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1.
Stefano Raimondi Daniela Uccelletti Alberto Amaretti Alan Leonardi Claudio Palleschi Maddalena Rossi 《Applied microbiology and biotechnology》2010,86(3):871-878
The Kluyveromyces lactis Cu/Zn SOD gene (SOD1) was fused with the toxin K1 signal sequence to obtain extracellular production of superoxide dismutase. Kluyveromyces marxianus L3 and K. lactis MW98-8C strains were transformed and compared as hosts for the secretion. The effects of the media composition were evaluated:
In K. lactis, the highest volumetric activity was obtained in YKK synthetic medium in the presence of Cu2+/Zn2+ cofactors (9.6 kU l−1). In K. marxianus, active SOD was produced only in YPD medium supplemented with Cu2+ and Zn2+ (8.8 kU l−1). In order to improve the production of secreted active SOD in K. lactis, the SOD1 copper carrier (CCS1) was overexpressed and targeted to the secretory apparatus. A positive effect was observed
only when K. lactis was grown in a medium without Cu2+/Zn2+ supplement. The best performing culture conditions for K. lactis and K. marxianus recombinant strains were successfully applied to two laboratory-scale fed-batch processes, and volumetric SOD activities
increased up to 19.4 and 24.1 kU l−1, respectively. 相似文献
2.
Effects of methanol on expression of an anticoagulant hirudin in recombinant Hansenula polymorpha 总被引:1,自引:0,他引:1
A series of batch, fed-batch, and continuous cultures was carried out to analyze the effects of methanol on the fermentation
characteristics of recombinant Hansenula polymorpha for the production of hirudin, an anticoagulant. Hirudin expression efficiencies were greatly influenced by the methanol
concentrations in continuous and fed-batch culture modes. At a steady state of continuous culture, an optimum methanol concentration
of 1.7 g l−1 was determined at a dilution rate of 0.18 h−1 with 1.8 mg l−1 h−1 hirudin productivity. Journal of Industrial Microbiology & Biotechnology (2001) 27, 58–61.
Received 21 September 2000/ Accepted in revised form 10 June 2001 相似文献
3.
Fluorescence-activated cell sorting (FACS) was used to isolate mutants of Lactococcus lactis LAC275, an indicator strain in GFPuv nisin bioassay. It harbors the GFPuv encoding gene under the nisA promoter and the nisin signal transduction nisRK genes whereby nisin concentration can be correlated to GFPuv fluorescence. The sorted L. lactis cells, which showed higher fluorescence intensities at low inducer concentration, were analysed for higher responsiveness
to low concentration of nisin. Two strains showed lower detection limits (0.2 pg ml−1) for nisin than the parent strain (10 pg ml−1). This showed that mutants of LAC275 could successfully be isolated using FACS. 相似文献
4.
There are remarkably few reports on d-arabitol production from lactose. Previous studies in our laboratory have shown that the osmophilic yeast Kluyveromyces lactis NBRC 1903 convert lactose to extracellular d-arabitol. The present study was undertaken to determine the participation of osmotic stress caused by lactose on d-arabitol production by K. lactis NBRC 1903 and to provide the information on the kinetics of d-arabitol production from lactose by K. lactis NBRC 1903. It was confirmed that d-arabitol production was triggered when an initial lactose concentration was above 278 mmol L−1. d-Arabitol yield increased with an increase in initial lactose concentration. The highest d-arabitol concentration of 79.5 mmol L−1 was achieved in the cultivation of K. lactis NBRC 1903 in a medium containing 555 mmol L−1 lactose and 40 g L−1 yeast extract. Lactose was found to play two important roles in d-arabitol production by K. lactis NBRC 1903 grown on lactose. First, lactose was assimilated as the substrate both for cell growth and d-arabitol production. Second, a high lactose concentration induced cellular response to high osmotic stress and up-regulated
the flow from d-glucose-6-phosphate to d-arabitol. The arrest of cell growth triggered d-arabitol production. 相似文献
5.
Aicha Nancib Nabil Nancib Joseph Boudrant 《World journal of microbiology & biotechnology》2009,25(8):1423-1429
The production of lactic acid from date juice by single and mixed cultures of Lactobacillus casei and Lactococcus lactis was investigated. In the present conditions, the highest concentration of lactic acid (60.3 g l−1) was obtained in the mixed culture system while in single culture fermentations of Lactobacillus casei or Lactococcus lactis, the maximum concentration of lactic acid was 53 and 46 g l−1, respectively. In the case of single Lactobacillus casei or Lactococcus lactis, the total percentage of glucose and fructose utilized were 82.2; 94.4% and 93.8; 60.3%, respectively, whereas in the case
of mixed culture, the total percentage of glucose and fructose were 96 and 100%, respectively. These results showed that the
mixed culture system gave better results than single cultures regarding lactic acid concentration, and sugar consumption. 相似文献
6.
