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1.
Tohru Kobayashi Kohsuke Uchimura Masayuki Miyazaki Yuichi Nogi Koki Horikoshi 《Extremophiles : life under extreme conditions》2009,13(1):121-129
A high-alkaline, salt-activated alginate lyase is produced by Agarivorans sp. JAM-A1m from a deep-sea sediment off Cape Nomamisaki on Kyushu Island, Japan. Purified to homogeneity, as judged by SDS-PAGE,
the enzyme (A1m) had a molecular mass of approximately 31 kDa. The optimal pH was around 10 in glycine–NaOH buffer, and the
activity was increased to 1.8 times by adding 0.2 M NaCl. However, when the optimal pH in the presence of 0.2 M NaCl was shifted
to pH 9.0, the activity was more than 10 times compared with that at pH 9 in the absence of NaCl. A1m showed the optimal temperature
at around 30°C and was stable to incubation between pH 6 and 9. The enzyme degraded favorably mannuronate–guluronate and guluronate-rich
fragments in alginate. Shotgun cloning and sequencing of the gene for A1m revealed a 930-bp open reading frame, which encoded
a mature enzyme of 289 amino acids (32,295 Da) belonging to polysaccharide lyase family 7. The deduced amino acid sequence
showed the highest similarity to that of a Klebsiella enzyme, with only 54% identity. 相似文献
2.
Purification and characterization of a serine alkaline protease from Bacillus clausii GMBAE 42 总被引:1,自引:0,他引:1
Kazan D Denizci AA Oner MN Erarslan A 《Journal of industrial microbiology & biotechnology》2005,32(8):335-344
An extracellular serine alkaline protease of Bacillus clausii GMBAE 42 was produced in protein-rich medium in shake-flask cultures for 3 days at pH 10.5 and 37°C. Highest alkaline protease
activity was observed in the late stationary phase of cell cultivation. The enzyme was purified 16-fold from culture filtrate
by DEAE-cellulose chromatography followed by (NH4)2SO4 precipitation, with a yield of 58%. SDS-PAGE analysis revealed the molecular weight of the enzyme to be 26.50 kDa. The optimum
temperature for enzyme activity was 60°C; however, it is shifted to 70°C after addition of 5 mM Ca2+ ions. The enzyme was stable between 30 and 40°C for 2 h at pH 10.5; only 14% activity loss was observed at 50°C. The optimal
pH of the enzyme was 11.3. The enzyme was also stable in the pH 9.0–12.2 range for 24 h at 30°C; however, activity losses
of 38% and 76% were observed at pH values of 12.7 and 13.0, respectively. The activation energy of Hammarsten casein hydrolysis
by the purified enzyme was 10.59 kcal mol−1 (44.30 kJ mol−1). The enzyme was stable in the presence of the 1% (w/v) Tween-20, Tween-40,Tween-60, Tween-80, and 0.2% (w/v) SDS for 1 h
at 30°C and pH 10.5. Only 10% activity loss was observed with 1% sodium perborate under the same conditions. The enzyme was
not inhibited by iodoacetate, ethylacetimidate, phenylglyoxal, iodoacetimidate, n-ethylmaleimidate, n-bromosuccinimide, diethylpyrocarbonate or n-ethyl-5-phenyl-iso-xazolium-3′-sulfonate. Its complete inhibition by phenylmethanesulfonylfluoride and relatively high k
cat value for N-Suc-Ala-Ala-Pro-Phe-pNA hydrolysis indicates that the enzyme is a chymotrypsin-like serine protease. K
m and k
cat values were estimated at 0.655 μM N-Suc-Ala-Ala-Pro-Phe-pNA and 4.21×103 min−1, respectively. 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.
