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1.
Metschnikowia reukaufii W6b isolated from marine environment was found to produce a cell-bound acid protease. The full-length cDNA (cDNASAP6 gene) of the acid protease (SAP6) from the marine-derived yeast M. reukaufii W6b was cloned. The insert was 1,755-bp long and contained an open reading frame of 1,527-bp encoding 508 amino acids. The
deduced amino acid sequence included a signal peptide of 16 amino acids. The consensus motifs contained a VLLDTGSSDLRM active
site and an ALLDSGTTITQF active site. The protein sequence deduced from the cDNASAP6 gene exhibited 12.9% overall identity with Cwp1 of Saccharomyces cerevisiae and a hydropathy profile characteristic of glycosylphosphatidylinositol cell-wall proteins. The cDNASAP6 gene without 48 bp encoding the signal peptide sequence was subcloned into an expression plasmid pET-24a (+) and fused with
a 6-His Tag and transformed into Escherichia coli BL21 (DE3) for recombinant expression of the protease. The expressed fusion protein was found to have a unique band with
molecular mass of about 54 kDa. The crude acid protease of the culture of the marine yeast strain W6b and the crude recombinant
acid protease had milk clotting activity. 相似文献
2.
Meijuan Xu Zhiming Rao Hong Xu Chunyan Lan Wenfang Dou Xiaomei Zhang Hongyu Xu Jian Jin Zhenghong Xu 《Applied biochemistry and biotechnology》2011,163(6):707-719
Corynebacterium crenatum SYPA 5-5 is an aerobic and industrial l-arginine producer. It was proved that the Corynebacterium glutamicum/Escherichia coli shuttle vector pJC1 could be extended in C. crenatum efficiently when using the chloramphenicol acetyltransferase gene (cat) as a reporter under the control of promoter tac. The expression system was applied to over-express the gene vgb coding Vitreoscilla hemoglobin (VHb) to further increase the dissolved oxygen in C. crenatum. As a result, the recombinant C. crenatum containing the pJC-tac-vgb plasmid expressed VHb at a level of 3.4 nmol g−1, and the oxygen uptake rates reached 0.25 mg A562−1 h−1 which enhanced 38.8% compared to the wild-type strain. Thus, the final l-arginine concentration of the batch fermentation reached a high level of 35.9 g L−1, and the biomass was largely increased to 6.45 g L−1, which were 17.3% and 10.5% higher than those obtained by the wild-type strain, respectively. To our knowledge, this is the
first report that the efficient expression system was constructed to introduce vgb gene increasing the oxygen and energy supply for l-arginine production in C. crenatum, which supplies a good strategy for the improvement of amino acid products. 相似文献
3.
Xiaofeng Wang Yongchuan Sun Feng Ke Heyun Zhao Tao Liu Li Xu Yun Liu Yunjun Yan 《Applied biochemistry and biotechnology》2012,166(5):1355-1367
A gene encoding Yarrowia lipolytica lipase LIP2 (YlLIP2) was cloned into a constitutive expression vector pGAPZαA and electrotransformed into the Pichia pastoris X-33 strain. The high-yield clones obtained by high copy and enzyme activity screening were chosen as the host strains for
shaking flask and fermentor culture. The results showed that glucose was the optimum carbon source for YlLIP2 production,
and the maximum hydrolytic activity of recombinant YlLIP2 reached 1,315 U/ml under the flask culture at 28 °C, pH 7.0, for
48 h. The fed-batch fermentation was carried out in 3- and 10-l bioreactors by continuously feeding glucose into the growing
medium for achieving high cell density and YlLIP2 yields. The maximum hydrolytic activity of YlLIP2 and cell density obtained
in the 3-l bioreactor were 10,300 U/ml and 116 g dry cell weight (DCW)/l, respectively. The peak hydrolytic activity of YlLIP2
and cell density were further improved in the 10-l fermentor where the values respectively attained were 13,500 U/ml and 120 g
DCW/l. The total protein concentration in the supernatant reached 3.3 g/l and the cell viability remained approximately 99%
after 80 h of culture. Furthermore, the recombinant YlLIP2 produced in P. pastoris pGAP and pAOX1 systems have similar content of sugar (about 12%) and biochemical characteristics. The above results suggest
that the GAP promoter-derived expression system of P. pastoris is effective for the expression of YlLIP2 by high cell density culture and is probably an alternative to the conventional
AOX1 promoter expression system in large-scale production of industrial lipases. 相似文献
4.
