共查询到20条相似文献,搜索用时 31 毫秒
1.
Horton CE Huang KX Bennett GN Rudolph FB 《Journal of industrial microbiology & biotechnology》2003,30(7):427-432
Esters are formed by the condensation of acids with alcohols. The esters isoamyl acetate and butyl butyrate are used for food and beverage flavorings. Alcohol acetyltransferase is one enzyme responsible for the production of esters from acetyl-CoA and different alcohol substrates. The genes ATF1 and
ATF2, encoding alcohol acetyltransferases from the yeast Saccharomyces cerevisiae have been sequenced and characterized. The production of acids and alcohols in mass quantities by the industrially important Clostridium acetobutylicum makes it a potential organism for exploitation of alcohol acetyltransferase activity. This report focuses on the heterologous expression of the alcohol acetyltransferases in Escherichia coli and C. acetobutylicum.
ATF1 and ATF2 were cloned and expressed in E. coli and ATF2 was expressed in C. acetobutylicum. Isoamyl acetate production from the substrate isoamyl alcohol in E. coli and C. acetobutylicum cultures was determined by head-space gas analysis. Alcohol acetyltransferase I produced more than twice as much isoamyl acetate as alcohol acetyltransferase II when expressed from a high-copy expression vector. The effect of substrate levels on ester production was explored in the two bacterial hosts to demonstrate the efficacy of utilizing ATF1and ATF2 in bacteria for ester production. 相似文献
2.
3.
Kurdrid P Subudhi S Cheevadhanarak S Tanticharoen M Hongsthong A 《Molecular biology reports》2007,34(4):261-266
When the gene desD encoding Spirulina Δ6-desaturase was heterologously expressed in E. coli, the enzyme was expressed without the ability to function. However, when this enzyme was co-expressed with an immediate electron
donor, i.e. the cytochrome b
5 domain from Mucor rouxii, the results showed the production of GLA (γ-linolenic acid), the product of the reaction catalyzed by Δ6-desaturase. The results revealed that in E. coli cells, where cytochrome b
5 is absent and ferredoxin, a natural electron donor of Δ6-desaturase, is present at a very low level, the cytochrome b
5 domain can complement for the function of ferredoxin in the host cells. In the present study, the Spirulina-ferredoxin gene was cloned and co-expressed with the Δ6-desaturase in E. coli. In comparison to the co-expression of cytochrome b
5
with the Δ6-desaturase, the co-expression with ferredoxin did not cause any differences in the GLA level. Moreover, the cultures containing
the Δ6-desaturase co-expressed with cytochrome b
5 and ferredoxin were exogenously supplied with the intermediate electron donors, NADPH (nicotinamide adenine dinucleotide
phosphate, reduced form) and FADH2 (flavin adenine dinucleotide, reduced form), respectively. The GLA level in these host cells increased drastically, by approximately
50%, compared to the cells without the intermediate electron donors. The data indicated that besides the level of immediate
electron donors, the level of intermediate electron donors is also critical for GLA production. Therefore, if the pools of
the immediate and intermediate electron donors in the cells are manipulated, the GLA production in the heterologous host will
be affected. 相似文献
4.
Soror SH Verma V Rao R Rasool S Koul S Qazi GN Cullum J 《Journal of industrial microbiology & biotechnology》2007,34(8):525-531
The genome sequence of Streptomyces coelicolor A3(2) contains 51 putative lipase and esterase genes mostly of unknown function. The gene estB (locus SCO 6966) was expressed as a His-tagged protein in E. coli. Esterase B was active at low temperatures exerting its maximum activity at 30°C and retaining more than 25% of its activity
at 4°C. The optimum pH was 8–8.5. The enzyme was active against short synthetic p-nitrophenylesters (C2–C10) with maximum activity towards the acetate ester (C2). The esterase was tested on 13 series of racemic esters of potential interest for the synthesis of chiral pharmaceutical
compounds. 4 of the series were substrates and a modest degree of enantioselectivity was observed (enantiomeric ratios of
1.1–1.9). 相似文献
5.