Dodia MS Rawal CM Bhimani HG Joshi RH Khare SK Singh SP 《Journal of industrial microbiology & biotechnology》2008,35(2):121-131
An alkaline protease secreting Haloalkaliphilic bacterium (Gene bank accession number EU118361) was isolated from the Saurashtra
Coast in Western India. The alkaline protease was purified by a single step chromatography on phenyl sepharose 6 FF with 28%
yield. The molecular mass was 40 kDa as judged by SDS-PAGE. The enzyme displayed catalysis and stability over pH 8–13, optimally
at 9–11. It was stable with 0–4 M NaCl and required 150 mM NaCl for optimum catalysis at 37 °C; however, the salt requirement
for optimal catalysis increased with temperature. While crude enzyme was active at 25–80 °C (optimum at 50 °C), the purified
enzyme had temperature optimum at 37 °C, which shifted to 80 °C in the presence of 2 M NaCl. The NaCl not only shifted the
temperature profile but also enhanced the substrate affinity of the enzyme as reflected by the increase in the catalytic constant
(K
cat). The enzyme was also calcium dependent and with 2 mM Ca+2, the activity reached to maximum at 50 °C. The crude enzyme was highly thermostable (37–90 °C); however, the purified enzyme
lost its stability above 50 °C and its half life was enhanced by 30 and sevenfold at 60 °C with 1 M NaCl and 50 mM Ca+2, respectively. The activity of the enzyme was inhibited by PMSF, indicating its serine type. While the activity was slightly
enhanced by Tween-80 (0.2%) and Triton X-100 (0.05%), it marginally decreased with SDS. In addition, the enzyme was highly
stable with oxidizing-reducing agents and commercial detergents and was affected by metal ions to varying extent. The study
assumes significance due to the enzyme stability under the dual extremities of pH and salt coupled with moderate thermal tolerance.
Besides, the facts emerged on the enzyme stability would add to the limited information on this enzyme from Haloalkaliphilic
bacteria. 相似文献
5.
Yada E Nagata H Noguchi Y Kodera Y Nishimura H Inada Y Matsushima A 《Marine biotechnology (New York, N.Y.)》2005,7(5):474-480
An arginine specific protease, Sp-protease, was purified by column chromatography from freeze-dried Spirulina platensis using a five-step process. Purified Sp-protease has a molecular weight of 80 kDa. It hydrolyzed the synthetic substrates
containing arginine residue in the P1 position but did not hydrolyze synthetic substrates containing other amino acid residues,
including lysine residue in the P1 position. Among the synthetic substrates tested, a substrate of plasminogen activator (Pyr-Gly-Arg-MCA)
was hydrolyzed most effectively with the enzyme (Km = 5.5 × 10−6 M), and fibrin gel was solubilized via activation of intrinsic plasminogen to plasmin with the enzyme. Activity was inhibited
completely with camostat mesilate (Ki = 1.1 × 10−8 M) and leupeptin (Ki = 3.9 × 10−8 M) but was not inhibited with Nα-tosyl-L-lysine chloromethyl ketone (TLCK). The optimum pH of the enzyme has a range of pH 9.0 to pH 11.0. The optimum temperature
was 50°C; the enzyme was stable at 0–50°C. 相似文献
6.
Basidiospore germination in an ectomycorrhizal ammonia fungus Hebeloma vinosophyllum was stimulated by 10–500 mM NH4Cl aqueous solution at pH 4.5–9.0, but not by pure water. The basidiospores germinated at 10°–35°C with an optimum at 25°–30°C.
The highest germination percentage (83.0%) was observed in 100 mM NH4Cl aqueous solution adjusted to pH 8.0 by KOH, when the basidiospores were incubated at a density of 106 spores/ml at 30°C for 14 days. The percent germination value decreased with the increased duration of storage under both
dry and wet conditions. Humidity and temperature affected the longevity of H. vinosophyllum basidiospores. The basidiospores maintained their germination ability longer under a dry condition than under a wet condition.
The greatest longevity was accomplished by storage at 15°C under a dry condition. 相似文献
7.
An extracellular polygalacturonase was isolated from 5-day culture filtrates of Thermoascus aurantiacus CBMAI-756 and purified by gel filtration and ion-exchange chromatography. The enzyme was maximally active at pH 5.5 and 60–65°C.