The protective antigen (PA) of Bacillus anthracis is a potent immunogen and an important candidate vaccine. In addition, it is used in monitoring systems like enzyme-linked
immunosorbent assay to assess antibodies against PA in immunized subjects. The low level of PA production in B. anthracis and the difficulty of separating it from other bacterial components have made the researchers do different studies with the
aim of producing recombinant PA (rPA). In this study, to produce rPA as a recombinant protein vaccine, the partial sequence
of protective antigen of B. anthracis, amino acids 175–764, as a potent immunogenic target was inserted in pET21b(+). This is a prokaryotic plasmid that carries
an N-terminal T7.tag sequence. The integrity of constructed plasmid was confirmed using restriction enzyme mapping. rPA was
expressed after induction with isopropyl β-d-1-thiogalactopyranoside in Escherichia coli BL21. Purification of rPA was done with an affinity system using anti T7.tag antibody. Electrophoresis and Western blotting
confirmed the specificity of the expressed protein. BALB/c mice were immunized with obtained PA protein and evaluation of
specific immunoglobulin G antibodies against PA in sera using Western blotting method and showed that rPA is immunogenic.
The challenge of immunized mice with virulent strain of B. anthracis showed that rPA is functional to protect against pathogenic strain. 相似文献
5.
6.
Chen Dong Guolin Li Zhuoqi Li Honglin Zhu Mingquan Zhou Zhongli Hu 《Applied biochemistry and biotechnology》2009,158(3):605-614
A rapid amplification cDNA end (RACE) assay was established to achieve the complete sequence of mitochondrial manganese-superoxide
dismutase (Mn-SOD) cDNA in Nelumbo nucifera. The obtained full-length cDNA of Mn-SOD was 926 bp and contained a 699-bp open reading frame encoding an Mn-SOD precursor of 233 amino acids. The recombinant of
Mn-SOD expressed by PET-32a vector in Escherichia coli BL21 was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blotting assays. A 3D structural
model of the Mn-SOD was constructed by homology modeling. Real-time polymerase chain reaction analysis revealed that Mn-SOD mRNA was expressed in young leaves, blossom, stems, and terminal buds during reproductive stage but with the highest expression
in young leaves. This significant difference demonstrated the differential expression of Mn-SOD in various organs of N. nucifera. 相似文献
7.
The 5-aminolevulinate (ALA) synthase gene (hemA) from Agrobacterium radiobacter zju-0121, which was cloned previously in our laboratory, contains several rare codons. To enhance the expression of this
gene, Escherichia coli Rosetta(DE3), which is a rare codon optimizer strain, was picked out as the host to construct an efficient recombinant strain.
Cell extracts of the recombinant E. coli were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under the appropriate conditions. The results
indicated that the activity of ALA synthase expressed in Rosetta(DE3)/pET-28a(+)-hemA was about 20% higher than that in E. coli BL21(DE3). Then the effects of precursors (glycine and succinate) and glucose, which is an inhibitor for ALA dehydratase
as well as the carbon sources for cell growth, on the production of 5-aminolevulinate were investigated. Based on an optimal
fed-batch culture system described in our previous work, up to 6.5 g/l (50 mM) ALA was produced in a 15-l fermenter. 相似文献
8.
Fatemeh Vahedi Mojtaba Sankian Malihe Moghadam Maryam Mohaddesfar Sirous Ghobadi Abdol Reza Varasteh 《Applied biochemistry and biotechnology》2011,163(7):895-905
Chenopodium album is a weedy annual plant in the genus Chenopodium. C. album pollen represents a predominant allergen source in Iran. The main C. album pollen allergens have been described as Che a 1, Che a 2, and Che a 3. The aim of this work was to clone the Che a 1 in Escherichia coli to establish a system for overproduction of the recombinant Che a 1 (rChe a 1). In order to clone this allergen, the pollens
were subjected to RNA extraction. A full-length fragment encoding Che a 1 was prepared by polymerase chain reaction amplification
of the first-strand cDNA synthesized from extracted RNA. Cloning was carried out by inserting the cDNA into the pET21b (+)
vector, thereafter the construct was transformed into E. coli Top10 cells and expression of the protein was induced by IPTG. The rChe a 1 was purified using histidine tag in recombinant
protein by means of Ni–NTA affinity chromatography. IgE immunoblotting, ELISA, and inhibition ELISA were done to evaluate
IgE binding of the purified protein. In conclusion, the cDNA for the major allergen of the C. album pollen, Che a 1, was successfully cloned and rChe a 1 was purified. Inhibition assays demonstrated allergic subjects sera
reacted with rChe a 1 similar to natural Che a 1 in crude extract of C. album pollen. This study is the first report of using the E. coli as a prokaryotic system for Che a 1 cloning and production of rChe a 1. 相似文献
9.
