共查询到20条相似文献,搜索用时 31 毫秒
1.
OsLEA4, a late embryogenesis abundant (LEA) protein gene from rice (Oryza sativa L.), contains a 312-bp open reading frame encoding a putative polypeptide of 103 amino acids with a calculated molecular
mass of 11.19 kDa and a theoretical pI of 10.04. OsLEA4 polypeptide is rich in Ala (22%), Lys (15%), Glu (9%), His (8%), Thr (8%), and Arg (7%) and lacking in Trp,
Cys, Asn, and Phe residues. OsLEA4 protein contains a Pfam:LEA_1 domain architecture at positions 1–73 with three α-helical
domains and without β-sheet domain. In silico predictions showed that OsLEA4 protein was strongly hydrophilic with the grand average of hydropathy value of −0.816 and instability index of 27.31. The
hydrophilic regions were found in the conserved motif of OsLEA4. OsLEA4 gene was introduced into Escherichia coli, and a fusion protein (∼29.4 kDa) was expressed after isopropylthio-β-d-galactoside inducting by sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis. OsLEA4 protein enhanced the
tolerance of E. coli recombinant to high salinity, heat, freezing, and UV radiation, which suggested that OsLEA4 protein may play a protective
role under stressed conditions. This is the first successful use of E. coli as a prokaryotic system for LEA production from rice. 相似文献
2.
F. Ceyda Dudak İsmail H. Boyacı Agnese Jurkevica Mahmud Hossain Zoraida Aquilar H. Brian Halsall Carl J. Seliskar William R. Heineman 《Analytical and bioanalytical chemistry》2009,393(3):949-956
A rapid and convenient assay system was developed to detect viable Escherichia coli in water. The target bacteria were recovered from solution by immunomagnetic separation and incubated in tryptic soy broth
with isopropyl-β-d-thiogalactopyranoside, which induces formation of β-galactosidase in viable bacteria. Lysozyme was used to lyse E. coli cells and release the β-galactosidase. β-Galactosidase converted 4-methylumbelliferyl-β-d-galactoside to 4-methylumbelliferone (4-MU), which was measured by fluorescence spectrophotometry using excitation and emission
wavelengths of 355 and 460 nm, respectively. Calibration graphs of 4-MU fluorescence intensity versus E. coli concentration showed a detection range between 8 × 104 and 1.6 × 107 cfu mL−1, with a total analysis time of less than 3 h. The advantage of this method is that it detects viable cells because it is
based on the activity of the enzyme intrinsic to live E. coli. 相似文献
3.
Bing Chen Li-Qiang Fan Jian-He Xu Jian Zhao Xian Zhang Li-Ming Ouyang 《Applied biochemistry and biotechnology》2010,162(3):744-756
The levo-lactonase gene of Fusarium proliferatum ECU2002 (EC3.1.1.25) was cloned and expressed in Escherichia coli JM109 (DE3) for biocatalytic resolution of industrially important chiral lactones, including DL-pantoyl lactone which was
a key precursor to calcium d-pantothenate. By increasing the biomass concentration and lowering the inducer (isopropyl-β-d-thiogalactoside) concentration and induction temperature, the lactonase production was significantly enhanced up to 20 kU/L,
which was 20 times higher than that of wild-type strain F. proliferatum ECU2002. The recombinant Fusarium lactonase was purified using immobilized metal affinity chromatography, and its SDS-PAGE revealed a molecular mass of 50 kDa
for the recombinant protein, suggesting that the enzyme was a simplex protein. Furthermore, biocatalytic properties of the
recombinant lactonase were investigated, including kinetic parameters, additive’s effect, and substrate specificity. The results
reported in this paper provide a feasible method to make the whole cells of E. coli JM109 (DE3) expressing lactonase gene to be a highly efficient and easy-to-make biocatalyst for asymmetric synthesis of chiral
compounds. 相似文献
4.
