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1.
Wei Zhao Aisheng Xiong Xiaoyan Fu Feng Gao Yongsheng Tian Rihe Peng 《Applied biochemistry and biotechnology》2010,162(8):2157-2165
To obtain a high level expression of phytase with favorable characteristics, a codon-optimized phytase gene from Citrobacter freundii was synthesized and transferred into Pichia pastoris. Small-scale expression experiments and activity assays were used to screen positive colonies. After purified by Ni2+–NTA agarose affinity column, the characterizations of the recombinant phytase were determined. The recombinant phytase (r-phyC)
had two distinct pH optima at 2.5 and 4.5 and an optimal temperature at 50 °C. It retained more than 80% activity after being
incubated under various buffer (pH 1.5–8.0) at 37 °C for 1 h. The specific activity, Km, and Vmax values of r-phyC for sodium phytate were 2,072 ± 18 U mg−1, 0.52 ± 0.04 mM, and 2,380 ± 84 U mg−1 min−1, respectively. The enzyme activity was significantly improved by 1 mM of K+, Ca2+, and Mg2+. These characteristics contribute to its potential application in feed industry. 相似文献
2.
Yun Wang Jin-Zhu Song Qian Yang Zhi-Hua Liu Xiao-Mei Huang Yan Chen 《Applied biochemistry and biotechnology》2010,162(3):843-854
A gene encoding chitin deacetylase was cloned by polymerase chain reaction from Aspergillus nidulans. Sequencing result showed 40% homology to the corresponding gene from Colletotrichum lindemuthianum. The complete gene contains an open reading frame of 747 nucleotides encoding a sequence of 249 amino acid residues. The
chitin deacetylase gene was subcloned into a pET28a expression vector and expressed in Escherichia coli BL21 and then purified by metal affinity chromatography using a His-bind column. The purified chitin deacetylase demonstrated
an activity of 0.77 U ml−1 for the glycol chitin substrates, and its specific activity was 4.17 U mg−1 for it. The optimal temperature and pH of the purified enzyme were 50 °C and 8.0, respectively. When glycol chitin was used
as the substrate, K
m was 4.92 mg ml−1, and K
cat showed 6.25 s−1, thus the ratio of K
cat and K
m was 1.27 ml s−1 mg−1. The activity of chitin deacetylase was affected by a range of metal ions and ethylenediaminetetraacetic acid. 相似文献
3.
A phosphite dehydrogenase gene (ptdhK) consisting of 1,011-bp nucleotides which encoding a peptide of 336 amino acid residues was cloned from Pseudomonas sp. K. gene ptdhK was expressed in Escherichia coli BL21 (DE3) and the corresponding recombinant enzyme was purified by metal affinity chromatography. The recombinant protein
is a homodimer with a monomeric molecular mass of 37.2 kDa. The specific activity of PTDH-K was 3.49 U mg−1 at 25 °C. The recombinant PTDH-K exhibited maximum activity at pH 3.0 and at 40 °C and displayed high stability within a
wide range of pHs (5.0 to 10.5). PTDH-K had a high affinity to its natural substrates, with K
m values for sodium phosphite and NAD of 0.475 ± 0.073 and 0.022 ± 0.007 mM, respectively. The activity of PTDH-K was enhanced
by Na+, NH4+, Mg2+, Fe2+, Fe3+, Co2+, and EDTA, and PTDH-K exhibited different tolerance to various organic solvents. 相似文献
4.
5.
Meijuan Xu Zhiming Rao Hong Xu Chunyan Lan Wenfang Dou Xiaomei Zhang Hongyu Xu Jian Jin Zhenghong Xu 《Applied biochemistry and biotechnology》2011,163(6):707-719
Corynebacterium crenatum SYPA 5-5 is an aerobic and industrial l-arginine producer. It was proved that the Corynebacterium glutamicum/Escherichia coli shuttle vector pJC1 could be extended in C. crenatum efficiently when using the chloramphenicol acetyltransferase gene (cat) as a reporter under the control of promoter tac. The expression system was applied to over-express the gene vgb coding Vitreoscilla hemoglobin (VHb) to further increase the dissolved oxygen in C. crenatum. As a result, the recombinant C. crenatum containing the pJC-tac-vgb plasmid expressed VHb at a level of 3.4 nmol g−1, and the oxygen uptake rates reached 0.25 mg A562−1 h−1 which enhanced 38.8% compared to the wild-type strain. Thus, the final l-arginine concentration of the batch fermentation reached a high level of 35.9 g L−1, and the biomass was largely increased to 6.45 g L−1, which were 17.3% and 10.5% higher than those obtained by the wild-type strain, respectively. To our knowledge, this is the
first report that the efficient expression system was constructed to introduce vgb gene increasing the oxygen and energy supply for l-arginine production in C. crenatum, which supplies a good strategy for the improvement of amino acid products. 相似文献
6.
