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1.
Hyaluronic acid production by recombinant <Emphasis Type="Italic">Lactococcus lactis</Emphasis> 总被引:1,自引:0,他引:1
A gene encoding a new xylanase, named xynZG, was cloned by the genome-walking PCR method from the nematophagous fungus Plectosphaerella cucumerina. The genomic DNA sequence of xynZG contains a 780 bp open reading frame separated by two introns with the sizes of 50 and 46 bp. To our knowledge, this would
be the first functional gene cloned from P. cucumerina. The 684 bp cDNA was cloned into vector pHBM905B and transformed into Pichia pastoris GS115 to select xylanase-secreting transformants on RBB-xylan containing plate. The optimal secreting time was 3 days at
25°C and enzymatic activities in the culture supernatants reached the maximum level of 362 U ml−1. The molecular mass of the enzyme was estimated to be 19 kDa on SDS-PAGE. The optimal pH and temperature of the purified
enzyme is 6 and 40°C, respectively. The purified enzyme is stable at room temperature for at least 10 h. The K
m and V
max values for birchwood xylan are 2.06 mg ml−1 and 0.49 mmol min−1mg−1, respectively. The inhibitory effects of various mental ions were investigated. It is interesting to note that Cu2+ ion, which strongly inhibits most other xylanases studied, reduces enzyme activity by only 40%. Furthermore, enzyme activity
is unaffected by EDTA even at a concentration of 5 mM. 相似文献
2.
Lee MH Yang SJ Kim JW Lee HS Kim JW Park KH 《Extremophiles : life under extreme conditions》2007,11(3):537-541
A gene that encodes the enzyme Pyrococcus furiosus cyclodextrin glucanotransferase (PFCGT) was cloned in Escherichia
coli. PFCGT was highly expressed in recombinant E. coli after compensation for codon usage bias using the pRARE plasmid. Purified PFCGT was extremely thermostable with an optimal
temperature and pH of 95°C and 5.0, respectively, retaining 97% of its activity at 100°C. Incubation at 60°C for 20 min during
the purification process led to a 1.5-fold increase in enzymatic activity. A time course assay of the PFCGT reaction with
starch indicated that cyclic α-1,4-glucans with DPs greater than 20 were produced at the beginning of the incubation followed
by an increase in β-CD. The major final product of PFCGT cyclization was β-CD, and thus the enzyme is a β-CGTase. 相似文献
3.
Jorge CD Sampaio MM Hreggvidsson GO Kristjánson JK Santos H 《Extremophiles : life under extreme conditions》2007,11(1):115-122
Trehalases play a central role in the metabolism of trehalose and can be found in a wide variety of organisms. A periplasmic
trehalase (α,α-trehalose glucohydrolase, EC 3.2.1.28) from the thermophilic bacterium Rhodothermus marinus was purified and the respective encoding gene was identified, cloned and overexpressed in Escherichia coli. The recombinant trehalase is a monomeric protein with a molecular mass of 59 kDa. Maximum activity was observed at 88°C
and pH 6.5. The recombinant trehalase exhibited a K
m
of 0.16 mM and a V
max of 81 μmol of trehalose (min)−1 (mg of protein)−1 at the optimal temperature for growth of R. marinus (65°C) and pH 6.5. The enzyme was highly specific for trehalose and was inhibited by glucose with a K
i
of 7 mM. This is the most thermostable trehalase ever characterized. Moreover, this is the first report on the identification
and characterization of a trehalase from a thermophilic bacterium. 相似文献
4.
Filipa M. Lupi Helena M. L Fernandes Isabel Sá-Correia Júlio M. Novais 《Journal of applied phycology》1991,3(1):35-42
Temperature profiles (range 20–33 °C) were obtained for growth and exopolysaccharide (EPS) biosynthesis of the microalga Botryococcus braunii strain UC 58 under photoautotrophic conditions. The maximum temperature for growth was 32 °C and the temperature dependence
of the specific growth rate was described by the Hinshelwood equation based on the Arrhenius relationship. The optimal range
of temperatures for growth and extracellular EPS synthesis (25–30 °C) concurred and production of 4.5–5 g l−1 of EPS was obtained routinely, leading to high broth viscosities. Below 23 °C EPS biosynthesis was negligible, although the
specific growth rate maintained high values. At supraoptimal temperatures EPS biosynthesis decreased, accompanying the increase
in doubling time.
