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1.
Zhiqiang Wu Guoliang Jiang 《International journal of peptide research and therapeutics》2008,14(2):75-80
Using N-α-benzoyl-l-arginine p-nitroanilide (BApNA) as substrate, trypsin-like enzymes (TLEs) were purified from mysis (Neomysis japonica) following two chromatographic steps, Sephacryl S100 HR gel filtration and Benzamidine-Sepharose 4B affinity. They presented
a high stability in the raw material, retaining over 45% of the initial activity after 30 days of storage at pH 8.0, 45 °C.
The purified TLEs had relative molecular mass between 32 kDa and 33 kDa. With higher stability and greater activity, they
had similar stability and activity profiles (pH 6.0–11.0, 15–65 °C) as bovine trypsin but had a different optimum temperature
(35 °C for trypsin and 45 °C for TLEs). Similar to bovine trypsin, the purified TLEs could be activated by Ca2+ and Mg2+. And the purified TLEs also showed similar inhibitory profiles as bovine trypsin with the exception of chicken egg ovomucoid
(CEOM), an effective inhibitor of bovine trypsin but less so for purified TLEs. Having TLEs with physiological efficiency
3.6 times that of bovine trypsin, the use of mysis as a source for commercial production of TLEs is discussed. 相似文献
2.
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
3.
Two genes that encode proteins which share 30–35% sequence identity with yeast OYE (Old Yellow Enzyme, an NAD(P)H FMN-oxidoreductase),
the well-studied archetype of the OYE protein family, have been identified in Gluconobacter oxydans M5. The two genes are localized in the chromosome and plasmid, respectively. Comparison of the deduced amino acid sequences
of the enzymes with database entries revealed 75.1% similarity and 64.9% identity to that of the Pseudomonas syringae pv. glycinea NAD(P)H-dependent 2-cyclohexen-1-one reductase. The two proteins were expressed as His-tag fusion proteins in Escherichia coli and purified. The ability of the purified proteins to hydrogenate citral was identified. The results showed that the α,β-double
bond of citral cis-isomer ‘neral’ could be stereoselectively reduced to produce citronellal by the purified OYE homologues. 相似文献
4.
Monodehydroascorbate reductase (MDHAR; EC 1.6.5.4) catalyses the reduction of the monodehydroascorbate (MDHA) radical to ascorbate,
using NADH or NADPH as an electron donor, and is believed to be involved in maintaining the reactive oxygen scavenging capability
of plant cells. This key enzyme in the ascorbate-glutathione cycle has been studied here in the moss Physcomitrella patens, which is tolerant to a range of abiotic stresses and is increasingly used as a model plant. In the present study, three
cDNAs encoding different MDHAR isoforms of 47 kDa were identified in P. patens, and found to exhibit enzymic characteristics similar to MDHARs in vascular plants despite low-sequence identity and a distant
evolutionary relationship between the species. The three cDNAs for the P. patens MDHAR enzymes were expressed in Escherichia coli and the active enzymes were purified and characterized. Each recombinant protein displayed an absorbance spectrum typical
of flavoenzymes and contained a single non-covalently bound FAD coenzyme molecule. The K
m and k
cat values for the heterologously expressed PpMDHAR enzymes ranged from 8 to 18 μM and 120–130 s−1, respectively, using NADH as the electron donor. The K
m values were at least an order of magnitude higher for NADPH. The K
m values for the MDHA radical were ∼0.5–1.0 μM for each of the purified enzymes, and further kinetic analyses indicated that
PpMDHARs follow a ‘ping–pong’ kinetic mechanism. In contrast to previously published data, site-directed mutagenesis indicated
that the conserved cysteine residue is not directly involved in the reduction of MDHA. 相似文献
5.
Nguyen VN Oh IJ Kim YJ Kim KY Kim YC Park RD 《Journal of industrial microbiology & biotechnology》2009,36(2):195-203
Two extracellular chitinases were purified from Paecilomyces variotii DG-3, a chitinase producer and a nematode egg-parasitic fungus, to homogeneity by DEAE Sephadex A-50 and Sephadex G-100 chromatography.
