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1.
Recombinant Zantedeschia aethiopica agglutinin (ZAA) was expressed in Escherichia coli as N-terminal His-tagged fusion. After induction with isopropylthio-β-d-galactoside (IPTG), the recombinant ZAA was purified by metal-affinity chromatography. The purified ZAA protein was applied
in anti-fungal assay and the result showed that recombinant ZAA had anti-fungal activity towards leaf mold (Fulvia fulva), one of the most serious phytopathogenic fungi causing significant yield loss of crops. This study suggests that ZAA could
be an effective candidate in genetic engineering of plants for the control of leaf mold. 相似文献
2.
Liangfan Zhou Qingping Lin Baocun Li Nannan Li Shuangquan Zhang 《Biotechnology letters》2009,31(3):437-441
The antimicrobial peptide CM4 is a 35-residue cationic peptide. To explore a new approach for the expression and purification
of CM4 in Escherichia coli, the CM4 gene was cloned into the vector pET32a to construct an expression vector pET32a-CM4. The fusion protein Trx-CM4,
purified by Ni2+-chelating chromatography, was cleaved by hydroxylamine hydrochloride to release recombinant CM4. Purification of recombinant
CM4 was achieved by reverse HPLC chromatography, and about 1.4 mg/l active recombinant CM4 with the purity more than 98% was
obtained. The recombinant CM4 showed antimicrobial activities that were similar to synthetic one.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.
An erratum to this article can be found at 相似文献
3.
The gene encoding a cold-active and xylose-stimulated β-glucosidase of Marinomonas MWYL1 was synthesized and expressed in Escherichia coli. The recombinant enzyme (reBglM1) was purified and characterized. The molecular mass of the purified reBglM1 determined by
SDS-PAGE agree with the calculated values (50.6 Da). Optima of temperature and pH for enzyme activity were 40°C and 7.0, respectively.
The enzyme exhibited about 20% activity at 5°C and was stable over the range of pH 5.5–10.0. The presence of xylose significantly
enhanced enzyme activity even at higher concentrations up to 600 mM, with maximal stimulatory effect (about 1.45-fold) around
300 mM. The enzyme is active with both glucosides and galactosides and showed high catalytic efficiency (k
cat = 500.5 s−1) for oNPGlc. These characterizations enable the enzyme to be a promising candidate for industrial applications. 相似文献
4.
María D. Frade-Pérez Arturo Hernández-Cervantes Arturo Flores-Carreón Héctor M. Mora-Montes 《Antonie van Leeuwenhoek》2010,98(3):291-298
Protein glycosylation is one of the most common post-translational modifications present in the eukaryotic cell. The N-linked glycosylation is a biosynthetic pathway where an oligosaccharide is added to asparagine residues within the endoplasmic
reticulum. Upon addition of the N-linked glycan to nascent proteins, α-glucosidase I removes the outermost α1,2-glucose unit from the N-linked core Glc3Man9GlcNAc2. We have previously demonstrated that the endoplasmic reticulum α-glucosidase I is required for normal cell wall composition,
and virulence of the human pathogen Candida albicans. In spite of the importance of this enzyme for normal cell biology, little is known about its structure and the amino acids
participating in enzyme catalysis. Here, a DNA fragment corresponding to the 3′-end fragment of C. albicans
CWH41, the encoding gene for α-glucosidase I, was expressed in a bacterial system and the recombinant peptide showed α-glucosidase
activity, despite lacking 419 amino acids from the N-terminal end. The biochemical characterisation of the recombinant enzyme
showed that presence of hydroxyl groups at carbons 3 and 6, and orientation of hydroxyl moiety at C-2 are important for glucose
recognition. Additionally, results suggest that cysteine rather than histidine residues are involved in the catalysis by the
recombinant enzyme. 相似文献
5.
Nadiawati Alias Nor Muhammad Mahadi Abdul Munir Abdul Murad Farah Diba Abu Bakar Nik Azmi Nik Mahmood Rosli Md Illias 《World journal of microbiology & biotechnology》2009,25(4):561-572
A gene encoding endochitinase from Trichoderma virens UKM-1 was cloned and expressed in E. coli BL21 (DE3). Both the endochitinase gene and its cDNA sequences were obtained. The endochitinase gene encodes 430 amino acids
from an open reading frame comprising of 1,690 bp nucleotide sequence with three introns. The endochitinase was expressed
as soluble and active enzyme at 20°C when induced with 1 mM IPTG. Maximum activity was observed at 4 h of post-induction time.