The polymers formed at temperatures within the optimal range for production, when dissolved in water, produced solutions (2
gl−1) with the highest viscosity, suggesting that their molecular weight showed the highest values. The degree of polymerization
of the EPS synthesized at suboptimal and supraoptimal temperatures was significantly below the values within the optimal range. 相似文献
5.
Anaerobic fungi belonging to the family Neocallimastigaceae are native inhabitants in the rumen of the most herbivores, such
as cattle, sheep and goats. A member of this unique group, Neocallimastix sp. GMLF2 was isolated from cattle feces and screened for its xylanase encoding gene using polymerase chain reaction. The
gene coding for a xylanase (xyn2A) was cloned in Escherichia coli and expression was monitored. To determine the enzyme activity, assays were conducted for both fungal xylanase and cloned
xylanase (Xyl2A) for supernatant and cell-associated activities. Optimum pH and temperature of the enzyme were found to be
6.5 and 50°C, respectively. The enzyme was stable at 40°C and 50°C for 20 min but lost most of its activity when temperature
reached 60°C for 5-min incubation time. Rumen fungal xylanase was mainly released to the supernatant of culture, while cloned
xylanase activity was found as cell-associated. Multiple alignment of the amino acid sequences of Xyl2A with published xylanases
from various organisms suggested that Xyl2A belongs to glycoside hydrolase family 11. 相似文献
6.
The rpiB gene, encoding ribose-5-phosphate isomerase (RpiB) from Clostridium thermocellum, was cloned and expressed in Escherichia coli. RpiB converted d-psicose into d-allose but it did not convert d-xylose, l-rhamnose, d-altrose or d-galactose. The production of d-allose by RpiB was maximal at pH 7.5 and 65°C for 30 min. The half-lives of the enzyme at 50°C and 65°C were 96 h and 4.7 h,
respectively. Under stable conditions of pH 7.5 and 50°C, 165 g d-allose l−1
was produced without by-products from 500 g d-psicose l−1 after 6 h. 相似文献
7.
A heat shock was applied to Saccharomyces cerevisiae: a change from 18°C to 45°C over 5 min and then maintenance at later temperature for 20 min followed by cooling to 18°C.
Such a treated inoculum, when used in an alcoholic fermentation of Welsch Riesling grape must at 18°C, gave up to 12 g glycerol l−1 This is a new and easy method for high glycerol production in large scale wine production. 相似文献
8.
9.
The marcoalga Ulva pertusa was cultured under (20 ± 2)°C, (20 ± 4)°C, (20 ± 6)°C, (20 ± 8)°C and (20 ± 10)°C circadian rhythms of fluctuating temperature
conditions, and constant temperature of 20°C was used as the control. The growth rate of macroalga at (20 ± 2)°C, (20 ± 4)°C
and (20 ± 6)°C were significantly higher than that at constant temperature of 20°C, while growth rate at (20 ± 8)°C and (20
± 10)°C were significantly lower than that at constant temperature of 20°C. The growth rate of macroalga was a quadratic function
of the thermal amplitude. Such a growth model can be described by G = β
0 + β
1(TA) + β
2(TA)2, where G represents the relative growth rate, TA is thermal amplitude in degree Celsius, β
0 is the intercept on the G axis, and β
1 and β
2 are the regression coefficients. The optimal thermal amplitude for the growth of thallus at mean temperature of 20°C was
estimated to be ± 3.69°C. Analysis of biochemical composition at the final stages of thaulls growth revealed that diel fluctuating
temperature caused various influences (P < 0.05). The content of chlorophyll, protein and total solute carbohydrate at (20 ± 2)°C and (20 ± 4)°C were slightly higher
than those at constant temperature of 20°C, however no statistically significant differences were found among them (P > 0.05). While osmolytes (total solute carbohydrate and free proline) at (20 ± 10)°C were significantly higher than that at
20°C (P < 0.05). Therefore, more chlorophyll and carbohydrate production might account for the enhancement in the growth of macroalga
at the diel fluctuating temperatures in the present study.
Handling editor: S. M. Thomaz 相似文献
10.