Barboza-Corona JE Reyes-Rios DM Salcedo-Hernández R Bideshi DK 《Molecular biotechnology》2008,39(1):29-37
An endochitinase gene (chiA-HD73) from the insecticidal bacterium Bacillus thuringiensis subsp. kurstaki HD-73 was cloned, sequenced, and expressed in Escherichia coli DH5αF′. The chitinase activity of the encoded protein was studied in assays with different fluorogenic substrates. The chiA-HD73 gene contained an open-reading frame that encoded an endochitinase with a deduced molecular weight and an isoelectric
point of, respectively, 74.5 kDa and 5.75. A putative signal peptide with cleavage sites for both Gram-positive and Gram-negative
bacteria was identified. Comparison of ChiA-HD73 with other chitinases revealed a modular structure composed of a catalytic
domain and a putative chitin-binding domain. ChiA-HD73 hydrolyzed both tetrameric and trimeric fluorogenic substrates, but
not a chitobiose analog substrate, suggesting that the activity of ChiA-HD73 is mainly endochitinolytic. In addition, ChiA-HD73
showed high enzymatic activity within a broad pH range (pH 4–10), with a peak activity at pH 6.5. The optimal temperature
for enzymatic activity was observed at 55°C. Its activity in a broad range of temperatures and pH suggests ChiA-HD73 could
have biotechnological applications in insect control, particularly in synergizing the insecticidal crystal protein toxins
of B. thuringiensis. 相似文献
6.
S A Wardwell Y T Yang H Y Chang K Y San F B Rudolph G N Bennett 《Journal of industrial microbiology & biotechnology》2001,27(4):220-227
Acetoin reductase catalyzes the production of 2,3-butanediol from acetoin. The gene encoding the acetoin reductase of Klebsiella pneumoniae CG21 was cloned and expressed in Escherichia coli and Clostridium acetobutylicum ATCC 824. The nucleotide sequence of the gene encoding the enzyme was determined to be 768 bp long. Expression of the K. pneumoniae acetoin reductase gene in E. coli revealed that the enzyme has a molecular mass of about 31,000 Da based on sodium dodecyl sulfate polyacrylamide gel electrophoresis
analysis. The K. pneumoniae acetoin reductase gene was cloned into a clostridial/E. coli shuttle vector, and expression of the gene resulted in detectable levels of acetoin reductase activity in both E. coli and C. acetobutylicum. While acetoin, the natural substrate of acetoin reductase, is a typical product of fermentation by C. acetobutylicum, 2,3-butanediol is not. Analysis of culture supernatants by gas chromatography revealed that introduction of the K. pneumoniae acetoin reductase gene into C. acetobutylicum was not sufficient for 2,3-butanediol production even though the cultures were producing acetoin. 2,3-Butanediol was produced
by cultures of C. acetobutylicum containing the gene only when commercial acetoin was added. Journal of Industrial Microbiology & Biotechnology (2001) 27, 220–227.
Received 12 September 2000/ Accepted in revised form 26 June 2001 相似文献
7.
Characterization of a New Stearoyl-acyl Carrier Protein Desaturase Gene from Jatropha curcas 总被引:2,自引:0,他引:2
Tong L Shu-Ming P Wu-Yuan D Dan-Wei M Ying X Meng X Fang C 《Biotechnology letters》2006,28(9):657-662
A new full-length cDNA of stearoyl-acyl carrier protein desaturase was obtained by RT-PCR and RACE techniques from developing
seeds of Jatropha curcas. Sequence alignment showed that its deduced amino acid sequence had high similarity with other stearoyl-acyl carrier protein
desaturases. The gene was functionally expressed in E. coli and the desaturating activity of recombinant protein was easily detected when assayed in vitro with added spinach ferredoxin. Southern blot analysis indicated that the gene was a member of a small gene family. Northern
blot analysis revealed it was highly expressed in developing fruits of J. curcas.
Revisions requested 16 December 2005; Revisions received 6 February 2006 相似文献
8.
Ruiz-Sanchez A Cruz-Camarillo R Salcedo-Hernandez R Ibarra JE Barboza-Corona JE 《Molecular biotechnology》2005,31(2):103-111
An endochitinase gene from the Serratia marcescens Nima strain (chiA Nima) was cloned, sequenced, and expressed in Escherichia coli DH5αF′, and the recombinant protein (ChiA Nima) was purified by hydrophobic interaction chromatography. chiA Nima contains an open reading frame (ORF) that encodes an endochitinase with a deduced molecular weight and an isoelectric
point of 61 kDa and 6.84, respectively. A sequence at the 5′-end was identified as a signal peptide, recognized by Gram-negative
bacteria transport mechanism. Comparison of ChiA Nima with other chitinases revealed a modular structure formed by the catalytic
domain and a putative chitin-binding domain. The purified chitinase was able to hydrolyze both trimeric and tetrameric fluorogenic
substrates, but not a chitobiose analog substrate. ChiA Nima showed high enzymatic activity within a broad pH range (pH 4.0–10.0),
with a peak activity at pH 5.5. The optimal temperature for enzymatic activity was detected at 55°C. 相似文献
9.