Functional expression of a β-d-1,4 glucanase-encoding gene (egl1) from a filamentous fungus was achieved in both Escherichia coli and Saccharomyces cerevisiae using a modified version of pRS413. Optimal activity of the E. coli-expressed enzyme was found at incubation temperatures of 60°C, whereas the enzyme activity was optimal at 40°C when expressed
by S. cerevisiae. Enzyme activity at different pH levels was similar for both bacteria and yeast, being highest at 5.0. Yeast expression resulted
in a highly glycosylated protein of approx 60 kDa, compared to bacterial expression, which resulted in a protein of 30 kDa.
The hyperglycosylated protein had reduced enzyme activity, indicating that E. coli is a preferred vehicle for production scale-up. 相似文献
5.
Ah-Reum Park Hye-Jung Kim Jung-Kul Lee Deok-Kun Oh 《Applied biochemistry and biotechnology》2010,160(8):2236-2247
We expressed a putative β-galactosidase from Sulfolobus acidocaldarius in Escherichia coli and purified the recombinant enzyme using heat treatment and Hi-Trap ion-exchange chromatography. The resultant protein gave
a single 57-kDa band by SDS-PAGE and had a specific activity of 58 U/mg. The native enzyme existed as a dimer with a molecular
mass of 114 kDa by gel filtration. The maximum activity of this enzyme was observed at pH 5.5 and 90 oC. The half-lives of
the enzyme at 70, 80, and 90 oC were 494, 60, and 0.2 h, respectively. The hydrolytic activity with p-nitrophenyl(pNP) substrates followed the order p-nitrophenyl-β-d-fucopyranoside > pNP-β-d-glucopyranoside > pNP-β-d-galactopyranoside > pNP-β-d-mannopyranoside > pNP-β-d-xylopyranoside, but not toward aryl-α-glycosides or pNP-β-l-arabinofuranoside. Thus, the enzyme was actually a β-glycosidase. The β-glycosidase exhibited transglycosylation activity
with pNP-β-d-galactopyranoside, pNP-β-d-glucopyranoside, and pNP-β-d-fucopyranoside in decreasing order of activity, in the reverse order of its hydrolytic activity. The hydrolytic activity
was higher toward cellobiose than toward lactose, but the transglycosylation activity was lower with cellobiose than with
lactose. 相似文献
6.
A simple one-step synthesis of β-d-galactopyranosyl azide from 0-nitrophenyl-β-d-galactopyranoside and azide catalyzed by E461G-β-galactosidase is described. The synthesis is quantitative in the presence
of excess azide and only the β anomer is produced. The product was purified (71% yield) from the other reaction components
by extraction with ethyl acetate, silica gel chromatography, and crystallization. The purity was verified by GLC, TLC, and
NMR. Thus, E461G-β-galactosidase is able to specifically and quantitatively from β-d-galactopyranosyl-azide. The purified β-d-galactopyranosylazide inhibited the growth of Escherichia coli that express β-galactosidase but not of E. coli that do not. Growth is stopped because β-galactosidase catalyzes the hydrolysis of the β-galactopyranosyl-azide, and the
azide that is produced inhibits cell growth. This selective inhibition of growth has potential application in molecular biology
screening. 相似文献
7.
Xu Z Li S Fu F Li G Feng X Xu H Ouyang P 《Applied biochemistry and biotechnology》2012,166(4):961-973
d-tagatose is a ketohexose that can be used as a novel functional sweetener in foods, beverages, and dietary supplements. This
study was aimed at developing a high-yielding d-tagatose production process using alginate immobilized Lactobacillus fermentum CGMCC2921 cells. For the isomerization from d-galactose into d-tagatose, the immobilized cells showed optimum temperature and pH at 65 °C and 6.5, respectively. The alginate beads exhibited
a good stability after glutaraldehyde treatment and retained 90% of the enzyme activity after eight cycles (192 h at 65 °C)
of batch conversion. The addition of borate with a molar ratio of 1.0 to d-galactose led to a significant enhancement in the d-tagatose yield. Using commercial β-galactosidase and immobilized L. fermentum cells, d-tagatose was successfully obtained from lactose after a two-step biotransformation. The relatively high conversion rate and
productivity from d-galactose to d-tagatose of 60% and 11.1 g l−1 h−1 were achieved in a packed-bed bioreactor. Moreover, lactobacilli have been approved as generally recognized as safe organisms,
which makes this L. fermentum strain an attracting substitute for recombinant Escherichia coli cells among d-tagatose production progresses. 相似文献
8.