Wan-Chi Liang Xuan-Hui Wang Min-Guan Lin Long-Liu Lin 《Indian journal of microbiology》2009,49(2):151-160
The heat shock protein 70 (Hsp70/DnaK) gene of Bacillus licheniformis is 1,839 bp in length encoding a polypeptide of 612 amino acid residues. The deduced amino acid sequence of the gene shares
high sequence identity with other Hsp70/DnaK proteins. The characteristic domains typical for Hsps/DnaKs are also well conserved
in B. licheniformis DnaK (BlDnaK). BlDnaK was overexpressed in Escherichia coli using pQE expression system and the recombinant protein was purified to homogeneity by nickel-chelate chromatography. The
optimal temperature for ATPase activity of the purified BlDnaK was 40°C in the presence of 100 mM KCl. The purified BlDnaK had a V
max of 32.5 nmol Pi/min and a K
M of 439 μM. In vivo, the dnaK gene allowed an E. coli dnaK756-ts mutant to grow at 44°C, suggesting that BlDnaK should be functional for survival of host cells under environmental changes especially higher temperature. We also described
the use of circular dichroism to characterize the conformation change induced by ATP binding. Binding of ATP was not accompanied
by a net change in secondary structure, but ATP together with Mg2+ and K+ ions had a greater enhancement in the stability of BlDnaK at stress temperatures. Simultaneous addition of DnaJ, GrpE, and NR-peptide (NRLLLTG) synergistically stimulates the
ATPase activity of BlDnaK by 11.7-fold. 相似文献
11.
Yin Li Zhiqiang Liu Fengjie Cui Yingying Xu Hui Zhao 《World journal of microbiology & biotechnology》2007,23(6):837-843
A new strain of Penicillium sp. ZH-30 that produces xylanase was isolated from soil. According to the morphology and comparison of internal transcribed
spacer (ITS) rDNA gene sequence, the strain Penicillium sp. ZH-30 was identified as a strain of Penicillium oxalicum. When xylan or wheat bran was used as substrate at 30°C for 3 days under submerged cultivation, xylanase production was 5.3
and 13.3 U ml−1, respectively. The temperature and pH for optimum activity were 50°C and 5.0–6.0, respectively. 相似文献
12.
A truncated Escherichia coli Novablue γ-glutamyltranspeptidase (EcGGT) gene lacking the first 48-bp coding sequence for part of the signal sequence was amplified by polymerase chain reaction and cloned into expression vector pQE-30 to generate pQE-EcGGT. The maximum production of His6-tagged enzyme by E. coli M15 (pQE-EcGGT) was achieved with 0.1 mM IPTG induction for 12 h at 20 °C. The overexpressed enzyme was purified to homogeneity by nickel-chelate chromatography to a specific transpeptidase activity of 4.25 U/mg protein and a final yield of 83%. The molecular masses of the subunits of the purified enzyme were estimated to be 41 and 21 kDa respectively by SDS-PAGE, indicating EcGGT still undergoes the post-translational cleavage even in the truncation of signal sequence. The optimum temperature and pH for the recombinant enzyme were 40 °C and 9, respectively. The apparent K
m and V
max values for γ-glutamyl-p-nitroanilide as γ-glutamyl donor in the transpeptidation reaction were 37.9 μM and 53.7 × 10−3 mM min−1, respectively. The synthesis of L-theanine was performed in a reaction mixture containing 10 mM
L-Gln, 40 mM ethylamine, and 1.04 U His6-tagged EcGGT/ml, pH 10, and a conversion rate of 45% was obtained. 相似文献
13.
Victor E. Balderas Hernández Luz M. T. Paz Maldonado Emilio Medina Rivero Ana P. Barba de la Rosa Leandro G. Ordoñez Acevedo Antonio De León Rodríguez 《Biotechnology and Bioprocess Engineering》2008,13(1):7-13
The production of human interferon gamma (hIFN-γ) using a synthetic gene in Escherichia coli BL21-SI was optimized by response surface methodology (RSM) and a Box-Behnken design. The process variables studied were
temperature, bio-mass concentration at induction time and the NaCl concentration as inducer. According to the Box-Behnken
design, a second order response function was developed. The optimal expression conditions were a temperature of 32.6°C, induction
biomass of 0.31 g/L and 0.3 M NaCl in minimal medium. The model prediction for the maximum hIFN-γ production was 77.3 mg/L,
which corresponded satisfactorily with the experimental data. The hIFN-γ concentration attained under optimized conditions
was 13-times higher than that obtained using the non-optimized conditions. We conclude that RSM is an effective method for
the optimization of recombinant protein expression using synthetic genes in E. coli. 相似文献
14.
15.