Wei Zhao Aisheng Xiong Xiaoyan Fu Feng Gao Yongsheng Tian Rihe Peng 《Applied biochemistry and biotechnology》2010,162(8):2157-2165
To obtain a high level expression of phytase with favorable characteristics, a codon-optimized phytase gene from Citrobacter freundii was synthesized and transferred into Pichia pastoris. Small-scale expression experiments and activity assays were used to screen positive colonies. After purified by Ni2+–NTA agarose affinity column, the characterizations of the recombinant phytase were determined. The recombinant phytase (r-phyC)
had two distinct pH optima at 2.5 and 4.5 and an optimal temperature at 50 °C. It retained more than 80% activity after being
incubated under various buffer (pH 1.5–8.0) at 37 °C for 1 h. The specific activity, Km, and Vmax values of r-phyC for sodium phytate were 2,072 ± 18 U mg−1, 0.52 ± 0.04 mM, and 2,380 ± 84 U mg−1 min−1, respectively. The enzyme activity was significantly improved by 1 mM of K+, Ca2+, and Mg2+. These characteristics contribute to its potential application in feed industry. 相似文献
10.
The triosephosphate isomerase of Leishmania donovani (LdTIM) was expressed at high level in Escherichia coli. The TIM gene was cloned in expression vector pET-23(a) with C-terminal 6× His tag fused in frame, and expressed as a 27.6-kDa
protein in E. coli as inclusion bodies. The recombinant LdTIM from E. coli lysate was solubilized in 6 M guanidine hydrochloride and purified by Ni-NTA chromatography. In the present study, the effect
of bovine serum albumin on the reactivation of TIM was investigated. Furthermore, 8-anilino-1-naphthalene sulfonic acid was
used to detect the structural changes induced by bovine serum albumin (BSA). Here, we conclude that BSA assists in the refolding
and regain of LdTIM enzyme activity by providing framework for structure formation. This study indicates that numerous protein–protein
contacts are constantly occurring inside the cell that leads to the formation of native protein. 相似文献
11.
Nie Z Xu J Chen J Lv Z Wang D Sheng Q Wu Y Wang X Wu X Zhang Y 《Applied biochemistry and biotechnology》2009,158(1):59-71
A recombinant Bombyx mori profilin protein (rBmPFN) was overexpressed in Escherichia coli BL21. Purified rBmPFN was used to generate anti-BmPFN polyclonal antibody, which were used to determine the subcellular localization
of BmPFN. Immunostaining indicated that profilin can be found in both the nucleus and cytoplasm but is primarily located in
the cytoplasm. Real-time RT-PCR and Western blot analyses indicated that, during the larvae stage, profilin expression levels
are highest in the silk gland, followed by the gonad, and are lowest in the fatty body. Additionally, BmPFN expression begins
during the egg stage, increases during the larvae stage, reaches a peak during the pupa stage, and decreases significantly
in the moth. Therefore, we propose that BmPFN may play an important role during larva stage development, especially in the
silk gland. 相似文献
12.
Qiong-ming Xu Quan Li Yan-li Liu Yu-lin Feng Shi-lin Yang Xiao-ran Li 《Chemistry of Natural Compounds》2010,46(3):366-369
In the search for platelet-activating-factor (PAF) antagonists, two new lignan compounds were isolated from the leaves of
Syringa reticulata Hara var. mandshurica. Their structures were elucidated as (7R,8S, 8'S)-3,4,3',4'-dimethylenedioxy-8,9-dihydroxy-8.8', 7-O-9'-lignan (mandshuricol A) and (7R,8S,8'S)-3',4'methylenedioxy-4-methoxy-3,8,9-trihydroxy-8.8', 7-O-9'-lignan (mandshuricol B), Mandshuricol A and B showed antagonistic activity on PAF in the [3H] PAF receptor binding assay with IC50 values of 4.8 × 10–5 M and 3.5 × 10–5 M, respectively. 相似文献
13.
14.
Yao Guo Fengxia Lu Haizhen Zhao Yanchong Tang Zhaoxin Lu 《Applied biochemistry and biotechnology》2010,162(2):498-509
A gene of glucose oxidase (GOD) from Aspergillus niger Z-25 was cloned and sequenced. The entire open reading frame (ORF) consisted of 1,818 bp and encoded a putative peptide of
605 amino acids. The gene was fused to the pPICZαA plasmid and overexpressed in Pichia pastoris SMD1168. The recombinant GOD (rGOD) was secreted into the culture using MF-α factor signal peptide under the control of the
AOX1 promoter. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that rGOD exhibited a single band at around
94 kDa. The maximal GOD activity of approximately 40 U/mL was achieved in shake flask by induction under optimal conditions
after 7 days. rGOD was purified by ammonium sulfate precipitate leading to a final specific activity of 153.46 U/mg. The optimum
temperature and pH of the purified enzyme were 40 °C and 6.0, respectively. Over 88% of maximum activity was maintained below
40 °C. And the recombinant enzyme displayed a favorable stability in the pH range from 4.0 to 8.0. The Lineweaver–Burk plotting
revealed that rGOD exhibited a K
m value of 16.95 mM and a K
cat value of 484.26 s−1. 相似文献
15.