Inui M Suda M Kimura S Yasuda K Suzuki H Toda H Yamamoto S Okino S Suzuki N Yukawa H 《Applied microbiology and biotechnology》2008,77(6):1305-1316
A recombinant butanol pathway composed of Clostridium acetobutylicum ATCC 824 genes, thiL, hbd, crt, bcd-etfB-etfA, and adhe1 (or adhe) coding for acetyl-CoA acetyltransferase (THL), β-hydroxybutyryl-CoA dehydrogenase (HBD), 3-hydroxybutyryl-CoA dehydratase
(CRT), butyryl-CoA dehydrogenase (BCD), butyraldehyde dehydrogenase (BYDH), and butanol dehydrogenase (BDH), under the tac promoter control was constructed and was introduced into Escherichia coli. The functional expression of these six enzymes was proved by demonstrating the corresponding enzyme activities using spectrophotometric,
high performance liquid chromatography and gas chromatography analyses. The BCD activity, which was not detected in E. coli previously, was shown in the present study by performing the procedure from cell extract preparation to activity measurement
under anaerobic condition. Moreover, the etfA and etfB co-expression was found to be essential for the BCD activity. In the case of BYDH activity, the adhe gene product was shown to have higher specificity towards butyryl-CoA compared to the adhe1 product. Butanol production from glucose was achieved by the highly concentrated cells of the butanologenic E. coli strains, BUT1 with adhe1 and BUT2 with adhe, under anaerobic condition, and the BUT1 and BUT2 strains were shown to produce 4 and 16-mM butanol with 6- and 1-mM butyrate
as a byproduct, respectively. This study reports the novel butanol production by an aerobically pregrown microorganism possessing
the genes of a strict anaerobe, Clostridium acetobutylicum. 相似文献
10.
The major enzyme in Clostridium acetobutylicum ATCC 824 leading to transformation of TNT has been reported to be the Fe-only hydrogenase. In this study, we examine the effect of inhibitors of hydrogenase on TNT reduction by Clostridial extracts. These experiments further demonstrate the major role of hydrogenase in TNT transformation. The C. acetobutylicum hydrogenase is closely related to that of C. pasteurianum; and can be fitted to the X-ray crystal structure with a root mean square deviation of 1.18 Å for the Cα atoms of the generated 3D simulation model. The Hyd1, Hyd2, and Hyd3 antibodies generated against hydrogenase reacted with both the hydrogenase in cell extracts and with C. acetobutylicum hydrogenase expressed in Escherichia coli. Inhibition studies using antibodies against Fe-only hydrogenase from C. acetobutylicum indicated that the transformation of TNT by crude cell extracts was completely inhibited by Hyd2 antibody (to amino acid 415–428) whereas antibodies Hyd1 (to residues 1–16) and Hyd3 (to amino acid 424–448) inhibited less effectively. The TNT transforming activity of the cell extract was retained when Hyd2 antibody pretreated with purified but enzymatically inactive recombinant hydrogenase was added to the extract. Addition of the transition metal Cu (2+) to extracts completely inhibited the transformation of TNT suggesting the destruction of [4Fe–4S] centers which are essential for transfer of electrons from the H2-activating site to TNT. Growth of C. acetobutylicum was also inhibited by 0.5 mM Cu(2+) and Hg(2+) ions. The triazine dye, procion red and the nitroimidazole drug, metronidazole inhibit TNT reduction. The inhibition studies using antibodies, procion red, metronidazole, and transition metals suggest that different portions of hydrogenase are required for effective TNT reduction. 相似文献
11.