Properties of the extracellular amylase produced by the psychrotrophic bacterium, Arthrobacter psychrolactophilus, were determined for crude preparations and purified enzyme. The hydrolysis of soluble starch by concentrated crude preparations
was found to be a nonlinear function of time at 30 and 40 °C. Concentrates of supernatant fractions incubated without substrate
exhibited poor stability at 30, 40, or 50 °C, with 87% inactivation after 21 h at 30 °C, 45% inactivation after 40 min at
40 °C and 90% inactivation after 10 min at 50 °C. Proteases known to be present in crude preparations had a temperature optimum
of 50 °C, but accounted for a small fraction of thermal instability. Inactivation at 30, 40, or 50 °C was not slowed by adding
20 mg/ml bovine serum albumin or protease inhibitor cocktail to the preparations or the assays to protect against proteases.
Purified amylase preparations were almost as thermally sensitive in the absence of substrate as crude preparations. The temperature
optimum of the amylase in short incubations with Sigma Infinity Amylase Reagent was about 50 °C, and the amylase required
Ca+2 for activity. The optimal pH for activity was 5.0–9.0 on soluble starch (30 °C), and the amylase exhibited a K
m with 4-nitrophenyl-α-D-maltoheptaoside-4,6-O-ethylidene of 120 μM at 22 °C. The amylase in crude concentrates initially hydrolyzed raw starch at 30 °C at about the same
rate as an equal number of units of barley α-amylase, but lost most of its activity after only a few hours. 相似文献
16.
The endochitinase DNA and cDNA from Trichoderma sp. were cloned, sequenced and expressed. The cloned DNA and cDNA sequences were 1,476 and 1,275 bp in length, respectively.
There were three introns in DNA sequence in comparison with the cDNA sequence. The endochitinase protein contained three regions:
the signal peptide, the prepro-region and the mature protein region. The gene fragment encoding the mature endochitinase was
ligated into the expression vector pET-28a+, yielding pET-1. The plasmid pET-1 was transformed into the Escherichia coli BL21 (DE3). The clone bearing pET-1 was picked and cultured at 30°C for the expression of endochitinase. SDS-PAGE analysis
showed that the endochitinase was expressed in the periplasmic space and the purified protein showed a single band. The activity
of 70.2 U/mg was obtained from the cellular extract of the recombinant strain. The activity of endochitinase was 2.5-fold
higher at 24 h than at 16 h in the periplasmic space. The optimal pH and temperature of the recombinant endochitinase were
determined to be 7.0 and 35°C, respectively. It was relatively stable within the pH range of 5–8. Significant activity stimulation
by 1 mM Mg2+ and 5 mM Fe2+ and inhibition by 5 mM Co2+ and 5 mM Hg2+ were observed. The kinetic constants Km, Vmax and Kcat for the hydrolysis of the colloidal chitin were 1.5 mM, 1.37 μmol min−1 and 6.23 min−1, respectively. 相似文献
17.
The gene encoding pectate lyase (PL) from Bacillus subtilis WSHB04-02 was amplified by PCR, fused with a periplasmic secretion signal peptide sequence, pelB, from pET22b(+), cloned and expressed in Escherichia coli cells using a temperature control vector, pHsh. The recombinant E. coil was grown in a 5 l fermentor. PL was secreted in broth at 22 U l−1 after 20 h when temperature was increased from 30°C to 42°C. The recombinant enzyme was purified to homogeneity as judged
by SDS-PAGE. It was optimally active at pH 9.4 and 50°C over 30 min. Analysis of polygalacturonic acid (PGA) degradation products
by electrospray ionization (ESI)-mass spectrometry (MS) indicated that PL produced a mixture of unsaturated oligo-galacturonides
including unsaturated tri-galacturonic acid and unsaturated bi-galacturonic acid but not unsaturated mono-galacturonic acid. 相似文献
18.
<Emphasis Type="Italic">Thermotoga maritima</Emphasis> TM0298 is a highly thermostable mannitol dehydrogenase 总被引:1,自引:1,他引:0
Song SH Ahluwalia N Leduc Y Delbaere LT Vieille C 《Applied microbiology and biotechnology》2008,81(3):485-495
Thermotoga maritima TM0298 is annotated as an alcohol dehydrogenase, yet it shows high identity and similarity to mesophilic mannitol dehydrogenases.