A phosphite dehydrogenase gene (ptdhK) consisting of 1,011-bp nucleotides which encoding a peptide of 336 amino acid residues was cloned from Pseudomonas sp. K. gene ptdhK was expressed in Escherichia coli BL21 (DE3) and the corresponding recombinant enzyme was purified by metal affinity chromatography. The recombinant protein
is a homodimer with a monomeric molecular mass of 37.2 kDa. The specific activity of PTDH-K was 3.49 U mg−1 at 25 °C. The recombinant PTDH-K exhibited maximum activity at pH 3.0 and at 40 °C and displayed high stability within a
wide range of pHs (5.0 to 10.5). PTDH-K had a high affinity to its natural substrates, with K
m values for sodium phosphite and NAD of 0.475 ± 0.073 and 0.022 ± 0.007 mM, respectively. The activity of PTDH-K was enhanced
by Na+, NH4+, Mg2+, Fe2+, Fe3+, Co2+, and EDTA, and PTDH-K exhibited different tolerance to various organic solvents. 相似文献
16.
S. X. Xiao J. J. Zhang X. Li L. J. Ye H. W. Gu N. Ren 《Journal of Thermal Analysis and Calorimetry》2010,102(2):813-817
A ternary binuclear complex of dysprosium chloride hexahydrate with m-nitrobenzoic acid and 1,10-phenanthroline, [Dy(m-NBA)3phen]2·4H2O (m-NBA: m-nitrobenzoate; phen: 1,10-phenanthroline) was synthesized. The dissolution enthalpies of [2phen·H2O(s)], [6m-HNBA(s)], [2DyCl3·6H2O(s)], and [Dy(m-NBA)3phen]2·4H2O(s) in the calorimetric solvent (VDMSO:VMeOH = 3:2) were determined by the solution–reaction isoperibol calorimeter at 298.15 K to be
\Updelta\texts H\textmq \Updelta_{\text{s}} H_{\text{m}}^{\theta } [2phen·H2O(s), 298.15 K] = 21.7367 ± 0.3150 kJ·mol−1,
\Updelta\texts H\textmq \Updelta_{\text{s}} H_{\text{m}}^{\theta } [6m-HNBA(s), 298.15 K] = 15.3635 ± 0.2235 kJ·mol−1,
\Updelta\texts H\textmq \Updelta_{\text{s}} H_{\text{m}}^{\theta } [2DyCl3·6H2O(s), 298.15 K] = −203.5331 ± 0.2200 kJ·mol−1, and
\Updelta\texts H\textmq \Updelta_{\text{s}} H_{\text{m}}^{\theta } [[Dy(m-NBA)3phen]2·4H2O(s), 298.15 K] = 53.5965 ± 0.2367 kJ·mol−1, respectively. The enthalpy change of the reaction was determined to be
\Updelta\textr H\textmq = 3 6 9. 4 9 ±0. 5 6 \textkJ·\textmol - 1 . \Updelta_{\text{r}} H_{\text{m}}^{\theta } = 3 6 9. 4 9 \pm 0. 5 6 \;{\text{kJ}}\cdot {\text{mol}}^{ - 1} . According to the above results and the relevant data in the literature, through Hess’ law, the standard molar enthalpy of
formation of [Dy(m-NBA)3phen]2·4H2O(s) was estimated to be
\Updelta\textf H\textmq \Updelta_{\text{f}} H_{\text{m}}^{\theta } [[Dy(m-NBA)3phen]2·4H2O(s), 298.15 K] = −5525 ± 6 kJ·mol−1. 相似文献
17.
T. F. Ibragimov M. G. Levkovich V. A. Saprykina Kh. M. Shakhidoyatov 《Chemistry of Natural Compounds》2010,46(5):767-770
N-Chloroacetylcytisine was synthesized by acylation of (–)-cytisine. Stable Z- and E-conformers with respect to rotational isomerism around the N-12–CO bond were found in PMR spectra at room temperature. The
point at which PMR resonances of the Z- and E-conformers coalesced upon heating was measured. The transition barrier between the conformers was estimated. 相似文献
18.
