11 was immobilized on various carriers by covalent linkage using bifunctional agent glutaraldehyde. Among tested carriers,
alumina proved to be the best carrier for immobilization. The effects of several parameters on the activation of the support
and on the immobilization of enzyme were optimized. The best preparation of immobilized CGTase retained 31.2% of its original
activity. After immobilization, the enzymatic properties were investigated and compared with those of the free enzyme. The
optimum pH of the immobilized CGTase was shifted from 6.0 to 7.0 whereas optimum temperature remained unaltered (60°C). Free
and immobilized CGTase showed similar pH stability profile but the thermal stability of the immobilized CGTase was 20% higher.
Kinetic data (K
M and V
max) for the free and immobilized enzymes were determined from the rate of β-CD formation and it was found that the immobilized
form had higher K
M and lower V
max. The immobilized CGTase also exhibited higher stability when stored at both 4°C and 25°C for 2 months. The enzyme immobilized
on alumina was further used in a batch production of 2-O-α-glucopyranosyl-l-ascorbic acid (AA-2G) from ascorbic acid and β-cyclodextrin. The yield of AA-2G was 2.92% and the immobilized CGTase retained
its activity up to 74.4% of the initial catalytic activity after being used for 3 cycles. The immobilized CGTase would have
a promising application in the production of various transglycosylated compounds and in the production of cyclodextrin by
the hydrolysis of starch. 相似文献