The purified enzymes were a monomer with an apparent molecular mass of 32 kDa (Chi32) and 46 kDa (Chi46), respectively, and
showed chitinase activity bands with 0.01% glycol chitin as a substrate after SDS-PAGE. The first 20 and 15 N-terminal amino
acid sequences of Chi32 and Chi46 were determined to be Asp-Pro-Typ-Gln-Thr-Asn-Val-Val-Tyr-Thr-Gly-Gln-Asp-Phe-Val-Ser-Pro-Asp-Leu-Phe
and Asp-Ala-X-X-Tyr-Arg-Ser-Val-Ala-Tyr-Phe-Val-Asn-Trp-Ala, respectively. Optimal temperature and pH of the Chi32 and Chi46
were found to be both 60°C, and 2.5 and 3.0, respectively. Chi32 was almost inhibited by metal ions Ag+ and Hg2+ while Chi46 by Hg2+ and Pb2+ at a 10 mM concentration but both enzymes were enhanced by 1 mM concentration of Co2+. On analyzing the hydrolyzates of chitin oligomers [(GlcNAc)
n
, n = 2–6)], it was considered that Chi32 degraded chitin oligomers as an exo-type chitinase while Chi46 as an endo-type chitinase. 相似文献
6.
Wang SL Lin YT Liang TW Chio SH Ming LJ Wu PC 《Journal of industrial microbiology & biotechnology》2009,36(1):65-73
A lipase-producing bacterium was isolated and identified as Pseudomonas monteilii TKU009. A lipase (F2) and lipase-like materials (F1) were purified from the culture supernatant of P. monteilii TKU009 with soybean powder as the sole carbon/nitrogen source. The molecular mass of F1 and F2 was estimated to be 44 kDa
by SDS-PAGE and gel filtration. The optimum pH, optimum temperature, and pH and thermal stabilities of F2 were 7, 40°C, 8–11,
and 50°C; and of F1 were 6, 40°C, 6–7, and 50°C, respectively. F2 was completely inhibited by EDTA and slightly by Mg2+, Fe2+, Mn2+, and SDS. F1 was completely inhibited by EDTA and Fe2+ and strongly by Zn2+, Mn2+, Ca2+, Mg2+, and SDS. The activities of both the enzymes were enhanced by the addition of non-ionic surfactants Triton X–100 and Tween
40, especially for F1. F2 preferably acted on substrates with a long chain (C10–C18) of fatty acids, while F1 showed a broad
spectrum on those with chain length of C4–C18. The marked activity of F2 in organic solvents makes it an ideal choice for
application in a water-restricted medium including organic synthesis.
Li-June Ming is a visiting Professor at the National Cheng Kung University. 相似文献
7.
Nack-Shick Choi Jae Jun Song Dong-Min Chung Young Jae Kim Pil Jae Maeng Seung-Ho Kim 《Journal of industrial microbiology & biotechnology》2009,36(3):417-426
A novel fibrinolytic enzyme (AJ) was purified from Staphylococcus sp. strain AJ screened from Korean salt-fermented Anchovy-jeot. Relative molecular weight of AJ was determined as 26 kDa by using SDS-PAGE and fibrin zymography. Based on a 2D gel, AJ
was found to consist of three active isoforms (pI 5.5–6.0) with the same N-terminal amino acid sequence. AJ exhibited optimum pH and temperature at 2.5–3.0 and 85°C, respectively.
AJ kept 85% of the initial activity after heating at 100°C for 20 min on the zymogram gel. The Michaelis constant (K
m) and K
cat values of AJ towards α-casein were 0.38 mM and 19.73 s−1, respectively. AJ cleaved the Aα-chain of fibrinogen but did not affect the Bβ- and γ-chains, indicating that it is an α-fibrinogenase.
The fibrinolytic activity was inhibited by diisopropyl fluorophosphate, indicating AJ is a serine protease. Interestingly,
AJ was very stable at acidic condition, SDS, and heat (100°C), whereas it was easily degraded at neutral and alkaline conditions.
In particular, AJ formed an active homo-dimer in the pH range from 7.0 to 8.0. To our knowledge, a similar combination of
acid and heat stability has not yet been reported for other fibrinolytic enzymes. 相似文献
8.
