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1.
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
2.
Root segments from seedlings of Panax ginseng produced adventitious roots directly when cultured on 1/2 MS solid medium lacking NH4NO3 and containing 3.0 mg l−1 IBA. Using this adventitious root formation, we developed rapid and efficient transgenic root formation directly from adventitious
root segments in P. ginseng. Root segments were co-cultivated with Agrobacterium tumefaciens (GV3101) caring β-glucuronidase (GUS) gene. Putative transgenic adventitious roots were formed directly from root segments on medium with 400 mg l−1 cefotaxime and 50 mg l−1 kanamycin. Kanamycin resistant adventitious roots were selected and proliferated as individual lines by subculturing on medium
with 300 mg l−1 cefotaxime and 50 mg l−1 kanamycin at two weeks subculture interval. Frequency of transient and stable expression of GUS gene was enhanced by acetosyringon (50 mg l−1) treatment. Integration of transgene into the plants was confirmed by the X-gluc reaction, PCR and Southern analysis. Production
of transgenic plants was achieved via somatic embryogenesis from the embryogenic callus derived from independent lines of
adventitious roots. The protocol for rapid induction of transgenic adventitious roots directly from adventitious roots can
be applied for a new Agrobacterium tumefaciens-mediated genetic transformation protocol in P. ginseng. 相似文献
3.
Heike Helmholz Blair D. Johnston Christiane Ruhnau Andreas Prange 《Hydrobiologia》2010,645(1):223-234
Scyphozoan medusae are very successful foragers which occasionally occur in high abundances in boreal waters and may impact
many different groups in the marine ecosystem by means of a variety of toxins. A rainbow trout gill cell line, RTgill-W1,
was tested for its suitability as quantitative indicator of the cytotoxicity of Cyanea capillata and Aurelia aurita; the major scyphozoan species in the North and Baltic seas. Cultures of rainbow trout gill cells were exposed to whole venoms
extracted from fishing tentacles and oral arms at increasing protein concentrations. The venom caused detachment, clumping
and lysis of cells, as well as a drop in vitality, in a dose-dependent manner. Morphological changes in the cells were evident
within 1 h after venom addition. The damage to gill cells was quantified by measuring the metabolic activity of the cells
by means of the fluorescence of resorufin derived from the nonfluorescent substrate, resazurin. In general, a decrease in
the metabolic activity of the cells was detected at a venom (protein) concentration above 2.0 μg ml−1 (corresponding to 0.2 μg 104 cells−1), and a total loss of activity was observed above 40.0 μg ml−1 (corresponding to 4.0 μg 104 cells−1). C. capillata venoms had increased cytotoxic activity as compared to A. aurita venoms at the same concentration. Cnidocyst extracts from oral arms of A. aurita induced an 85% loss of gill cell viability at concentrations of 0.2 μg 104 cells−1, whereas crude venoms from fishing tentacles reduced cell viability by 18% at the same concentration. Gel electrophoresis
of the venoms indicated that these consist of a large number of proteins in a fairly wide size range, from 6 to 200 kDa, including
some that are the same size as those found in cubomedusae. It also appears that larger (i.e., older) medusae have more complex
venoms and, in some cases, more potent venoms than smaller animals. 相似文献
4.
5.
Abdul Ghaffar Sher Afzal Khan Zahid Mukhtar Muhammad Ibrahim Rajoka Farooq Latif 《Molecular biology reports》2011,38(5):3227-3233
We studied heterologous expression of xylanase 11A gene of Chaetomium thermophilum in Pichia pastoris and characterized the thermostable nature of the purified gene product. For this purpose, the xylanase 11A gene of C. thermophilum was cloned in P. pastoris GS115 under the control of AOX1 promoter. The maximum extracellular activity of recombinant xylanase (xyn698: gene with intron) was 15.6 U ml−1 while that of recombinant without intron (xyn669) was 1.26 U ml−1 after 96 h growth. The gene product was purified apparently to homogeneity level. The optimum temperature of pure recombinant
xylanase activity was 70°C and the enzyme retained its 40.57% activity after incubation at 80°C for 10 min. It exhibited quite
lower demand of activation energy, enthalpy, Gibbs free energy, entropy, and xylan binding energy during substrate hydrolysis
than that required by that of the donor, thus indicating its thermostable nature. pH-dependent catalysis showed that it was
quite stable in a pH range of 5.5–8.5. This revealed that gene was successfully processed in P. pastoris and remained heat stable and may qualify for its potential use in paper and pulp and animal feed applications. 相似文献
6.
7.