The apparent K
m with citrus pectin was 1.46 mg/ml and the V
max was 2433.3 μmol/min/mg. The apparent molecular weight of the enzyme was 30 kDa. The enzyme was 100% stable at 50°C for 1 h
and showed a half-life of 10 min at 60°C. Polygalacturonase was stable at pH 5.0–5.5 and maintained 33% of initial activity
at pH 9.0. Metal ions, such as Zn+2, Mn+2, and Hg+2, inhibited 50, 75 and 100% of enzyme activity. The purified polygalacturonase was shown to be an endo/exo-enzyme, releasing
mono, di and tri-galacturonic acids within 10 min of hydrolysis. 相似文献
8.
Karbalaei-Heidari HR Amoozegar MA Hajighasemi M Ziaee AA Ventosa A 《Journal of industrial microbiology & biotechnology》2009,36(1):21-27
The production of a protease was investigated under conditions of high salinity by the moderately halophilic bacterium Halobacillus karajensis strain MA-2 in a basal medium containing peptone, beef extract, maltose and NaCl when the culture reached the stationary
growth phase. Effect of various temperatures, initial pH, salt and different nutrient sources on protease production revealed
that the maximum secretion occurred at 34°C, pH 8.0–8.5, and in the presence of gelatin. Replacement of NaCl by various concentrations
of sodium nitrate in the basal medium also increased the protease production. The secreted protease was purified 24-fold with
68% recovery by a simple approach including a combination of acetone precipitation and Q-Sepharose ion exchange chromatography.
The enzyme revealed a monomeric structure with a relative molecular mass of 36 kDa by running on SDS-PAGE. Maximum caseinolytic
activity of the enzyme was observed at 50°C, pH 9.0 and 0.5 M NaCl, although at higher salinities (up to 3 M) activity still
remained. The maximum enzyme activity was obtained at a broad pH range of 8.0–10.0, with 55 and 50% activity remaining at
pH 6 and 11, respectively. Moreover, the enzyme activity was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF), Pefabloc
SC and EDTA; indicating that it probably belongs to the subclass of serine metalloproteases. These findings suggest that the
protease secreted by Halobacillus karajensis has a potential for biotechnological applications from its haloalkaline properties point of view. 相似文献
9.
M. Vidyasagar S. Prakash S. K. Jayalakshmi K. Sreeramulu 《World journal of microbiology & biotechnology》2007,23(5):655-662
An extremely halophilic Chromohalobacter sp. TVSP101 was isolated from solar salterns and screened for the production of extracellular halothermophilic protease.
Identification of the bacterium was done based upon biochemical tests and the 16S rRNA sequence. The partially purified enzyme
displayed maximum activity at pH 8 and required 4.5 M of NaCl for optimum proteolytic activity. In addition, this enzyme was
thermophilic and active in broad range of temperature 60–80°C with 80°C as optimum. The Chromohalobacter sp. required 4 M NaCl for its optimum growth and protease secretion and no growth was observed below 1 M of NaCl. The initial
pH of the medium for growth and enzyme production was in the range 7.0–8.0 with optimum at pH 7.2. Various cations at 1 mM
concentration in the growth medium had no significant effect in enhancing the growth and enzyme production but 0.5 M MgCl2 concentration enhanced enzyme production. Casein or skim milk powder 1% (w/v) along with 1% peptone proved to be the best
nitrogen sources for maximum biomass and enzyme production. The carbon sources glucose and glycerol repressed the protease
secretion. Immobilization of whole cells in absence of NaCl proved to be useful for continuous production of halophilic protease. 相似文献
10.