Wanli Liu Pengjun Shi Qiang Chen Peilong Yang Guozeng Wang Yaru Wang Huiying Luo Bin Yao 《Applied biochemistry and biotechnology》2010,162(1):1-12
A xylanase-encoding gene, xyn11F63, was isolated from Penicillium sp. F63 CGMCC1669 using degenerated polymerase chain reaction (PCR) and thermal asymmetric interlaced (TAIL)-PCR techniques.
The full-length chromosomal gene consists of 724 bp, including a 73-bp intron, and encodes a 217 amino acid polypeptide. The
deduced amino acid sequence of xyn11F63 shows the highest identity of 70% to the xylanase from Penicillium sp. strain 40, which belongs to glycosyl hydrolases family 11. The gene was overexpressed in Pichia pastoris, and its activity in the culture medium reached 516 U ml−1. After purification to electrophoretic homogeneity, the enzyme showed maximal activity at pH 4.5 and 40°C, was stable at
acidic buffers of pH 4.5–9.0, and was resistant to proteases (proteinase K, trypsin, subtilisin A, and α-chymotrypsin). The
specific activity, K
m, and V
max for oat spelt xylan substrate was 7,988 U mg−1, 22.2 mg ml−1, and 15,105.7 μmol min−1 mg−1, respectively. These properties make XYN11F63 a potential economical candidate for use in feed and food industrial applications. 相似文献
7.
Glucose 2-oxidase (pyranose oxidase, pyranose:oxygen-2-oxidoreductase, EC 1.1.3.10) from Coriolus versicolor catalyses the oxidation of d-glucose at carbon 2 in the presence of molecular O2 producing d-glucosone (2-keto-glucose and d-arabino-2-hexosulose) and H2O2. It was used to convert d-glucose into d-glucosone at moderate pressures (i.e. up to 150 bar) with compressed air in a modified commercial batch reactor. Several
parameters affecting biocatalysis at moderate pressures were investigated as follows: pressure, [enzyme], [glucose], pH, temperature,
nature of fluid and the presence of catalase. Glucose 2-oxidase was purified by immobilized metal affinity chromatography
on epoxy-activated Sepharose 6B-IDA-Cu(II) column at pH 6.0. The rate of bioconversion of d-glucose increased with the pressure since an increase in the pressure with compressed air resulted in higher rates of conversion.
On the other hand, the presence of catalase increased the rate of reaction which strongly suggests that H2O2 acted as inhibitor for this reaction. The rate of bioconversion of d-glucose by glucose 2-oxidase in the presence of either nitrogen or supercritical CO2 at 110 bar was very low compared with the use of compressed air at the same pressure. The optimum temperature (55°C) and
pH (5.0) of d-glucose bioconversion as well as kinetic parameters for this enzyme were determined under moderate pressure. The activation
energy (E
a) was 32.08 kJ mol−1 and kinetic parameters (V
max, K
m, K
cat and K
cat/K
m) for this bioconversion were 8.8 U mg−1 protein, 2.95 mM, 30.81 s−1 and 10,444.06 s−1 M−1, respectively. The biomass of C. versicolor as well as the cell-free extract containing glucose 2-oxidase activity were also useful for bioconversion of d-glucose at moderate pressures. The enzyme was apparently stable at moderate pressures since such pressures did not affect
significantly the enzyme activity. 相似文献
8.
Rym Agrebi Noomen Hmidet Mohamed Hajji Nawrez Ktari Anissa Haddar Nahed Fakhfakh-Zouari Moncef Nasri 《Applied biochemistry and biotechnology》2010,162(1):75-88
In this study, Mirabilis jalapa tuber powder (MJTP) was used as a new complex organic substrate for the growth and production of fibrinolytic enzymes by
a newly isolated Bacillus amyloliquefaciens An6. Maximum protease activity (1,057 U/ml) with casein as a substrate was obtained when the strain was grown in medium containing
(grams per liter) MJTP 30, yeast extract 6, CaCl2 1, K2HPO4 0.1, and K2HPO4 0.1. The strain was also found to grow and produce extracellular proteases in a medium containing only MJTP, indicating that
it can obtain its carbon, nitrogen, and salts requirements directly from MJTP. The B. amyloliquefaciens An6 fibrinase (BAF1) was partially purified, and fibrinolytic activity was assayed in a test tube with an artificial fibrin
clot. The molecular weight of the partially purified BAF1 fibrinolytic protease was estimated to be 30 kDa by sodium dodecyl
sulfate polyacrylamide gel electrophoresis and gel filtration. The optimum temperature and pH for the caseinolytic activity
were 60 °C and 9.0, respectively. The enzyme was highly stable from pH 6.0 to 11.0 and retained 62% of its initial activity
after 1 h incubation at 50 °C. However, the enzyme was inactivated at higher temperatures. The activity of the enzyme was
totally lost in the presence of phenylmethylsulfonyl fluoride, suggesting that BAF1 is a serine protease. 相似文献
9.