Farshad Sohbatzadeh Abasalt Hosseinzadeh Colagar Saeed Mirzanejhad Somayeh Mahmodi 《Applied biochemistry and biotechnology》2010,160(7):1978-1984
We developed and employed a new geometrical structure of dielectric barrier discharge in atmospheric pressure for bacterial
broad spectrum sterilization. We utilized a plasma source having an AC power supply at 50 HZ and 5,400 V (rms value). We prepared
suspensions of the Gram-negative bacteria species (Escherichia coli, Pseudomonas aeruginosa) and a Gram-positive of Bacillus cereus with Luria–Bertani broth media up to OD600 nm = 0.25 of McFarland standard. Afterglow of non-thermal atmospheric pressure plasma treated these suspensions. The influence
of the atmospheric plasma afterglow on the species was assayed in different time durations 5, 10, and 15 min. The spectroscopic
results of this investigation indicated that the survival reduction of the species can reach to 100% for P. aeruginosa in an exposure time of 10 min, E. coli and B. cereus in an exposure time of 15 min. 相似文献
19.
Glucose 2-oxidase (pyranose oxidase, pyranose:oxygen-2-oxidoreductase, EC 1.1.3.10) from Coriolus versicolor catalyses the oxidation of d-glucose at carbon 2 in the presence of molecular O2 producing d-glucosone (2-keto-glucose and d-arabino-2-hexosulose) and H2O2. It was used to convert d-glucose into d-glucosone at moderate pressures (i.e. up to 150 bar) with compressed air in a modified commercial batch reactor. Several
parameters affecting biocatalysis at moderate pressures were investigated as follows: pressure, [enzyme], [glucose], pH, temperature,
nature of fluid and the presence of catalase. Glucose 2-oxidase was purified by immobilized metal affinity chromatography
on epoxy-activated Sepharose 6B-IDA-Cu(II) column at pH 6.0. The rate of bioconversion of d-glucose increased with the pressure since an increase in the pressure with compressed air resulted in higher rates of conversion.
On the other hand, the presence of catalase increased the rate of reaction which strongly suggests that H2O2 acted as inhibitor for this reaction. The rate of bioconversion of d-glucose by glucose 2-oxidase in the presence of either nitrogen or supercritical CO2 at 110 bar was very low compared with the use of compressed air at the same pressure. The optimum temperature (55°C) and
pH (5.0) of d-glucose bioconversion as well as kinetic parameters for this enzyme were determined under moderate pressure. The activation
energy (E
a) was 32.08 kJ mol−1 and kinetic parameters (V
max, K
m, K
cat and K
cat/K
m) for this bioconversion were 8.8 U mg−1 protein, 2.95 mM, 30.81 s−1 and 10,444.06 s−1 M−1, respectively. The biomass of C. versicolor as well as the cell-free extract containing glucose 2-oxidase activity were also useful for bioconversion of d-glucose at moderate pressures. The enzyme was apparently stable at moderate pressures since such pressures did not affect
significantly the enzyme activity. 相似文献
20.
Rym Agrebi Noomen Hmidet Mohamed Hajji Nawrez Ktari Anissa Haddar Nahed Fakhfakh-Zouari Moncef Nasri 《Applied biochemistry and biotechnology》2010,162(1):75-88
In this study, Mirabilis jalapa tuber powder (MJTP) was used as a new complex organic substrate for the growth and production of fibrinolytic enzymes by
a newly isolated Bacillus amyloliquefaciens An6. Maximum protease activity (1,057 U/ml) with casein as a substrate was obtained when the strain was grown in medium containing
(grams per liter) MJTP 30, yeast extract 6, CaCl2 1, K2HPO4 0.1, and K2HPO4 0.1. The strain was also found to grow and produce extracellular proteases in a medium containing only MJTP, indicating that
it can obtain its carbon, nitrogen, and salts requirements directly from MJTP. The B. amyloliquefaciens An6 fibrinase (BAF1) was partially purified, and fibrinolytic activity was assayed in a test tube with an artificial fibrin
clot. The molecular weight of the partially purified BAF1 fibrinolytic protease was estimated to be 30 kDa by sodium dodecyl
sulfate polyacrylamide gel electrophoresis and gel filtration. The optimum temperature and pH for the caseinolytic activity
were 60 °C and 9.0, respectively. The enzyme was highly stable from pH 6.0 to 11.0 and retained 62% of its initial activity
after 1 h incubation at 50 °C. However, the enzyme was inactivated at higher temperatures. The activity of the enzyme was
totally lost in the presence of phenylmethylsulfonyl fluoride, suggesting that BAF1 is a serine protease. 相似文献