Muthu Manikandan Lejla Pašić Vijayaraghavan Kannan 《World journal of microbiology & biotechnology》2009,25(12):2247-2256
An extremely halophilic archaeon Haloferax lucentensis VKMM 007, isolated from a solar saltern, was found to produce a protease. This extracellular enzyme consisted of a single
polypeptide chain of 57.8 kDa as determined by SDS–PAGE and was purified by a combination of ultrafiltration, bacitracin–Sepharose
affinity chromatography and Sephadex G-100 gel filtration. The purified protein was stable in a wide range of temperatures
(20–70°C), NaCl concentrations (0.85–5.13 M) and pH (5.0–9.0) with maximal activity observed at 60°C, 4.3 M NaCl and pH 8.0.
Proteolytic activity was enhanced by Ca2+, K+, Mg2+, Na+, and Fe2+ ions and the protein was classified as a trypsin-like serine protease. Further assays indicated highest degree of specificity
when hemoglobin was used as an enzyme substrate. Most importantly, the proteolytic activity remained stable or only marginally
inhibited in the presence of various polar and non-polar solvents, surfactants and reducing agents thus emphasizing the biotechnological
potential of this novel halophilic protease. 相似文献
11.
The importance of various parameters such as sugarcane juice concentration, pH of the medium, and effects of different solid
supports for maximum secretion of pectin lyase from Penicillium citrinum MTCC 8897 has been studied. The enzyme was purified to homogeneity by Sephadex G-100 and DEAE-cellulose chromatography. The
molecular mass determined by SDS-PAGE was 31 kDa. The K
m and k
cat values were found to be 1 mg/ml and 76 sec−1, respectively. The optimum pH of the purified pectin lyase was 9.0, though it retains activity in the pH 9.0–12.0 range when
exposed for 24 h. The optimum temperature was 50°C, and the pectin lyase was found to be completely stable up to 40°C when
exposed for 1 h. The purified pectin lyase was found efficient in retting of Linum usitatissimum, Cannabis sativa, and Crotalaria juncea.
Published in Russian in Biokhimiya, 2009, Vol. 74, No. 7, pp. 985–992. 相似文献
12.
W. Chungool W. Thongkam P. Raweesri A. Thamchaipenet P. Pinphanichakarn 《World journal of microbiology & biotechnology》2008,24(4):549-556
Acetyl esterase was produced by Streptomyces sp. PC22 at comparable levels of about 0.3 U ml−1 using either 1.0% (w/v) birchwood xylan or 1.5% (w/v) corn husks as a carbon source and cultivating at 45 °C, at pH 9 for
3 or 2 days, respectively. The enzyme was purified from culture filtrate to about 54-fold purity by ammonium sulfate precipitation,
followed by consecutive chromatography using a Macro-Prep DEAE, t-butyl hydrophobic interaction and hydroxyapatite, respectively. The approximate molecular weight of the purified enzyme was
155 kDa as analyzed by gel filtration, and it contained four identical 34 kDa subunits, as assessed by SDS-PAGE. It had K
m and V
max values for p-nitrophenyl acetate of 0.43 mM and 70.78 U mg−1 and 7.8 mM and 1,027 U mg−1 for α-naphthyl acetate, respectively. Its optimal pH and temperature were 6.5–7.0 and 50 °C, respectively. It was stable for 30 min
at a broad range of pH values, from 5.0 to 9.0, and at temperatures up to 60 °C. The purified enzyme had no other xylanolytic
activities. It showed cooperative action on birchwood xylan degradation, when used in combination with xylanase from the same
strain and β-xylosidase from Streptomyces sp. CH7. Enhancement was 1.4-fold, compared to the expected amount of individual enzymes alone. This indicates that the enzyme
has potential industrial applications, especially for utilizing renewable hemicelluloses containing acetyl xylan for the production
of biofuels or other fermentation products. 相似文献
13.
Ling Zhao Yongming Bao Jingyun Wang Boshi Liu Lijia An 《World journal of microbiology & biotechnology》2009,25(1):57-64
A mutant designated as UV-3 was obtained from wild-type Enterobacter aerogenes 10293 through u.v. radiation. The activities of α-acetolactate decarboxylase (Ald), lactate dehydrogenase (Ldh) and diacetyl
reductase (Dr) in UV-3 were strongly attenuated, with the lowest activities at pH 7.0–7.5, and temperature between 36 and
39°C. Compared to the wild-type, the yield of diacetyl by UV-3 was increased 18.7-fold, up to 1.05 ± 0.01 g l−1. Acetoin and ethanol productions were decreased by 48.4 and 71.4%, respectively, but acetate yield was increased by 34.6%.