Yali Xu Amrita Yasin Thomas Wucherpfennig C. Perry Chou 《World journal of microbiology & biotechnology》2008,24(12):2827-2835
Functional expression of heterologous Pseudozyma antarctica lipase B (PalB) in the periplasm of Escherichia coli was explored using four fusion tags, i.e. DsbC, DsbA, maltose-binding protein (MBP), and FLAG in the sequence of increasing
expression efficacy. Amongst these fusion tags, FLAG and MBP appear to be the most effective ones in terms of boosting enzyme
activity and enhancing solubility of PalB, respectively. Overexpression of these PalB fusions often resulted in concomitant
formation of insoluble inclusion bodies. Coexpression of a selection of periplasmic folding factors, including DegP (and its
mutant variant of DegPS210A), FkpA, DsbA, DsbC, and a cocktail of SurA, FkpA, DsbA, and DsbC, could improve the expression performance. Coexpression
of DsbA appeared to be the most effective in reducing the formation of inclusion bodies for all the four PalB fusions, implying
that functional expression of PalB could be limited by initial bridging of disulfide bonds. Culture performance was optimized
by overexpressing FLAG-PalB with DsbA coexpression, resulting in a high volumetric PalB activity of 360 U/L. 相似文献
8.
Nathaniel Liddy Peter E. Molloy Alan D. Bennett Jonathan M. Boulter Bent K. Jakobsen Yi Li 《Molecular biotechnology》2010,45(2):140-149
Previously, we have described the use of phage display to generate high affinity disulfide bond-linked T cell receptors (TCRs).
The affinities of the mutant TCRs were analysed after refolding of separately expressed α and β chains from Escherichia coli inclusion bodies. This approach is only suitable for the analysis of small numbers of TCR variants. An attractive alternative
would be soluble expression within the bacterial periplasm, but the generic production of TCRs within the E. coli periplasm has so far not proved successful. Here we show that functional, soluble TCR can be produced within the cytoplasm
of trxB gor mutant E. coli strains, with maximum yields of 3.4 mg/l. We also investigated the effect of coexpressing the folding modulators Skp and
DsbC finding that the TCR expression levels were largely unaffected by these chaperones. Importantly, we demonstrated that
the amount of protein purified from 50 ml starter cultures was sufficient to show functionality of the TCR by specific antigen
binding in both ELISA and surface plasmon resonance (SPR) assays. This TCR production method has the potential to allow rapid
and medium throughput analysis of affinity-matured TCRs selected from TCR phage display libraries. 相似文献
9.
Recently, the gene coding for a new beta-glucuronidase enzyme has been identified and cloned from Streptococcus equi subsp. zooepidemicus. This is another report of a beta-glucuronidase gene cloned from bacterial species. The ORF Finder analysis of a sequenced
DNA (EMBL, AJ890474) revealed a presence of 1,785 bp large ORF potentially coding for a 594 aa protein. Three protein families
in (Pfam) domains were identified using the Conserved Domain Database (CDD) analysis: Pfam 02836, glycosyl hydrolases family
2, triose phosphate isomerase (TIM) barrel domain; Pfam 02837, glycosyl hydrolases family 2, sugar binding domain; and Pfam
00703, glycosyl hydrolases family 2, immunoglobulin-like beta-sandwich domain. To gain more insight into the enzymatic activity,
the domains were used to generate a bootstrapped unrooted distance tree using ClustalX. The calculated distances for two domains,
TIM barrel domain, and sugar-binding domain were comparable and exhibited similarity pattern based on function and thus being
in accordance with recently published works confirming beta-glucuronidase activity of the enzyme. The calculated distances
and the tree arrangement in the case of centrally positioned immonoglobulin-like beta-sandwich domain were somewhat higher
when compared to other two domains but clustering with other beta-glucuronidases was rather clear. Nine proteins, including
beta-glucuronidases, beta-galactosidase, and mannosidase were selected for multiple alignment and subsequent distance tree
creation. 相似文献
10.
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
11.
Dong Cao Wensheng Hou Shikui Song Hongbo Sun Cunxiang Wu Yongsheng Gao Tianfu Han 《Plant Cell, Tissue and Organ Culture》2009,96(1):45-52
Agrobacterium rhizogenes-mediated transformation has become a powerful tool for studying gene function and root biology due to its quick and simple
methodology. This transformation method is particularly suitable for those plants, including legumes, whose transformation
using Agrobacterium tumefaciens has been challenging. Although there are some reports on A. rhizogenes-mediated transformation of legumes to produce ‘composite’ plants, conditions influencing A. rhizogenes-mediated transformation of soybean [Glycine max (L.) Merr.] have not been yet fully investigated. To better understand A. rhizogenes-mediated root transformation in soybean, we have evaluated the impact of genotype, plant age for infection, bacterial inoculating
concentration, inoculation temperature, and other factors on transformation of soybean. The results have shown that there
are significant differences among soybean genotypes in their susceptibility to A. rhizogenes. Soybean cv. Zigongdongdou is the most susceptible to A. rhizogenes strain K599 among 10 genotypes tested. The effects of seedling age have been evaluated, and 1-day-old plantlets are found
to be optimal for hairy root induction. There are no significant differences in hairy root induction for bacterial suspension
from OD600 = 0.2 to OD600 = 1.2. Under 16 h photoperiod, hairy roots can be induced both at 23°C/20°C and 28°C/25°C, but not at 33°C/30°C as day/night
temperature regimes. Using this transformation protocol, almost 100% of the composite plants formed hairy roots within 2 weeks,
and based on GUS histochemical analysis, 94.2% transformation frequency is obtained. Transgene integration has been also confirmed
by Southern blot analysis.