7.
Octavio Carvajal-Zarrabal Cirilo Nolasco-Hipólito Kopli B. Bujang Ayaaki Ishizaki 《Journal of industrial microbiology & biotechnology》2009,36(3):409-415
A membrane bioreactor for production of nisin Z was constructed using Lactococcus lactis IO-1 in continuous culture using hydrolyzed sago starch as carbon source. A strategy used to enhance the productivity of
nisin Z was to maintain the cells in a continuous growth at high cell concentration. This resulted in a volumetric productivity
of nisin Z, as 50,000 IU l−1 h−1 using a cell concentration of 15 g l−1, 30°C, pH 5.5 and a dilution rate of 1.24 h−1. Adding 10 g l−1 YE and 2 g l−1 polypeptone, other inducers were unnecessary to maintain production of nisin. The operating conditions of the reactor removed
nisin and lactate, thus minimizing their effects which allowed the maintenance of cells in continuous exponential growth phase
mode with high metabolic activity. 相似文献
8.
Mashitha Pise Jaishree Rudra Sunita Bundale Deovrat Begde Nandita Nashikkar Avinash Upadhyay 《In vitro cellular & developmental biology. Plant》2012,48(1):85-91
Asparagus racemosus is an important monocot medicinal plant that is in great demand for its steroidal saponins called shatavarins. This study
was initiated to optimize the conditions for production of shatavarins in cell cultures of A. racemosus in a modified Murashige and Skoog (MS) medium supplemented with six different combinations of growth regulators. Biomass
accumulation was correlated with saponin production over a 30-d culture cycle. Biomass and saponin accumulation patterns were
dependent on combinations of growth regulators and the pH of the medium. Maximum levels of saponin and biomass accumulation
were recorded on day 25 of the culture cycle within a pH range of 3.4 to 5.6. Total saponin produced by the in vitro cultures was 20-fold higher than amounts produced by cultivated plants. Saponin accumulation was not a biomass-associated
phenomenon; cultures which showed the highest biomass accumulation were not the highest saponin accumulators. Maximum biomass
(28.30 ± 0.29 g l−1) and maximum levels of shatavarin IV(11.48 ± 0.61 mg g−1) accumulation was found using a medium containing 2.0 mg l−1 2,4-D, 2 g l−1 casein hydrolysate and 0.005% pectinase. The highest levels of sarsapogenin, secreted and intracellular (4.02 ± 0.09 mg g−1), accumulated using a medium containing 1.0 mg l−1 NAA, 1.0 mg l−1 2,4-D, 0.5 mg l−1 BAP, 2 g l−1 casein hydrolysate and 0.005% pectinase, after 25 d. Shatavarins were secreted into the medium and can be isolated easily
for further purification. 相似文献
9.
Yihan Liu Fuping Lu Guanqun Chen Crystal L. Snyder Jing Sun Yu Li Jianling Wang Jing Xiao 《Biotechnology letters》2010,32(1):119-124
Alpha-amylases are important industrial enzymes with a wide range of applications. Although medium-temperature alpha amylase
(AmyE) has some practical advantages, its low yield has limited its applications. When an amyE gene from Bacillus subtilis BF768 was cloned into vector pWB980 and over-expressed in B. subtilis WB600, high activities (723 U ml−1) of secreted AmyE were produced. Recombinant AmyE was purified to a specific activity of 36 U mg−1 having optimal activity at pH 6.0 and 60°C. 相似文献
10.