Optimum medium for diacetyl production by UV-3 contained 10% glucose, 0.5% peptone, 0.5% yeast extract powder, 0.01% (NH4)2SO4, 0.1% citric acid, 0.2% MnSO4 and 0.2% MgSO4, and this was determined by one-factor-at-a-time approach. Data from the five level central composite designs demonstrated
that initial pH of 7.0, temperature of 37°C and rotational speed of 180 rev/min were optimum processing parameters for diacetyl
production. The maximum yield of diacetyl could reach 1.35 g l−1 in a 5-l bioreactor. These results showed an enhancement of the non-enzymatic oxidative decarboxylation of α-acetolactate
and a decrease in the activities of Ald, Ldh and Dr as a consequence of diacetyl accumulation in UV-3. 相似文献
14.
The gene encoding Lentinula edodes glucoamylase (GLA) was cloned into Saccharomyces cerevisiae, expressed constitutively and secreted in an active form. The enzyme was purified to homogeneity by (NH4)2SO4 fractionation, anion exchange and affinity chromatography. The protein had a correct N-terminal sequence of WAQSSVIDAYVAS,
indicating that the signal peptide was efficiently cleaved. The recombinant enzyme was glycosylated with a 2.4% carbohydrate
content. It had a pH optimum of 4.6 and a pH 3.4–6.4 stability range. The temperature optimum was 50°C with stability ≤50°C.
The enzyme showed considerable loss of activity when incubated with glucose (44%), glucosamine (68%), galactose (22%), and
xylose (64%). The addition of Mn++ activated the enzyme by 45%, while Li+, Zn++, Mg++, Cu+, Ca++, and EDTA had no effect. The enzyme hydrolyzed amylopectin at rates 1.5 and 8.0 times that of soluble starch and amylose,
respectively. Soluble starch was hydrolyzed 16 and 29 times faster than wheat and corn starch granules, respectively, with
the hydrolysis of starch granules using 10× the amount of GLA. Apparent Km and Vmax for soluble starch were estimated to be 3.0 mg/ml and 0.13 mg/ml/min (40°C, pH 5.3), with an apparent kcat of 2.9×105 min−1. 相似文献
15.
Zhiqiang Wu Guoliang Jiang Peng Xiang Honglei Xu 《International journal of peptide research and therapeutics》2008,14(2):113-120
An anionic trypsin (TRY-EP) was purified from North Pacific krill (Euphausia pacifica) by ammonium sulfate precipitation, ion-exchange and gel-filtration chromatography. The purified enzyme was identified as
a trypsin by LC-ESI-MS/MS analysis. The relative molecular mass of TRY-EP was 33 kDa, with isoelectric point of 4.5. The histidine,
tryptophan, arginine, lysine, aspartic acid and glutamic acid residues were functional groups to TRY-EP. TRY-EP was activated
by Ca2+ and Mg2+ and inhibited by some heavy metal ions (Zn2+, Cu2+, Pb2+ and Hg2+), organic solvents (ethanol, glycerin, DMSO and acetone) and specific trypsin inhibitors (benzamidine, CEOM, SBTI and TLCK).
TRY-EP was active over a wide pH (6.0–11.0) and temperature (10–70°C) range, with optimum of pH 9.0 and 40–50°C. TRY-EP was
stable between pH 6.0 and 11.0 and below 30°C. Compared with some trypsins from the Temperate and Tropical Zone organisms,
TRY-EP and other trypsins from the Frigid Zone organisms have higher affinity to substrate and 2–42-fold physiological efficiency. 相似文献
16.