The enzyme aspartate racemase from Pyrococcus horikoshii OT3 catalyzes the interconversion between l- and d-Asp. In this work, we employed the hybrid QM/MM approach with the self-consistent charge-density functional tight binding
(SCC-DFTB) model to study the catalytic mechanism for the conversion of l-Asp into d-Asp. The molecular dynamics simulation showed that the substrate l-Asp forms an extensive network of interactions with the active-site residues of the aspartate racemase through its side chain
carboxylate, ammonium group, and α-carboxylate. The potential of mean force calculations confirmed that the racemization reaction
involves two proton transfers (from the α-carbon to Cys194 and from Cys82 to the α-carbon), which occurs in a concerted way,
although highly asynchronous. The calculated free energy of activation is 17.5 kcal/mol, which is consistent with the reaction
rate measured from experiment. An electrostatic interaction analysis was performed to estimate the key role played by individual
residues in stabilizing the transition state. The docking study on the binding of l-Asp and d-Asp to aspartate racemase indicates that this enzyme employs a “two-base” mechanism not a “one-base” mechanism. 相似文献
9.
Hong Kui Leung Yun Chung Kwok Sui Yi Law Kin Ho Lo Wai Hung Chua Hong Yu Peter Hoi Fu 《Applied biochemistry and biotechnology》2000,84(1-9):381-390
Construction and comparison of recombinant Escherichia coli strains harboring the polyhydroxybutyrate (PHB) operon from Ralstonia entropha using vectors possessing different promotors, as well as the production of PHB from soy waste by the recombinant strain,
are reported. The lac promotor was the most efficient on expression of the phb operon among the three promotors studied: i.e., lac promotor, T7 promotor and the normal σ70 promotor. The pKS/PHB was the most efficient plasmid for phboperon expression among the three plasmids used: i.e., pKS−, pAED4, and pJM9131. It was observed that isopropyl-β-d-thiogalactopyranoside was not required for the induction of the expression of phb operon. The cell dry wt and polyhydroxyalkan cote content by E. coli XL-1 Blue (pKS/PHB) were 3.025 g/L and 27.83%, respectively. 相似文献
10.
Wild type (WT) DNA sequence, which encoded a mature β-mannanase of Aspergillus sulphureus, composed of 1,152 nucleotides (nt), was amplified from pUCm-T-mann by polymerase chain reaction (PCR). Based on this DNA
fragment, mutants designated as E206G and E314G were constructed by overextension PCR (OE-PCR). Glutamic acids of the 206th and 314th sites in the amino acid sequence of
β-mannanase were separately replaced by glycine in these two mutants. The WT and mutant genes were ligated into prokaryotic
vector pET-28a (+) and transformed into the Escherichia coli BL21 strain, respectively. The recombinant enzyme proteins were expressed by IPTG induction and detected by Western blot.
The recombinant proteins purified with Ni-NTA column were dialyzed to correctly refold. The WT recombinant β-mannanase showed
optimal activity at 50 °C and pH 2.4. The kinetic parameters of K
m and V
max for purified β-mannanase were 1.38 mg/ml and 72.99 U/mg, respectively. However, the mutant proteins did not show any activity.
It was demonstrated that E206 and E314 were the catalytic residues of β-mannanase. 相似文献
11.