D. Cao and W. Hou contributed equally to this work. 相似文献
12.
Young-Min Kim Byung-Hoon Kim Joon-Seob Ahn Go-Eun Kim Sheng-De Jin Thanh-Hanh Nguyen Doman Kim 《Biotechnology letters》2009,31(9):1433-1438
Alkyl glucosides were synthesized by the reaction of Leuconostoc mesenteroides dextransucrase with sucrose and various alcohols. Alkyl α-d-glucosides were obtained with a yield of 30% (mol/mol) with primary alcohols, but secondary alcohols or tertiary alcohols
gave yields below 5%. The optimal yield was 50% using 1-butyl α-d-glucoside with 0.9 M 1-butanol. The acceptor products of methanol or ethanol were confirmed as methyl α-d-glucopyranoside and ethyl α-d-glucopyranoside via MALDI-TOF MS and NMR analysis. Thus, methyl or ethyl α-d-glucoside constituted half the emulsification activities of Triton X-100 as commercially available surfactants.
Young-Min Kim and Byung-Hoon Kim contributed equally to this work. 相似文献
13.
Akhilesh Kumar Amrita Chakraborty Srijani Ghanta Sharmila Chattopadhyay 《Plant Cell, Tissue and Organ Culture》2009,96(2):117-126
Morphologically identical transgenic mint (Mentha arvensis L.) with bacterial glutathione synthetase gene has been developed. Transformed plants were obtained by co-cultivation of
leaf disks with Agrobacterium tumefaciens strain LBA 4404 harbouring a binary vector pCAMBIA-CpGS that carried E. coli glutathione synthetase (GS), β-glucuronidase as reporter gene and nptII as selective marker gene for kanamycin resistance. Using a constitutive double CaMV 35S promoter and an rbcS transit peptide, we successfully addressed CpGS to the chloroplasts through pJIT 117 vector. Preculture and the presence of AS in the co-cultivation medium played a significant
role in enhancing transformation frequency. The highest transformation frequency was achieved with MS selection medium supplemented
with 25% coconut water, 1.12 mg l−1 BAP, 0.2 mg l−1 NAA, 50 mg l−1 kanamycin and 125 mg l−1 cefotaxime. Robust rooting of regenerated shoots was obtained in half-strength liquid MS medium containing 0.2 mg l−1 NAA and 50 mg l−1 kanamycin. The presence and expression of transgenes in transgenics (T0) was evidenced by GUS histoenzymatic assay, PCR and RT-PCR analysis of nptII and the gene of interest, i.e., GS of putative transgenic leaves. Chromosomal integration of GS gene was confirmed by Southern blot analysis. Transgenic plants were successfully acclimatized in the greenhouse. An overall
transformation frequency of 15% was achieved in approximately 3 months of time period. These results are discussed in relation
to heavy metal trafficking pathways in higher plants and to the interest of using plastid expression of PCS for biotechnological applications.
Akhilesh Kumar and Amrita Chakraborty contributed equally. 相似文献
14.
15.
Heterologous expression of genes involved in the biosynthesis of various products is of increasing interest in biotechnology and in drug research and development. Microbial cells are most appropriate for this purpose. Availability of more microbial genomic sequences in recent years has greatly facilitated the elucidation of metabolic and regulatory networks and helped gain overproduction of desired metabolites or create novel production of commercially important compounds. Saccharomyces cerevisiae, as one of the most intensely studied eukaryotic model organisms with a rich density of knowledge detailing its genetics, biochemistry, physiology, and large-scale fermentation performance, can be capitalized upon to enable a substantial increase in the industrial application of this yeast. In this review, we describe recent efforts made to produce commercial secondary metabolites in Saccharomyces cerevisiae as pharmaceuticals. As natural products are increasingly becoming the center of attention of the pharmaceutical and nutraceutical industries, such as naringenin, coumarate, artemisinin, taxol, amorphadiene and vitamin C, the use of S. cerevisiae for their production is only expected to expand in the future, further allowing the biosynthesis of novel molecular structures with unique properties. 相似文献
16.
Konstantin V. Kiselev Anna V. Turlenko Yuri N. Zhuravlev 《Plant Growth Regulation》2009,59(3):237-243
17.