Petri-Jaan Lahtvee Kaspar Valgepea Ranno Nahku Kristo Abner Kaarel Adamberg Raivo Vilu 《Antonie van Leeuwenhoek》2009,96(4):487-496
Growth space of Lactococcus lactis subsp. lactis IL1403 was studied at constant growth rate using D-stat cultivation technique. Starting from steady state conditions in a
chemostat culture (μ = 0.2 h−1), the pH and/or temperature were continuously changed in the range of 5.4–6.4 and 26–34°C, respectively, followed by the
return to the initial environmental conditions. Based on substrate consumption and product formation yields and expression
changes of 1,920 genes, it was shown that changes of physiological state were not dependent on the direction of movement (from
pH 6.3 to 5.4 or from 5.4 to 6.3), showing that quasi steady state values in D-stat corresponded to the steady state values
in chemostats. Relative standard deviation of growth characteristics in triplicate D-stat experiments was below 10%. Continuing
the experiment and reestablishing initial growth conditions revealed in average 7% difference (hysteresis) in growth characteristics
when comparing chemostat steady state cultures prior and after the change of environmental conditions. Similarly, shifts were
also seen at gene expression levels. The large amount of quantitatively reliable data obtained in this study provided a new
insight into dynamic properties of bacterial physiology, and can be used for describing the growth space of microorganisms
by modeling cell metabolism. 相似文献
11.
Okano K Kimura S Narita J Fukuda H Kondo A 《Applied microbiology and biotechnology》2007,75(5):1007-1013
To achieve direct and efficient lactic acid production from starch, a genetically modified Lactococcus lactis IL 1403 secreting α-amylase, which was obtained from Streptococcus bovis 148, was constructed. Using this strain, the fermentation of soluble starch was achieved, although its rate was far from
efficient (0.09 g l−1 h−1 lactate). High-performance liquid chromatography revealed that maltose accumulated during fermentation, and this was thought
to lead to inefficient fermentation. To accelerate maltose consumption, starch fermentation was examined using L. lactis cells adapted to maltose instead of glucose. This led to a decrease in the amount of maltose accumulation in the culture,
and, as a result, a more rapid fermentation was accomplished (1.31 g l−1 h−1 lactate). Maximum volumetric lactate productivity was further increased (1.57 g l−1 h−1 lactate) using cells adapted to starch, and a high yield of lactate (0.89 g of lactate per gram of consumed sugar) of high
optical purity (99.2% of l-lactate) was achieved. In this study, we propose a new approach to lactate production by α-amylase-secreting L. lactis that allows efficient fermentation from starch using cells adapted to maltose or starch before fermentation. 相似文献
12.
Srinivas Ramisetti Hyun Ah Kang Sang Ki Rhee Chul Ho Kim 《Biotechnology and Bioprocess Engineering》2003,8(3):183-186
The artificial gene coding for anticoagulant hirudin was placed under the control of theGAL10 promoter and expressed in the galactokinase-deficient strain (Δgal1) ofSaccharomyces cereivisiae, which uses galactose only as a gratuitous inducer in order to avoid its consumption. For efficient production of recombinant
hirudin, a carbon source other than galactose should be provided in the medium to support growth of the Δgal1 strain. Here we demonstrate the successful use of glucose in the fed-batch fermentation of the Δgal1 strain to achieve efficient production of recombinant hirudin, with a yield of up to 400 mg hirudin/L. 相似文献
13.
The Kluyveromyces marxianus strains CBS 6556, CBS 397 and CBS 712T were cultivated on a defined medium with either glucose, lactose or sucrose as the sole carbon source, at 30 and 37°C. The
aim of this work was to evaluate the diversity within this species, in terms of the macroscopic physiology. The main properties
evaluated were: intensity of the Crabtree effect, specific growth rate, biomass yield on substrate, metabolite excretion and
protein secretion capacity, inferred by measuring extracellular inulinase activity. The strain Kluyveromyces lactis CBS 2359 was evaluated in parallel, since it is the best described Kluyveromyces yeast and thus can be used as a control for the experimental setup. K. marxianus CBS 6556 presented the highest specific growth rate (0.70 h−1) and the highest specific inulinase activity (1.65 U mg−1 dry cell weight) among all strains investigated, when grown at 37°C with sucrose as the sole carbon source. The lowest metabolite
formation and highest biomass yield on substrate (0.59 g dry cell weight g sucrose−1) was achieved by K. marxianus CBS 712T at 37°C. Taken together, the results show a systematic comparison of carbon and energy metabolism among three of the best
known K. marxianus strains, in parallel to K. lactis CBS 2359. 相似文献
14.