To investigate this enzyme further, its gene was cloned and expressed in Escherichia coli. The purified recombinant enzyme was most active on fructose and mannitol, making it the first known hyperthermophilic mannitol
dehydrogenase. T. maritima mannitol dehydrogenase (TmMtDH) is optimally active between 90 and 100 °C and retains 63% of its activity at 120 °C but shows
no detectable activity at room temperature. Its kinetic inactivation follows a first-order mechanism, with half-lives of 57 min
at 80 °C and 6 min at 95 °C. Although TmMtDH has a higher V
max with NADPH than with NADH, its catalytic efficiency is 2.2 times higher with NADH than with NADPH and 33 times higher with
NAD+ than with NADP+. This cofactor specificity can be explained by the high density of negatively charged residues (Glu193, Asp195, and Glu196)
downstream of the NAD(P) interaction site, the glycine motif. We demonstrate that TmMtDH contains a single catalytic zinc
per subunit. Finally, we provide the first proof of concept that mannitol can be produced directly from glucose in a two-step
enzymatic process, using a Thermotoga neapolitana xylose isomerase mutant and TmMtDH at 60 °C. 相似文献
19.
Liu JR Duan CH Zhao X Tzen JT Cheng KJ Pai CK 《Applied microbiology and biotechnology》2008,81(2):225-233
A gene encoding a xylanase, named xynS20, was cloned from the ruminal fungus Neocallimastix patriciarum. The DNA sequence of xynS20 revealed that the gene was 1,008 bp in size and encoded amino acid sequences with a predicted molecular weight of 36 kDa.
The amino acid sequence alignment showed that the highest sequence identity (28.4%) is with insect gut xylanase XYL6805. According
to the sequence-based classification, a putative conserved domain of glycosyl hydrolase family 11 was detected at the N-terminus
of XynS20 and a putative conserved domain of family 1 carbohydrate-binding module (CBM) was observed at the C-terminus of
XynS20. An Asn-rich linker sequence was found between the N-terminal catalytic domain and the C-terminal CBM of XynS20. To
examine the activity of the gene product, xynS20 gene was cloned as an oleosin-fused protein, expressed in Escherichia coli, affinity-purified by formation of artificial oil bodies, released from oleosin by intein-mediated peptide cleavage, and
finally harvested by concentration of the supernatant. The specific activity of purified XynS20 toward oat spelt xylan was
1,982.8 U mg−1. The recombinant XynS20 was stable in the mild acid pH range from 5.0 to 6.0, and the optimum pH was 6.0. The optimal reaction
temperature of XynS20 was 45°C; at temperatures below 30 and above 55°C, enzyme activity was less than 50% of that at the
optimal temperature. 相似文献
20.
Mao-hong Zhou Jian-she Ma Jun Li Hai-ren Ye Ke-xin Huang Xiao-wei Zhao 《Biotechnology and Bioprocess Engineering》2008,13(5):545-551
A bacterial strain able to produce κ-carrageenase, designated WZUC10, was isolated from a live specimen of the red alga Plocamium telfainae collected in the East China Sea. The phylogenetic evidence and phenotypic features indicate that this strain belongs to the
genus Pseudoalteromonas. WZUC10 requires NaCl for growth and κ-carrageenan to induce κ-carrageenase synthesis; galactose and lactose do not induce
it. The optimal growth temperature is 23∼27°C. The secreted enzyme, which has a molecular mass of 45 kDa, breaks down κ-carrageenan
into κ-neocarratetraose sulfate and larger oligosaccharides with a repeating β-D-Galp4S-(1→4)-α-D-AnGalp structure, but cannot degrade κ-neocarratetraose sulfate or κ-neocarrahexaose sulfate into κ-neocarrabiose sulfate. The enzyme
retains 90% of its activity after 2 h at 40°C and is completely inactivated after 7.5 min at 70°C. The enzyme’s optimal temperature
is 30°C and its optimal pH is 7.5. The enzyme-catalyzed reaction follows Michaelis-Menten kinetics, with the Michaelis constant
(K
m) and the turnover number (k) being 0.015 mM and 125 s−1, respectively. WZUC10 produces 50 U/mL κ-carrageenase after cultivation at 25°C for 35 h on a medium containing 80 g/L glucose,
5 g/L corn steep liquor, 3 g/L κ-carrageenan, and 15 g/L NaCl. κ-Neocarratetraose sulfate was prepared simply with precipitation
by ethanol:water (5:1, v/v). 相似文献