Anna Szczerbakowa Justyna Tarwacka Michał Oskiera Henryka Jakuczun Bernard Wielgat 《Acta Physiologiae Plantarum》2010,32(5):867-873
Interspecific somatic hybrids between a diploid potato clone DG 81-68 susceptible to Phytophthora infestans (Mont.) de Bary and a resistant diploid tuber-bearing species Solanum × michoacanum were generated and analyzed. About 30 regenerants displaying an intermediate morphology were obtained as a result of three
separate PEG-mediated fusion experiments. The RAPD analysis confirmed the hybridity of all the regenerants. About 50% of the
hybrid plants exhibited vigorous growth and were stable in culture, while the rest of them rooted poorly and grew slowly in
vitro. Most of the hybrid clones were at the tetraploid level (70%), while 30% of the clones examined were at the hexaploid
level. The S. × michoacanum (+) DG 81-68 hybrids with growth anomalies were aneuploid. The variation in late blight resistance of the hybrid clones was
found in detached leaflet tests, with enhanced resistance characteristic for three tetraploid hybrids. 相似文献
18.
Many different processes have an impact on the shape of plant karyotype. Recently, cytogenetic examination of Lolium species has revealed the occurrence of spontaneous fragile sites (FSs) associated with 35S rDNA regions. The FSs are defined as the chromosomal regions that are sensitive to forming gaps or breaks on chromosomes. The shape of karyotype can also be determined by interstitial telomeric sequences (ITSs), what was recognized for the first time in this paper in chromosomes of Festuca pratensis × Lolium perenne hybrids. Both FSs and ITSs can contribute to genome instabilities and chromosome rearrangements. To evaluate whether these cytogenetic phenomena have an impact on karyotype reshuffling observed in Festuca × Lolium hybrids, we examined F1 F. pratensis × L. perenne plants and generated F2-F9 progeny by fluorescent in situ hybridization (FISH) using rDNA sequences, telomere and centromere probes, as well as by genomic in situ hybridization (GISH). Analyses using a combination of FISH and GISH revealed that intergenomic rearrangements did not correspond to FSs but overlapped with ITSs for several analyzed genotypes. It suggests that internal telomeric repeats can affect the shape of F. pratensis × L. perenne karyotypes. However, other factors that are involved in rearrangements and have a more crucial impact could exist, but they are still unknown. 相似文献
19.
Transformation of 2,4,6-trinitrotoluene (TNT) by <Emphasis Type="Italic">Raoultella</Emphasis> <Emphasis Type="Italic">terrigena</Emphasis> 总被引:1,自引:0,他引:1
Claus H Bausinger T Lehmler I Perret N Fels G Dehner U Preuss J König H 《Biodegradation》2007,18(1):27-35
Manufacture of nitroorganic explosives generates toxic wastes leading to contamination of soils and waters, especially groundwater.
For that reason bacteria living in environments highly contaminated with 2,4,6-trinitrotoluene (TNT) and other nitroorganic
compounds were investigated for their capacity for TNT degradation. One isolate, Raoultella terrigena strain HB, removed TNT at concentrations between 10 and 100 mg l−1 completely from culture supernatants under optimum aerobic conditions within several hours. Only low concentrations of nutrient
supplements were needed for the cometabolic transformation process. Radioactivity measurements with ring-labelled 14C–TNT detected about 10–20% of the initial radioactivity in the culture supernatant and the residual 80–90% as water-insoluble
organic compounds in the cellular pellet. HPLC analysis identified aminodinitrotoluenes (2-ADNT, 4-ADNT) and diaminonitrotoluenes
(2,4-DANT) as the metabolites which remained soluble in the culture medium and azoxy-dimers as the main products in the cell
extracts. Hence, the new isolate could be useful for the removal of TNT from contaminated waters. 相似文献
20.
Özge Özden William M. Ciesla Wayne J. Fuller David J. Hodgson 《Biodiversity and Conservation》2008,17(12):2821-2832
We analysed the influence of contemporary geography on butterfly diversity for islands in the Mediterranean Basin. We found
that island size and distance from the mainland has a significant effect on the number of species. We also used butterflies
as an indicator group to identify the importance of forest habitats for biodiversity conservation in the island of Cyprus.
To understand the relative importance of local vegetation characteristics of butterflies in the Pentadaktylos mountains transect
counts were used to assess the abundance and butterfly diversity in two different forest types. A total of 1,602 butterflies
and 23 species were recorded during this research. We observed highly significant effects of forest type on abundance and
species richness of butterflies. For example, number of butterflies was significantly higher in old forest than young pine
forest. Also, the abundance of endemic butterflies was highest in old forest habitats. Therefore, the survival of the majority
of endemic butterflies in Cyprus may depend on conservation of old forests and their understorey plants. 相似文献