SDS-PAGE showed that the purified endochitinase exhibited a single band with molecular weight of 42 kDa. Biochemical characterization
of the enzyme displayed a near neutral pH characteristic with an optimum pH at 6.0 and optimum temperature at 50°C. The enzyme
is stable between pH 3.0–7.0 and is able to retain its activity from 30 to 60°C. The presence of Mg2+ and Ca2+ ions increased the enzyme activity up to 20%. The purified enzyme has a strong affinity towards colloidal chitin and low
effect on ethyl cellulose and D-cellubiose which are non-chitin related substrates. HPLC analysis from the chitin hydrolysis
showed the release of (GlcNAc)3, (GlcNAc)2 and GlcNAc, in which (GlcNAc)2 was the main product. 相似文献
6.
An expression plasmid containing the agdA gene encoding Aspergillus oryzae ZL-1 α-glucosidase was constructed and expressed in Pichia pastoris X-33. The molar mass of the purified protein was estimated by SDS-PAGE. HPLC analysis showed that the purified enzyme has
a transglucosylating activity with maltose as substrate. The main component of the enzyme products was panose, while amounts
of isomaltose and isomaltotriose were very low or absent. pH 5.2 and temperature of 37 °C were optimum for enzyme activity. 相似文献
7.
Rhodococcus equi is an intracellular pathogen of macrophages, causing disease in young foals, humans, and sporadically other animals. Although R. equi is easy to grow and manipulate, the analysis of virulence is hampered by a lack of molecular tools. This paper describes the development of a number of versatile plasmids for use in R. equi. Plasmids pREV2 and pREV5 use origins of replication derived from the Mycobacterium fortuitum plasmids pAL5000 and pMF1. These plasmids and their derivatives are compatible in R. equi, allowing their use for analysis of gene function in trans. The stability of these plasmids in R. equi in the absence of selection for the plasmid borne antibiotic resistance markers, and their integrity following passage through Escherichia coli and R. equi was determined. 相似文献
8.
Background
Thymosin α1 (Tα1), a 28-amino acid N α -acetylated peptide, has a powerful general immunostimulating activity. Although biosynthesis is an attractive means of large-scale manufacture, to date, Tα1 can only be chemosynthesized because of two obstacles to its biosynthesis: the difficulties in expressing small peptides and obtaining N α -acetylation. In this study, we describe a novel production process for N α -acetylated Tα1 in Escherichia coli. 相似文献9.
Background
Cyclodextrin glycosyltransferases (CGTases) catalyze the synthesis of cyclodextrins, which are circular α-(1,4)-linked glucans used in many applications in the industries related to food, pharmaceuticals, cosmetics, chemicals, and agriculture, among others. Economic use of these CGTases, particularly γ-CGTase, requires their efficient production. In this study, the effects of chemical chaperones, temperature and inducers on cell growth and the production of soluble γ-CGTase by Escherichia coli were investigated.Results
The yield of soluble γ-CGTase in shake-flask culture approximately doubled when β-cyclodextrin was added to the culture medium as a chemical chaperone.When a modified two-stage feeding strategy incorporating 7.5 mM β-cyclodextrin was used in a 3-L fermenter, a dry cell weight of 70.3 g·L??1 was achieved. Using this cultivation approach, the total yield of γ-CGTase activity (50.29 U·mL??1) was 1.71-fold greater than that observed in the absence of β-cyclodextrin (29.33 U·mL??1).Conclusions
Since β-cyclodextrin is inexpensive and nontoxic to microbes, these results suggest its universal application during recombinant protein production. The higher expression of soluble γ-CGTase in a semi-synthetic medium showed the potential of the proposed process for the economical production of many enzymes on an industrial scale.10.