A cDNA fragment encoding the A catalytic domain of the Neocallimastix frontalis endoxylanase XYN3 was amplified and cloned by the polymerase chain reaction technique. The xyn3A DNA fragment was inserted between the Saccharomyces cerevisiae phosphoglycerate kinase gene promoter and terminator sequences on a multicopy episomal plasmid for Kluyveromyces lactis. The XYN3A domain was successfully expressed in K. lactis and functional endoxylanase was secreted by the yeast cells with the K. lactis killer toxin secretion signal. The XYN3A domain was also expressed in a strain of Penicillium roqueforti as a fusion protein (ShBLE::XYN3A) of the phleomycin-resistance gene product and the endoxylanase. Active endoxylanase was
efficiently secreted from the fungal cells with the Trichoderma viride cellobiohydrolase (CBH1) secretion signal and processed by a related KEX2 endoprotease of the secretion pathway. Several
differently glycosylated forms of the recombinant enzymes were secreted by the yeast and the filamentous fungus.
Received: 10 November 1998 / Received revision: 8 March 1999 / Accepted: 14 March 1999 相似文献
15.
Jing Yu Jiaxi Jiang Zian Fang Yuyang Li Hong Lv Jianping Liu 《Biotechnology letters》2010,32(4):507-512
Inulinase gene (Kcinu) derived from Kluyveromyces cicerisporus was expressed extracellularly in Kluyveromyces lactis using an episomal vector directed by Kcinu promoter. The influence of hap1 gene disruption on the expression of inulinase was studied. Inulinase activity in the supernatant of the recombinant Klhap1Δ strain was 391 U ml−1 after cultured 120 h, which was 2.2-fold that of the wild type host. The relative inulinase mRNA level of the Klhap1Δ strain was 11.3-fold that of the wild type strain, and the expression plasmid was more stable in the mutant host. Based on
these results, the disruption of hap1 facilitated the high and stable expression of inulinase controlled by Kcinu promoter in K. lactis. 相似文献
16.
Amore A Amoresano A Birolo L Henrissat B Leo G Palmese A Faraco V 《Applied microbiology and biotechnology》2012,94(4):995-1006
An α-l-arabinofuranosidase produced by Pleurotus ostreatus (PoAbf) during solid state fermentation on tomato pomace was identified and the corresponding gene and cDNA were cloned and
sequenced. Molecular analysis showed that the poabf gene carries 26 exons interrupted by 25 introns and has an open reading frame encoding a protein of 646 amino acid residues,
including a signal peptide of 20 amino acid residues. The amino acid sequence similar to the other α-l-arabinofuranosidases indicated that the enzyme encoded by poabf can be classified as a family 51 glycoside hydrolase. Heterologous recombinant expression of PoAbf was carried out in the
yeasts Pichia pastoris and Kluyveromyces lactis achieving the highest production level of the secreted enzyme (180 mg L−1) in the former host. rPoAbf produced in P. pastoris was purified and characterized. It is a glycosylated monomer with a molecular weight of 81,500 Da in denaturing conditions.
Mass spectral analyses led to the localization of a single O-glycosylation site at the level of Ser160. The enzyme is highly specific for α-l-arabinofuranosyl linkages and when assayed with p-nitrophenyl α-l-arabinofuranoside it follows Michaelis–Menten kinetics with a K
M of 0.64 mM and a k
cat of 3,010 min−1. The optimum pH is 5 and the optimal temperature 40°C. It is worth noting that the enzyme shows a very high stability in
a broad range of pH. The more durable activity showed by rPoAbf in comparison to the other α-l-arabinofuranosidases enhances its potential for biotechnological applications and increases interest in elucidating the molecular
bases of its peculiar properties. 相似文献
17.
Meiru Li Hongqing Li Xiaoying Hu Xiaoping Pan Guojiang Wu 《Plant Cell, Tissue and Organ Culture》2011,106(3):363-371
Saussurea involucrata is a valuable traditional Chinese medicinal herb. This is the first report of a successful genetic transformation protocol
for S. involucrata using Agrobacterium tumefaciens. Leaf explants were incubated with A. tumefaciens strain EHA105 harboring the binary vector pCAMBIA 1301, which contains the hpt gene as a selectable marker for hygromycin resistance and an intron-containing β-glucuronidase gene as a reporter gene. Following
co-cultivation, about 23.7% of the explants produced hygromycin-resistant calli on MS basal medium (Murashige and Skoog in
Physiol Plant 15: 473–497, 1962) supplemented with 1 mg l−1 benzyladenine (BA), 0.1 mg l−1 α-naphthaleneacetic acid (NAA), 0.1 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D), 20 mg l−1 hygromycin, and 500 mg l−1 cefotaxime. Shoots were regenerated following transfer of the resistant calli to shoot induction medium containing 1.5 mg l−1 BA, 0.1 mg l−1 NAA, 0.25 mg l−1 gibberellic acid (GA3), 20 mg l−1 hygromycin, and 250 mg l−1 cefotaxime, and about 67.5% of the resistant calli differentiated into shoots. Finally, 80% of the hygromycin-resistant shoots
rooted on MS media supplemented with 0.2 mg l−1 NAA, 20 mg l−1 hygromycin, and 250 mg l−1 cefotaxime. The transgenic nature of the transformants was demonstrated by detection of β-glucuronidase activity in the primary
transformants and by Southern blot hybridization analysis. About 16% of the total inoculated leaf explants produced transgenic
plants after approximately 5 months. Using this optimized transformation system, a rice ortholog of the Arabidopsis FLOWERING LOCUS T gene, Hd3a, was transferred into S. involucrata. Introduction of this gene caused an early-flowering phenotype in S. involucrata. 相似文献
18.