Gulelat D. Haki Alfredo J. Anceno Sudip K. Rakshit 《World journal of microbiology & biotechnology》2008,24(11):2517-2524
Bacillus sp. GRE1 isolated from an Ethiopian hyperthermal spring produced raw-starch digesting, Ca2+-independent thermostable α-amylase. Enzyme production in shake flask experiments using optimum nutrient supplements and environmental
conditions was 2,360 U l−1. Gel filtration chromatography yielded a purification factor of 33.6-fold and a recovery of 46.5%. The apparent molecular
weight of the enzyme was 55 kDa as determined by SDS-PAGE. Presence or absence of Ca2+ produced similar temperature optima of 65–70°C. The optimum pH was in the range of 5.5–6.0. The enzyme maintained 50% of
its original activity after 45 min of incubation at 80°C and was stable at a pH range of 5.0–9.0. The V
max and K
m values for soluble starch were 42 mg reducing sugar min−1 and 4.98 mg starch ml−1, respectively. Strong inhibitors of enzyme activity included Cu2+, Zn2+ and Fe2+. The enzyme coding gene and the deduced protein translation revealed a characteristic but markedly atypical homology to Bacillus species α-amylase sequences. The enzyme hydrolyzed wheat, corn and tapioca starch granules efficiently below their gelatinization
temperatures. Rather than the higher oligosaccharides normally produced by Bacillus α-amylases operating at high temperatures, maltose was the major hydrolysis product with the present enzyme. 相似文献
17.
Kurata A Uchimura K Shimamura S Kobayashi T Horikoshi K 《Applied microbiology and biotechnology》2007,77(2):311-319
The acpI gene encoding an alkaline protease (AcpI) from a deep-sea bacterium, Alkalimonas collagenimarina AC40T, was shotgun-cloned and sequenced. It had a 1,617-bp open reading frame encoding a protein of 538 amino acids. Based on analysis
of the deduced amino acid sequence, AcpI is a subtilisin-like serine protease belonging to subtilase family A. It consists
of a prepropeptide, a catalytic domain, and a prepeptidase C-terminal domain like other serine proteases from the genera Pseudomonas, Shewanella, Alteromonas, and Xanthomonas. Heterologous expression of the acpI gene in Escherichia coli cells yielded a 28-kDa recombinant AcpI (rAcpI), suggesting that both the prepropeptide and prepeptidase C-terminal domains
were cleaved off to give the mature form. Analysis of N-terminal and C-terminal amino acid sequences of purified rAcpI showed
that the mature enzyme would be composed of 273 amino acids. The optimal pH and temperature for the caseinolytic activity
of the purified rAcpI were 9.0–9.5 and 45°C in 100 mM glycine–NaOH buffer. Calcium ions slightly enhanced the enzyme activity
and stability. The enzyme favorably hydrolyzed gelatin, collagen, and casein. AcpI from A. collagenimarina AC40T was also purified from culture broth, and its molecular mass was around 28 kDa, indicating that the cleavage manner of the
enzyme is similar to that in E. coli cells. 相似文献
18.