A chitinase (VpChiA) and its C-terminal truncated G589 mutant (VpChiAG589) of Vibrio parahaemolyticus were cloned by polymerase chain reaction (PCR) techniques. To study the role of the C-terminal 30 amino acids of VpChiA in
the enzymatic hydrolysis of chitin, both the recombinant VpChiA and VpChiAG589 encoded in 1,881 and 1,791 bp DNA fragments,
respectively, were expressed in Escherichia coli using the pET-20b(+) expression system. The His–Tag affinity purified VpChiA and VpChiAG589 enzymes had a calculated molecular
mass of 65,713 and 62,723 Da, respectively. The results of biochemical characterization including kinetic parameters, spectroscopy
of fluorescence and circular dichroism, chitin-binding and hydrolysis, and thermostability, both VpChiA and VpChiAG589, had
very similar physicochemical properties such as the optimum pH (6), temperature (40°C), and kinetic parameters of Km and kcat against the 4MU–(GlcNAc)2 or 4MU–(GlcNAc)3 soluble substrates. The significant increase of thermostability and the drastic decrease of the hydrolyzing ability of VpChiAG589
toward the insoluble α-chitin substrate suggested that a new role could be played by the C-terminal 30 amino acids. 相似文献
9.
Kazuoka T Oikawa T Muraoka I Kuroda S Soda K 《Extremophiles : life under extreme conditions》2007,11(2):257-267
An NAD+-dependent alcohol dehydrogenase of a psychrotorelant from Antarctic seawater, Flavobacterium frigidimaris KUC-1 was purified to homogeneity with an overall yield of about 20% and characterized enzymologically. The enzyme has an
apparent molecular weight of 160k and consists of four identical subunits with a molecular weight of 40k. The pI value of the enzyme and its optimum pH for the oxidation reaction were determined to be 6.7 and 7.0, respectively. The enzyme
contains 2 gram-atoms Zn per subunit. The enzyme exclusively requires NAD+ as a coenzyme and shows the pro-R stereospecificity for hydrogen transfer at the C4 position of the nicotinamide moiety of NAD+. F. frigidimaris KUC-1 alcohol dehydrogenase shows as high thermal stability as the enzymes from thermophilic microorganisms. The enzyme is
active at 0 to over 85°C and the most active at 70°C. The half-life time and k
cat value at 60°C were calculated to be 50 min and 27,400 min−1, respectively. The enzyme also shows high catalytic efficiency at low temperatures (0–20°C) (k
cat/K
m at 10°C; 12,600 mM−1 min−1) similar to other cold-active enzymes from psychrophiles. The alcohol dehydrogenase gene is composed of 1,035 bp and codes
344 amino acid residues with an estimated molecular weight of 36,823. The sequence identities were found with the amino acid
sequences of alcohol dehydrogenases from Moraxella sp. TAE123 (67%), Pseudomonas aeruginosa (65%) and Geobacillus stearothermophilus LLD-R (56%). This is the first example of a cold-active and thermostable alcohol dehydrogenase. 相似文献
10.
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
11.
Kasieczka-Burnecka M Kuc K Kalinowska H Knap M Turkiewicz M 《Applied microbiology and biotechnology》2007,77(1):77-89
Two extracellular tannin acyl hydrolases (TAH I and TAH II) produced by an Antarctic filamentous fungus Verticillium sp. P9 were purified to homogeneity (7.9- and 10.5-fold with a yield of 1.6 and 0.9%, respectively) and characterized. TAH
I and TAH II are multimeric (each consisting of approximately 40 and 46 kDa sub-units) glycoproteins containing 11 and 26%
carbohydrates, respectively, and their molecular mass is approximately 155 kDa. TAH I and TAH II are optimally active at pH
of 5.5 and 25 and 20°C, respectively. Both the enzymes were activated by Mg2+and Br− ions and 0.5–2.0 M urea and inhibited by other metal ions (Zn2+, Cu2+, K+, Cd2+, Ag+, Fe3+, Mn2+, Co2+, Hg2+, Pb2+ and Sn2+), anions, Tween 20, Tween 60, Tween 80, Triton X-100, sodium dodecyl sulphate, β-mercaptoethanol, α-glutathione and 4-chloromercuribenzoate.
Both tannases more efficiently hydrolyzed tannic acid than methyl gallate. E
a of these reactions and temperature dependence (at 0–30°C) of k
cat, k
cat/K
m, ΔG*, ΔH* and ΔS* for both the enzymes and substrates were determined. The k
cat and k
cat/K
m values (for both the substrates) were considerably higher for the combined preparation of TAH I and TAH II. 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.