Xin-chun Mo Chun-lan Chen Hao Pang Yi Feng Jia-xun Feng 《Applied microbiology and biotechnology》2010,87(6):2137-2146
A metagenomic library containing ca. 3.06 × 108 bp insert DNA was constructed from a rice straw degrading enrichment culture. A xylanase gene, umxyn10A, was cloned by screening the library for xylanase activity. The encoded enzyme Umxyn10A showed 58% identity and 73% similarity
with a xylanase from Thermobifida fusca YX. Sequence analyses showed that Umxyn10A contained a glycosyl hydrolase family 10 catalytic domain. The gene was expressed
in Escherichia coli, and the recombinant enzyme was purified and characterized biochemically. Recombinant Umxyn10A was highly active toward xylan.
However, the purified enzyme could slightly hydrolyze β-1,3/4-glucan and β-1,3/6-glucan. Umxyn10A displayed maximal activity
toward oat spelt xylan at a high temperature (75°C) and weak acidity (pH 6.5). The K
m
and V
max of Umxyn10A toward oat spelt xylan were 3.2 mg ml−1 and 0.22 mmol min−1 mg−1 and were 2.7 mg ml−1 and 1.0 mmol min−1 mg−1 against birchwood xylan, respectively. Metal ions did not appear to be required for the catalytic activity of this enzyme.
The enzyme Umxyn10A could efficiently hydrolyze birchwood xylan to release xylobiose as the major product and a negligible
amount of xylose. The xylanase identified in this work may have potential application in producing xylobiose from xylan. 相似文献
19.
Meiru Li Hongqing Li Xiaoying Hu Xiaoping Pan Guojiang Wu 《Plant Cell, Tissue and Organ Culture》2010,102(3):321-327
Zoysia tenuifolia Willd. ex Trin. is one of the most popularly cultivated turfgrass. This is the first report of successful plant regeneration
and genetic transformation protocols for Z. tenuifolia using Agrobacterium tumefaciens. Initial calli was induced from stem nodes incubated on a Murashige and Skoog (1962) (MS) medium supplemented with 2 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 mg l−1 6-benzyladenine (BA), with a frequency of 53%. Compact calli were selected and subcultured monthly on the fresh medium. Sixty-nine
percent of the calli could be induced to regenerate plantlets when the calli incubated on a MS medium supplemented with 0.2 mg l−1 BA under darkness. For genetic transformation, calli were incubated with A. tumefaciens strain EHA105 harboring the binary vector pCAMBIA 1301 which contains the hpt gene as a selectable marker for hygromycin resistance and an intron-containing β-glucuronidase gene (gus-int) as a reporter gene. Following co-cultivation, about 12% of the callus explants produced hygromycin resistant calli on
MS medium supplemented with 2 mg l−1 2,4-D, 1 mg l−1 BA, 50 mg l−1 hygromycin, 500 mg l−1 cefotaxime after 8 weeks. Shoots were regenerated following transfer of the resistant calli to shoot induction medium containing
0.2 mg l−1 BA, 50 mg l−1 hygromycin, and 250 mg l−1 cefotaxime, and about 46% of the resistant calli differentiated into shoots. Finally, all the resistant shoots were rooted
on 1/2 MS media supplemented with 50 mg l−1 hygromycin, 250 mg l−1 cefotaxime. The transgenic nature of the transformants was demonstrated by the detection of β-glucuronidase activity in the primary transformants and by PCR and Southern hybridization analysis. About 5% of the total
inoculated callus explants produced transgenic plants after approximately 5 months. The procedure described will be useful
for both, the introduction of desired genes into Z. tenuifolia and the molecular analysis of gene function. 相似文献