D. N. Karimova I. V. Manukhov E. Yu. Gnuchikh I. F. Karimov D. G. Deryabin 《Applied Biochemistry and Microbiology》2016,52(3):269-276
The effect of reactive oxygen and nitrogen species on lux-biosensors based on the Escherichia coli K12 MG1655 and Salmonella typhimurium LT2 host strains was investigated. The bioactivity of exogenous free radicals to the constitutively luminescent E. coli strain with plasmid pXen7 decreased in the order H2O2 > OCl– > NO? > RОO? > ONOO–> O2?- while the bioluminescence of S. typhimurium strain transformed with this plasmid decreased in the order NO? > H2O2 > ONOO– > RОO? > OCl– > O2?- The cross-reactivity of induced lux-biosensors to reactive oxygen and nitrogen species, the threshold sensitivity and the luminescence amplitude dependences from the plasmid specificity and the host strain were indicated. The biosensors with plasmid pSoxS′::lux possessed a wider range of sensitivity, including H2O2 and OCl–, along with O2?- and NO?. Among the used reactive oxygen and nitrogen species, H2O2 showed the highest induction activity concerning to the plasmids pKatG′::lux, pSoxS′::lux and pRecA′::lux. The inducible lux-biosensors based on S. typhimurium host strain possessed a higher sensitivity to the reactive oxygen and nitrogen species in comparison with the E. coli lux-biosensors. 相似文献
11.
A set of filamentous fungi (42 strains) was screened for alpha-N-acetylgalactosaminidase activity, and a series of inducers and different cultivation conditions were tested. Enzyme production by the best producer Aspergillus niger CCIM K2 was optimized and scaled up. alpha-N-Acetylgalactosaminidase was purified to apparent homogeneity by cation exchange chromatography, gel filtration, and chromatofocusing, and basic biochemical data of the enzyme were determined: The native molecular weight was estimated by gel filtration to be approximately 440 kDa, the molecular weight of the subunit was determined to be 76 kDa and the pI = 4.8. The K (M) was 0.73 mmol/l for o-nitrophenyl 2-acetamido-2-deoxy-alpha-D-galactopyranoside (o-NP-alpha-GalNAc), and optimum enzyme activity was achieved at pH 1.8 and 55 degrees C. This alpha-N-acetylgalactosaminidase is a retaining-type glycosidase, and it was N-deglycosylated without any loss of activity. 相似文献
12.
Wang L Zhou Q Chen H Chu Z Lu J Zhang Y Yang S 《Journal of industrial microbiology & biotechnology》2007,34(3):187-192
The gene encoding the Pyrococcus furiosus extracellular α-amylase (PFA) was amplified by PCR from P. furiosus genomic DNA and was highly expressed in Escherichia coli BL21-Codon Plus (DE3)-RIL. The recombinant α-amylase was mainly expressed in the form of insoluble inclusion bodies. An improved
purification method was established in this paper. The solubilization of the inclusion bodies was achieved by 90°C treatment
for 3 min in Britton–Robinson buffer at pH 10.5. The solubilized PFA was then diluted and subsequently purified by Phenyl
Sepharose chromatography. The overall yield of the new purification method was about 58,000 U/g wet cells, which is higher
than previously reported. 相似文献
13.
Escherichia coli BL21 (DE3) is commonly used for the overproduction of fusion proteins. Using this system, we recently reported the overproduction
of histidine-tagged mouse estrogen receptor (ER) α-ligand binding domain as an intact 30 kD protein and its inhibitory effect on the growth of bacteria. However, when GST-tagged
mouse ERα transactivation domain (TAD) was overproduced using this system, it showed no effect on the growth of bacteria but was specifically
degraded during its expression and purification. Here we report the expression of 47 kD GST-tagged mouse ERα-TAD protein, which was degraded partially and specifically into 46 and 43 kD fragments. This fusion protein was further degraded
into 37, 31, 29 and 26 kD fragments during its purification by affinity chromatography. Such specific degradation of GST-tagged
mouse ERα-TAD during its overproduction in E. coli and purification indicates the induction of specific protease and suggests the modification of expression system. 相似文献
14.
Carla Oliveira Sofia Costa José A. Teixeira Lucília Domingues 《Molecular biotechnology》2009,43(3):212-220
cDNA clones encoding frutalin, the α-d-galactose-binding lectin expressed in breadfruit seeds (Artocarpus incisa), were isolated and sequenced. The deduced amino acid sequences indicated that frutalin may be encoded by a family of genes.
The NCBI database searches revealed that the frutalin sequence is highly homologous with jacalin and mornigaG sequences. Frutalin
cDNA was re-amplified and cloned into the commercial expression vector pET-25b(+) for frutalin production in Escherichia coli. An experimental factorial design was employed to maximise the soluble expression of the recombinant lectin. The results
indicated that temperature, time of induction, concentration of IPTG and the interaction between the concentration of IPTG
and the time of induction had the most significant effects on the soluble expression level of recombinant frutalin. The optimal
culture conditions were as follows: induction with 1 mM IPTG at 22°C for 20 h, yielding 16 mg/l of soluble recombinant frutalin.