Gundllapalli SB Pretorius IS Cordero Otero RR 《Journal of industrial microbiology & biotechnology》2007,34(6):413-421
Enzyme engineering was performed to link the β-glucosidase enzyme (BGL1) from Saccharomycopsis fibuligera to the cellulose-binding domain (CBD2) of Trichoderma reesei cellobiohydrolase (CBHII) to investigate the effect of a fungal CBD on the enzymatic characteristics of this non-cellulolytic
yeast enzyme. Recombinant enzymes were constructed with single and double copies of CBD2 fused at the N-terminus of BGL1 to
mimic the two-domain organization displayed by cellulolytic enzymes in nature. The engineered S. fibuligera β-glucosidases were expressed in Saccharomyces cerevisiae under the control of phosphoglycerate-kinase-1 promoter (PGK1
P
) and terminator (PGK1
T
) and yeast mating pheromone α-factor secretion signal (MFα1
S
). The secreted enzymes were purified and characterized using a range of cellulosic and non-cellulosic substrates to illustrate
the effect of the CBD on their enzymatic activity. The results indicated that the recombinant enzymes of BGL1 displayed a
2–4-fold increase in their hydrolytic activity toward cellulosic substrates like avicel, amorphous cellulose, bacterial microcrystalline
cellulose, and carboxy methyl cellulose in comparison with the native enzyme. The organization of the CBD in these recombinant
enzymes also resulted in enhanced substrate affinity, molecular flexibility and synergistic activity, thereby improving the
ability of the enzymes to act on and hydrolyze cellulosic substrates, as characterized by adsorption, kinetics, thermal stability,
and scanning electron microscopic analyses. 相似文献
14.
A novel phytase gene appA, with upstream and downstream sequences from Citrobacter amalonaticus CGMCC 1696, was cloned by degenerate polymerase chain reaction (PCR), and thermal asymmetric interlaced (TAIL) PCR and was
overexpressed in Pichia pastoris. Sequence analysis revealed one open reading frame that consisted of 1311 bp encoding a 436–amino-acid protein, which had
a deduced molecular mass of 46.3 kDa. The phytase appA belongs to the histidine acid phosphatase family and exhibits the highest
identity (70.1%) with C. braakii phytase. The gene was overexpressed in P. pastoris. The secretion yield of recombinant appA protein was accumulated to approximately 4.2 mg·mL−1, and the enzyme activity level reached 15,000 U·mL−1, which is higher than any previous reports. r-appA was glycosylated, as shown by Endo H treatment. r-appA was purified and
characterized. The specific activity of r-appA for sodium phytate was 3548 U·mg−1. The optimum pH and temperature for enzyme activity were 4.5 and 55°C, respectively. r-appA was highly resistant to pepsin
or trypsin treatment. This enzyme could be an economic and efficient alternative to the phytases currently used in the feed
industry. 相似文献
15.
Bin Wu Licheng Wu Daijie Chen Zhijun Yang Minyu Luo 《Journal of industrial microbiology & biotechnology》2009,36(3):451-459
A novel fibrinolytic enzyme from Fusarium sp. CPCC 480097, named Fu-P, was purified to electrophoretic homogeneity using ammonium sulfate precipitation and ion exchange
and gel filtration chromatography. Fu-P, a single protein had a molecular weight of 28 kDa, which was determined by SDS-PAGE
and gel filtration chromatography. The isoelectric point of Fu-P determined by isoelectric focusing electrophoresis (IEF)
was 8.1, and the optimum temperature and pH value were 45°C and 8.5, respectively. Fu-P cleaved the α-chain of fibrin (ogen)
with high efficiency, and the β-chain and γ-γ (γ-)-chain with lower efficiency. Fu-P activity was inhibited by EDTA and PMSF,
and the enzyme exhibited a high specificity for the chymotrypsin substrate S-2586. Fu-P was therefore identified as a chymotrypsin-like
serine metalloprotease. The first 15 amino acids of the N-terminal sequence of Fu-P were Q-A-S–S-G-T-P-A-T-I-R-V-L-V–V and
showed no homology with that of other known fibrinolytic enzymes. This protease may have potential applications in thrombolytic
therapy and in thrombosis prevention. 相似文献
16.