Qilei Chen Youjia Hu Wenjie Zhao Chunbao Zhu Baoquan Zhu 《Applied biochemistry and biotechnology》2010,160(1):19-29
A gene encoding a novel (S)-specific NADH-dependent alcohol dehydrogenase (LK-ADH) was isolated from the genomic DNA of Lactobacillus kefir DSM 20587 by thermal asymmetric interlaced-polymerase chain reaction. The nucleotide sequence of (S)-LK-ADH gene (adhS) was determined, which consists of an open reading frame of 1,044 bp, coding for 347 amino acids with a molecular mass of
37.065 kDa. After a BLAST similarity search in GenBank database, the amino acid sequence of (S)-LK-ADH showed some homologies to several zinc containing medium-chain alcohol dehydrogenases. This novel gene was deposited
into GenBank with the accession number of EU877965. adhS gene was subcloned into plasmid pET-28a(+), and recombinant (S)-LK-ADH was successfully expressed in E. coli BL21(DE3) by isopropyl-β-d-1-thiogalactopyranoside induction. Purified enzyme showed a high enantioselectivity in the reduction of acetophenone to (S)-phenylethanol with an ee value of 99.4%. The substrate specificity and cofactor preference of recombinant (S)-LK-ADH were also tested. 相似文献
12.
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
13.
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
14.
Liang Xiang Feng Qi DaZhang Dai Chun Li YuanDa Jiang 《Applied biochemistry and biotechnology》2010,162(3):654-661
Effects of simulated microgravity (SMG) on bacteria have been studied in various aspects. However, few reports are available
about production of recombinant protein expressed by bacteria in SMG. In this study growth of E. coli BL21 (DE3) cells transformed with pET-28a (+)-pgus in double-axis clinostat that could model low shear SMG environment and
the recombinant β-d-glucuronidase (PGUS) expression have been investigated. Results showed that the cell dry weights in SMG were 16.47%, 38.06%,
and 28.79% more than normal gravity (NG) control, and the efficiency of the recombinant PGUS expression in SMG were 18.33%,
19.36%, and 33.42% higher than that in NG at 19 °C, 28 °C, and 37 °C, respectively (P < 0.05). 相似文献
15.
Han Seung Chae Kong-Hwan Kim Sun Chang Kim Pyung Cheon Lee 《Applied biochemistry and biotechnology》2010,162(8):2333-2344
Seven Escherichia coli strains, which were metabolically engineered with carotenoid biosynthetic pathways, were systematically compared in order
to investigate the strain-specific formation of carotenoids of structural diversity. C30 acyclic carotenoids, diaponeurosporene
and diapolycopene were well produced in all E. coli strains tested. However, the C30 monocyclic diapotorulene formation was strongly strain dependent. Reduced diapotorulene
formation was observed in the E. coli strain Top10, MG1655, and MDS42 while better formation was observed in the E. coli strain JM109, SURE, DH5a, and XL1-Blue. Interestingly, C40 carotenoids, which have longer backbones than C30 carotenoids,
also showed strain dependency as C30 diapotorulene did. Quantitative analysis showed that the SURE strain was the best producer
for C40 acyclic lycopene, C40 dicyclic β-carotene, and C30 monocyclic diapotorulene. Of the seven strains examined, the highest
volumetric productivity for most of the carotenoids structures was observed in the recombinant SURE strain. In conclusion,
we showed that recombinant hosts and carotenoid structures influenced carotenoid productions significantly, and this information
can serve as the basis for the subsequent development of microorganisms for carotenoids of interest. 相似文献
16.
Wu J Zhang L Lei J Cai G Zhu W Lu D Jin J 《Applied biochemistry and biotechnology》2009,157(2):299-310
This study was aimed at increasing the production of the recombinant human ADAM15 disintegrin domain (RADD) in Escherichia coli by releasing the rare codons and restricting amino acid residues. Three different strategies for increasing RADD production
were examined: to select the suitable host strain, to optimize the rare codons, and to delete the amino acids residues. The
total fusion protein glutathione-S-transferase (GST)-RADD concentration of 298 mg/l and 326 mg/l were achieved by selecting
E. coli Rosetta (DE3) as the host strain and by changing GGA to GGC at the GST-RADD cassette, respectively. The RADD concentration
was increased by 35.7% by eliminating the “Pro-Glu-Phe” residues at the GST–RADD junction. By combinational utilizing the
preferred codon introduction and amino acid sequence optimization in E. coli Rosetta (DE3), the highest RADD concentration of 68 mg/l was achieved. The proposed strategy may provide an alternative approach
for other enhanced recombinant protein production by E. coli. 相似文献
17.