Bordetella pertussis adenylate cyclase toxin-haemolysin (CyaA) can permeabilise erythrocytes by forming lytic pores. Here, a gene segment encoding
CyaA pore-forming (CyaA-PF) domain cloned from genomic DNA of B. pertussis Thai isolate was over-expressed in Escherichia coli as a 126-kDa soluble protein which cross-reacted with anti-RTX monoclonal antibody. By co-expressing with acyltransferase
CyaC, the CyaA-PF protein was found palmitoylated at Lys983. Unlike E. coli lysate with the non-acylated form, the lysate containing acylated CyaA-PF exhibited high haemolytic activity against sheep
erythrocytes. This study presents that the recombinant CyaA-PF protein comprising pore-forming domain can be expressed separately
as soluble native-folded precursor that conserves at least part of its functionality. 相似文献
12.
Microcystis viridis lectin (MVL), a sugar-binding protein originally isolated from freshwater blue-green algae Microcystis viridis, has been reported to have potent anti-HIV activity. In this paper, we described the expression and purification of recombinant-MVL
(R-MVL) gene in E. coli. The results demonstrated that the R-MVL in shake flask cultures was primarily expressed either in the form of inclusion
bodies at 37°C or in the soluble fraction at 23 °C. Secondly, a one-step purification based on nickel-affinity chromatography
was employed and 15 mg of highly purified (>95%) R-MVL from 1 l of cell cultures was yielded. The purified R-MVL was then
subjected to MALDI-TOF–MS analysis for protein identification. In conclusion, for the first time, the R-MVL was successfully
cloned and expressed in E. coli, which is useful for further study and large-scale cost-effective production of MVL protein. 相似文献
13.
14.
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
15.
Guerrini O Burlat B Léger C Guigliarelli B Soucaille P Girbal L 《Current microbiology》2008,56(3):261-267
In vivo hydrogen production in Clostridium acetobutylicum involves electron transfer between ferredoxin and [FeFe]-hydrogenase. Five C. acetobutylicum open reading frames were annotated as coding for putative ferredoxins. We focused our biophysical and biochemical investigations
on CAC0303 and CAC3527, which possess the sequence signature and length of classical 2[4Fe4S] clostridial ferredoxins but
differ significantly in theoretical pI. After cloning, heterologous expression in E. coli followed by in vitro Fe-S incorporation and purification, CAC0303 was shown to have a regular electron paramagnetic resonance
(EPR) signal for a classical 2[4Fe4S] clostridial ferredoxin, while CAC3527 displayed an unusual EPR signal and a quite low
reduction potential. Both ferredoxins were reduced in vitro by C. acetobutylicum [FeFe]-hydrogenase, but the CAC3527 reduction rate was 10-fold lower than that of CAC0303. These results are consistent with
the efficiency of intermolecular electron transfer being dictated by the redox thermodynamics, the contribution of the ferredoxin
global charge being only minor. The physiological function of CAC3527 is discussed. 相似文献
16.
Kurdrid P Subudhi S Hongsthong A Ruengjitchatchawalya M Tanticharoen M 《Molecular biology reports》2005,32(4):215-226
Spirulina-acyl-lipid desaturases are membrane-bound enzymes found in thylakoid and plasma membranes. These enzymes carry out the fatty
acid desaturation process of Spirulina to yield γ-linolenic acid (GLA) as the final desaturation product. In this study, Spirulina-Δ6 desaturase encoded by the desD gene was heterologously expressed and characterized in Saccharomyces cerevisiae. We then conducted site-directed mutagenesis of the histidine residues in the three histidine boxes to determine the role
of these amino acid residues in the enzyme function. Our results showed that while four mutants showed complete loss of Δ6-desaturase activity and two mutants showed only trace of the activity, the enzyme activity could be partially restored by
chemical rescue using exogenously provided imidazole. This study reveals that the histidine residues (which have imidazole
as their functional group) in the conserved clusters play a critical role in Δ6-desaturase activity, possibly by providing a di-iron catalytic center. In our previous study, this enzyme was expressed in
Escherichia coli. The results reveal that the enzyme can function only in the presence of an exogenous cofactor, ferredoxin, provided in vitro. This evidence suggests that baker’s yeast has a cofactor that can complement ferredoxin, thought to act as an electron donor
for the Δ6 desaturation in cyanobacteria, including Spirulina. The electron donor of the Spirulina-Δ6 desaturation in yeast is more likely to be cytochrome b5, which is absent in E. coli. This means that the enzyme expressed in S. cerevisiae can catalyze the biosynthesis of the product, GLA, in vivo. 相似文献
17.