Heng-Lin Cui Xia Gao Xin Yang Xue-Wei Xu 《Extremophiles : life under extreme conditions》2010,14(6):493-499
Two halophilic archaeal strains TBN4T and TBN5 were isolated from Taibei marine solar saltern in Jiangsu, China. Both strains showed light red-pigmented colonies
and their cells were rod, motile and Gram-stain-negative. They were able to grow at 25–50°C (optimum 37°C), at 1.4–4.3 M NaCl
(optimum 2.1 M NaCl), at 0–1.0 M MgCl2 (optimum 0.005 M MgCl2) and at pH 6.0–9.0 (optimum pH 7.0). Their cells lyse in distilled water and minimal NaCl concentration to prevent cell lysis
is 8% (w/v). The major polar lipids of the two strains were PG (phosphatidylglycerol), PGP-Me (phosphatidylglycerol phosphate
methyl ester), PGS (phosphatidylglycerol sulfate) and five glycolipids chromatographically identical to S-TGD-1 (sulfated
galactosyl mannosyl glucosyl diether), S-DGD-1 (sulfated mannosyl glucosyl diether), TGD-1 (galactosyl mannosyl glucosyl diether),
DGD-1 (mannosyl glucosyl diether) and DGD-2 (an unknown diglycosyl diether). Phylogenetic analysis revealed that TBN4T and strain TBN5 formed a distinct clade with genus Haladaptatus (showing 90.0–90.9% 16S rRNA gene similarities). The DNA G + C content of strain TBN4T and strain TBN5 are 66.1 and 65.4 mol%, respectively. The DNA–DNA hybridization value between strain TBN4T and strain TBN5 was 94.3%. The phenotypic, chemotaxonomic and phylogenetic properties suggest that strain TBN4T and strain TBN5 represent a novel species in a new genus within the family Halobacteriaceae, for which the name Halorussus rarus gen. nov., sp. nov. is proposed. The type strain is TBN4T (=CGMCC 1.10122T = JCM 16429T). 相似文献
19.
Subramanian Mohan Raj Chelladurai Rathnasingh Woo-Chel Jung Sunghoon Park 《Applied microbiology and biotechnology》2009,84(4):649-657
The stability and specific activity of endo-β-1,4-glucanase III from Trichoderma reesei QM9414 was enhanced, and the expression efficiency of its encoding gene, egl3, was optimized by directed evolution using error-prone PCR and activity screening in Escherichia coli RosettaBlue (DE3) pLacI as a host. Relationship between increase in yield of active enzyme in the clones and improvement
in its stability was observed among the mutants obtained in the present study. The clone harboring the best mutant 2R4 (G41E/T110P/K173M/Y195F/P201S/N218I)
selected in via second-round mutagenesis after optimal recombinating of first-round mutations produced 130-fold higher amount
of mutant enzyme than the transformant with wild-type EG III. Mutant 2R4 produced by the clone showed broad pH stability (4.4–8.8)
and thermotolerance (entirely active at 55°C for 30 min) compared with those of the wild-type EG III (pH stability, 4.4–5.2;
thermostability, inactive at 55°C for 30 min). k
cat of 2R4 against carboxymethyl-cellulose was about 1.4-fold higher than that of the wild type, though the K
m became twice of that of the wild type. 相似文献
20.
Chengtao Wang Yanping Cao Baoguo Sun Baoping Ji M. J. Robert Nout Ji Wang Yonghuan Zhao 《World journal of microbiology & biotechnology》2008,24(10):2149-2157
Rhodococcus sp. R14-2, isolated from Chinese Jin-hua ham, produces a novel extracellular cholesterol oxidase (COX). The enzyme was extracted from
fermentation broth and purified 53.1-fold based on specific activity. The purified enzyme shows a single polypeptide band
on SDS-PAGE with an estimated molecular weight of about 60 kDa, and has a pI of 8.5. The first 10 amino acid residues of the NH2-terminal sequence of the enzyme are A-P-P-V-A-S-C-R-Y-C, which differs from other known COXs. The enzyme is stable over a
rather wide pH range of 4.0–10.0. The optimum pH and temperature of the COX are pH 7.0 and 50°C, respectively. The COX rapidly
oxidizes 3β-hydroxysteroids such as cholesterol and phytosterols, but is inert toward 3α-hydroxysteroids. Thus, the presence
of a 3β-hydroxyl group appears to be essential for substrate activity. The Michaelis constant (Km) for cholesterol is estimated
at 55 μM; the COX activity was markedly inhibited by metal ions such as Hg2+ and Fe3+ and inhibitors such as p-chloromercuric benzoate, mercaptoethanol and fenpropimorph. Inhibition caused by p-chloromercuric benzoate, mercuric chloride, or silver nitrate was almost completely prevented by the addition of glutathione.
These suggests that -SH groups may be involved in the catalytic activity of the present COX. 相似文献