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
18.
Continuous production of L-aspartic acid 总被引:2,自引:0,他引:2
For the continuous production ofl-aspartic acid from fumaric acid and ammonia by the action of aspartase, the enzyme extracted fromEscherichia coli orE. coli cells having high aspartase activity were immobilized by various methods.
In 1973 we succeeded in the industrial production ofl-aspartic acid usingE. coli cells immobilized with polyacrylamide gel.
For the improvement of this process, we developed a novel technique using κ-carrageenan as the immobilizing matrix forE. coli cells. Further, EAPc-7 strain, having higher aspartase activity, was contracted from the parentE. coli by continuous cultivation with a definite medium. The aspartase activity was about seven times higher than that of the parent
cells. In 1982 we changed from the conventional method to the improved method, using EAPc-7 strain immobilized with κ-carrageenan. 相似文献
19.
Amperometric determination of live <Emphasis Type="Italic">Escherichia coli</Emphasis> using antibody-coated paramagnetic beads 总被引:2,自引:0,他引:2
Boyaci IH Aguilar ZP Hossain M Halsall HB Seliskar CJ Heineman WR 《Analytical and bioanalytical chemistry》2005,382(5):1234-1241
Detecting and enumerating fecal coliforms, especially Escherichia coli, as indicators of fecal contamination, are essential for the quality control of supplied and recreational waters. We have developed a sensitive, inexpensive, and small-volume amperometric detection method for E. coli -galactosidase by bead-based immunoassay. The technique uses biotin-labeled capture antibodies (Ab) immobilized on paramagnetic microbeads that have been functionalized with streptavidin (bead–Ab). The bead–Ab conjugate captures E. coli from solution. The captured E. coli is incubated in Luria Bertani (LB) broth medium with the added inducer isopropyl -D-thiogalactopyranoside (IPTG). The induced -galactosidase converts p-aminophenyl -D-galactopyranoside (PAPG) into p-aminophenol (PAP), which is measured by amperometry using a gold rotating disc electrode. A good linear correlation (R2=0.989) was obtained between log cfu mL–1 E. coli and the time necessary to product a specific concentration of PAP. Amperometric detection enabled determination of 2×106 cfu mL–1 E. coli within a 30 min incubation period, and the total analysis time was less than 1 h. It was also possible to determine as few as 20 cfu mL–1 E. coli under optimized conditions within 6–7 h. This process may be easily adapted as an automated portable bioanalytical device for the rapid detection of live E. coli. 相似文献
20.
Triblock cooligomers consisting of tri-O-methyl-glucopyranosyl and unmodified glucopyranosyl residues, methyl 2,3,4,6-tetra-O-methyl-β-d-glucopyranosyl-(1 → 4)-2,3,6-tri-O-methyl-β-d-glucopyranosyl-(1 → 4)-β-d-glucopyranosyl-(1 → 4)-β-d-glucopyranosyl-(1 → 4)-2,3,6-tri-O-methyl-β-d-glucopyranosyl-(1 → 4)-2,3,6-tri-O-methyl-α-d-glucopyranoside (1: ABA triblock cooligomer; DS = 2.1) and β-d-glucopyranosyl-(1 → 4)-2,3,6-tri-O-methyl-β-d-glucopyranosyl-(1 → 4)-2,3,6-tri-O-methyl-β-d-glucopyranosyl-(1 → 4)-2,3,6-tri-O-methyl-β-d-glucopyranosyl-(1 → 4)-2,3,6-tri-O-methyl-β-d-glucopyranosyl-(1 → 4)-d-glucopyranose (2: BAB triblock cooligomer; DS = 1.8) were prepared. Compound 1 dissolved both in distilled water and chloroform but compound 2 dissolved in distilled water not in chloroform, though compounds 1 and 2 consist of 4 tri-O-methyl-glucopyranosyl and 2 unmodified anhydro glucopyranosyl units. 相似文献