The gene for phospholipase D (PLD) of Streptomyces sp. YU100 was cloned from λ phage library and hetero-logously expressed in Escherichia coli. Using an amplified gene fragment based on the consensus sequences of streptomycetes PLDs, λ phage library of Streptomyces sp. YU100 chromosomal DNA was screened. The sequencing result of BamHI-digested 3.8 kb fragment in a positive phage clone revealed the presence of an open reading frame of a full sequence of
PLD gene encoding a 540-amino acid protein including 33-amino acid signal peptide. The deduced amino acid sequence showed
a high homology with other Streptomyces PLDs, having the highly conserved ‘HKD’ motifs. The PLD gene excluding signal peptide sequence was amplified and subcloned
into a pET-32b(+) expression vector in E. coli BL21(DE3). The recombinant PLD was purified by nickel affinity chromatography and compared the enzyme activity with wild-type
PLD. The results imply that the recombinant PLD produced by E. coli had the nearly same enzyme activity as PLD from Streptomyces sp. YU100. 相似文献
18.
The [2Fe-2S] cluster containing ferredoxin has attracted much attention in recent years. Genetic analyses show that it has
an essential role in the maturation of various iron–sulfur (Fe-S) proteins and functions as a component of the complex machinery
responsible for the biogenesis of Fe-S clusters. The gene of ferredoxin from A. ferrooxidans ATCC 23270 was cloned, successfully expressed in Escherichia coli, and purified by one-step affinity chromatography to homogeneity. The MALDI-TOF MS and spectra results of the recombinant
protein confirmed that the iron–sulfur cluster was correctly inserted into the active site of the protein. Site-directed mutagenesis
results revealed that Cys42, Cys48, Cys51, and Cys87 were ligating with the [Fe2S2] cluster of the protein. 相似文献
19.
Eun Kyung Cho 《Biotechnology and Bioprocess Engineering》2007,12(5):502-507
Members of the cyclophilin (Cyp) family are known to function as co-chaperones, interacting with chaperones such as heat shock
protein 90, and perform important roles in protein folding under high temperature stress. In addition, they have been isolated
from a wide range of organisms. However, there have been no reports on the functions of algal Cyps under other stress conditions.
To study the functions of the cDNAGjCyp-1 isolated from the red alga (Griffithsia japonica), a recombinant GjCyp-1 containing a hexahistidine tag at the amino-terminus was constructed and expressed inEscherichia coli. Most of the gene product expressed inE. coli was organized as aggregate insoluble particles known as inclusion bodies. Thus, the optimal time, temperature, and concentration
ofl(+)-arabinose for expressing the soluble and nonaggregated form of GjCyp-1 inE. coli were examined. The results indicate that the induction of Cyp, at 0.2%l(+)-arabinose for 2 h at 25°C, had a marked effect on the yield of the soluble and active form of the co-chaperone as PPlase.
An expressed fusion protein, H6GjCyp-1, maintained the stability ofE. coli proteins up to-75°C. In a functional bioassay of the recombinant H6GjCyp-1, the viability ofE. coli cells overexpressing H6GjCyp-1 was compared to that of cells not expressing H6GjCyp-1 at −75°C. For all the cycles of a freeze/thaw treatment, a significant increase in viability was observed in theE. coli cells overexpressing H6GjCyp-1. The results of the GjCyp-1 bioassays, as well asin vitro studies, strongly suggest that the algal Cyp confers freeze tolerance toE. coli. 相似文献
20.
Iron–sulfur clusters are one of the most common types of redox center in nature. Three proteins of IscS (a cysteine desulfurase),
IscU (a scaffold protein) and IscA (an iron chaperon) encoded by the operon iscSUA are involved in the iron–sulfur cluster assembly in Acidithiobacillus ferrooxidans. In this study the gene of IscS from A. ferrooxidans ATCC 23270 was cloned and expressed in Escherichia coli, the protein was purified by one-step affinity chromatography to homogeneity. The molecular mass of recombinant IscS was
46 kDa by SDS-PAGE. The IscS was a pyridoxal phosphate-containing protein, that catalyzed the elimination of S from l-cysteine to yield l-alanine and elemental sulfur or H2S, depending on whether or not a reducing agent was added to the reaction mixture.
Jia Zeng and Yanfei Zhang contributed equally to this work. 相似文献