SDS-PAGE and Western blot analysis revealed that recombinant frutalin was successfully expressed by bacteria with the expected
molecular weight (17 kDa). These analyses also showed that recombinant frutalin was mainly produced as insoluble protein.
Recombinant frutalin produced by bacteria revealed agglutination properties and carbohydrate-binding specificity similar to
the native breadfruit lectin. 相似文献
15.
Background
Whole cell-catalyzed biotransformation is a clear process option for the production of chiral alcohols via enantioselective reduction of precursor ketones. A wide variety of synthetically useful reductases are expressed heterologously in Escherichia coli to a high level of activity. Therefore, this microbe has become a prime system for carrying out whole-cell bioreductions at different scales. The limited capacity of central metabolic pathways in E. coli usually requires that reductase coenzyme in the form of NADPH or NADH be regenerated through a suitable oxidation reaction catalyzed by a second NADP+ or NAD+ dependent dehydrogenase that is co-expressed. Candida tenuis xylose reductase (CtXR) was previously shown to promote NADH dependent reduction of aromatic α-keto esters with high Prelog-type stereoselectivity. We describe here the development of a new whole-cell biocatalyst that is based on an E. coli strain co-expressing CtXR and formate dehydrogenase from Candida boidinii (CbFDH). The bacterial system was evaluated for the synthesis of ethyl R-4-cyanomandelate under different process conditions and benchmarked against a previously described catalyst derived from Saccharomyces cerevisiae expressing CtXR. 相似文献16.
17.
The lipase Lip2 of the edible basidiomycete, Pleurotus sapidus, is an extracellular enzyme capable of hydrolysing xanthophyll esters with high efficiency. The gene encoding Lip2 was expressed
in Escherichia coli TOP10 using the gene III signal sequence to accumulate proteins in the periplasmatic space. The heterologous expression under
control of the araBAD promoter led to the high level production of recombinant protein, mainly as inclusion bodies, but partially
in a soluble and active form. A fusion with a C-terminal His tag was used for purification and immunochemical detection of
the target protein. This is the first example of a heterologous expression and periplasmatic accumulation of a catalytically
active lipase from a basidiomycete fungus. 相似文献
18.
Fei Zhang Qing-Zhong Zheng Qing-Cai Jiao Jun-Zhong Liu Gen-Hai Zhao 《Biotechnology letters》2010,32(8):1147-1150
Glutamic acid γ-methyl ester (GAME) was used as substrate for theanine synthesis catalyzed by Escherichia coli cells possessing γ-glutamyltranspeptidase activity. The yield was about 1.2-fold higher than with glutamine as substrate. The reaction was optimal at pH 10 and 45°C, and the optimal substrate ratio of GAME to ethylamine was 1:10 (mol/mol). With GAME at 100 mmol, 95 mmol theanine was obtained after 8 h. 相似文献
19.
Picon A de Mattos MJ Postma PW 《Journal of industrial microbiology & biotechnology》2008,35(4):213-218
During Escherichia coli growth on glucose, uptake exceeds the requirement of flux to precursors and the surplus is excreted as acetate. Beside the loss of carbon source, the excretion of a weak acid may result in increased energetic demands and hence a decreased yield. The deletion of ptsG, the gene coding for one of the components (IICB(Glc)) of the glucose-phosphoenolpyruvate phosphotransferase system (Glc-PTS) reduced glucose consumption and acetate excretion. Induction of protein production at the onset of cultivation decreased growth rate and glucose consumption rate for both the WT and the mutant strains. The mutant strain produced beta-galactosidase at higher rates than the wild-type strain while directing more carbon into biomass and CO(2) and less into acetate. 相似文献
20.
C. violaceum appeared as important bacterium in different applications and mainly these aspects are related to the production of violacein.
This review discusses the last reports on biosynthetic pathways, production, genetic aspects, biological activities, pathological
effects, antipathogenic screening through quorum sensing, environmental effects and the products of C. violaceum with industrial interest. An important discussion is on biological applications in medicine and as industrial products such
as textile and in cosmetics. 相似文献