Shijin J. Wu Zhihang H. Hu Lili L. Zhang Xiang Yu Jianmeng M. Chen 《Applied microbiology and biotechnology》2009,82(4):731-740
Dichloromethane (DCM)-degrading bacterium strain wh22 (GenBank accession number FJ418643) was isolated and identified as Lysinibacillus sphaericus based on standard morphological and physiological properties, cellular fatty acid composition, mole percent guanine–cytosine
content, and nucleotide sequence analysis of enzymatically amplified 16S ribosomal deoxyribonucleic acid. The strain also
grew on many other halocarbons found in the waste gases of industrial effluents, such as 1,2-dichloroethane, chlorobromomethane,
methylene bromide, 1,1,1-trichloroethane, trichloroethylene, and hexachlorobenzene. The strain harbored a novel degradative
plasmid, pRC11 (48.8 kb). The genes coding for the metabolism of DCM were found to be plasmid-borne, and a physical map of
the plasmid has been established. The purified plasmid was transformed to dcm
−
Escherichia coli DH5 at a rate of 1.65 × 105. The transformed cells were able to grow on DCM at a concentration of 5–16 mM and can be further used as an excellent source
for genetic manipulations leading to the construction of genetically modified microbial strains or genetically engineered
microorganisms. 相似文献
17.
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
18.
Park SY Kim JT Kang SG Woo JH Lee JH Choi HT Kim SJ 《Applied microbiology and biotechnology》2007,77(1):107-115
Vibrio sp. GMD509, a marine bacterium isolated from eggs of the sea hare, exhibited lipolytic activity on tributyrin (TBN) plate, and the gene representing lipolytic activity was cloned. As a result,
an open reading frame (ORF) consisting of 1,017 bp (338 aa) was found, and the deduced amino acid sequence of the ORF showed
low similarity (<20%) to α/β hydrolases such as dienelactone hydrolases and esterase/lipase with G–X1–S–X2–G sequence conserved. Phylogenetic analysis suggested that the protein belonged to a new family of esterase/lipase together
with various hypothetical proteins. The enzyme was overexpressed in Escherichia coli and purified to homogeneity. The purified enzyme (Vlip509) showed the best hydrolyzing activity toward p-nitrophenyl butyrate (C4) among various p-nitrophenyl esters (C2 to C18), and optimal activity of Vlip509 occurred at 30°C and pH 8.5, respectively. Kinetic parameters toward p-nitrophenyl butyrate were determined as K
m (307 μM), k
cat (5.72 s−1), and k
cat/K
m (18.61 s−1 mM−1). Furthermore, Vlip509 preferentially hydrolyzed the S-enantiomer of racemic ofloxacin ester. Despite its sequence homology to dienelactone hydrolase, Vlip509 showed no dienelactone
hydrolase activity. This study represents the identification of a novel lipolytic enzyme from marine environment. 相似文献
19.
Erika Nahomy Marino-Marmolejo Antonio De León-Rodríguez Ana Paulina Barba de la Rosa Leticia Santos 《Molecular biotechnology》2009,42(1):61-67
Analysis of the Thermoplasma acidophilum DSM 1728 genome identified two putative alcohol dehydrogenase (ADH) open reading frames showing 50.4% identity against each
other. The corresponding genes Ta0841 and Ta1316 encode proteins of 336 and 328 amino acids with molecular masses of 36.48 and 36.01 kDa, respectively. The genes were expressed
in Escherichia coli and the recombinant enzymes were functionally assessed for activity. Throughout the study only Ta1316 ADH resulted active
in the oxidative reaction in the pH range 2–8 (optimal pH 5.0) and temperatures from 25 to 90°C (optimal 75°C). This ADH catalyzes
the oxidation of several alcohols such as ethanol, methanol, 2-propanol, butanol, and pentanol during the reduction of the
cofactor NAD+. The highest activity was found in the presence of ethanol producing optically pure acetaldehyde. The specific enzyme activity
of the purified Ta1316 ADH with ethanol as a substrate in the optimal conditions was 628.7 U/mg. 相似文献
20.
Rajan LA Joseph TC Thampuran N James R Chinnusamy V Bansal KC 《Archives of microbiology》2008,190(4):481-487
Ectoine, a cyclic tetrahydropyrimidine (2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid), is a natural compound, which
serves as a protective substance in many bacterial cells. In this study, the putative ectABC gene cluster from Bacillus halodurans was heterologously expressed in E. coli and the production of ectoine was confirmed by HPLC analysis. The activity of the enzymes coded by the ectA, B and C genes were found to be higher in induced transgenic cells compared to the uninduced cells. Phylogenetic analysis revealed
sequence identities ranging from 36–73% for ectA gene, 55–81% for ectB gene and 55–80% for ectC gene indicating that the enzymes are evolutionarily well conserved. 相似文献