Laccase activity was detected in a soil bacterium Stenotrophomonas maltophilia AAP56 identified by biochemical and molecular methods. It was produced in cells at the stationary growth phase in Luria Bertani
(LB) medium added by 0.4 mM copper sulfate. The addition of CuSO4 in culture medium improved production of laccase activity. However, one laccase enzyme was detected by native polyacrylamide
gel electrophoresis. The enzyme showed syringaldazine (K
m = 53 μM), 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (K
m = 700 μM), and pyrocatechol (K
m = 25 μM) oxidase activity and was activated by addition of 0.1% (v/v) Triton-X-100 in the reaction mixture. Moreover, the laccase activity was increased 2.6-fold by the addition of 10 mM copper
sulfate; the enzyme was totally inhibited by ethylenediaminetetraacetic acid (5 mM), suggesting that this laccase is a metal-dependant
one. Decolorization activity of some synthetic dyes (methylene blue, methyl green, toluidine blue, Congo red, methyl orange,
and pink) and the industrial effluent (SITEX Black) was achieved by the bacteria S. maltophilia AAP56 in the LB growth medium under shaking conditions. 相似文献
10.
Daniela de Araújo Viana Carolina de Albuquerque Lima Rejane Pereira Neves Cristina Souza Mota Keila Aparecida Moreira José Luiz de Lima-Filho Maria Taciana Holanda Cavalcanti Attilio Converti Ana Lúcia Figueiredo Porto 《Applied biochemistry and biotechnology》2010,162(3):830-842
Cow raw milk from dairy cooperatives was examined for its microbial composition. Among the isolates identified, 17.6% were
yeasts. The most frequent genus was Candida, although members belonging to the genera Brettanomyces, Dekkera, and Geotricum were also identified. Although qualitative and quantitative tests for extracellular proteolytic activity were positive for
all the species isolated, Candida buinensis showed the highest response (23.5 U/mg); therefore, it was selected for subsequent investigation. The results of fermentations
carried out at variable temperature, pH, and soybean flour concentration, according to a 23 full factorial design, demonstrated that this yeast ensured the highest production of extracellular proteases (573 U/mL)
when cultivated at 35 °C, pH 6.5, and using soybean flour concentrations in the range 0.1–0.5% (w/v). The cell-free supernatants showed the highest activity at 25 °C and pH 7.0, and satisfactory stability in the ranges 25–30 °C
and pH 7–9. The first-order rate constants of protease inactivation in the cell-free supernatants were calculated at different
temperatures from semi-log plots of the residual activity versus time and then used in Arrhenius and Eyring plots to estimate
the main thermodynamic parameters of thermoinactivation (E* = 40.0 kJ/mol; ΔH* = 37.3 kJ/mol; ΔS* = −197.5 J/mol K; ΔG* = 101 kJ/mol). 相似文献
11.
Qi Wu Chen Li Chenglei Li Hui Chen Liu Shuliang 《Applied biochemistry and biotechnology》2010,160(1):129-139
The collagenase, produced extracellular by Bacillus pumilus Col-J, was purified by ammonium sulfate precipitation followed by two gel filtrations, involving Sephadex G-100 column and
Sepharose Fast Flow column. Purified collagenase has a 31.53-fold increase in specific activity of 87.33 U/mg and 7.00% recovery.
The collagenase has a relative molecular weight of 58.64 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
The optimal temperature for the enzyme reaction was 45 °C. More than 50% of the original activity still remained after 5 min
of incubation at 70 °C or 10 min at 60 °C. The maximal enzyme activity of collagenase was obtained at pH 7.5, and it was stable
over a pH range of 6.5–8.0. The collagenase activity was strongly inhibited by Mn2+, Pb2+, ethylenediamine tetraacetic acid, ethylene glycol tetraacetic acid, and β-mercaptoethanol. However, Ca2+ and Mg2+ greatly increased its activity. The collagenase from B. pumilus Col-J showed highly specific activity towards the native collagen from calf skin. The K
m and V
max of the enzyme for collagen were 0.79 mg/mL and 129.5 U, respectively. 相似文献
12.
A 66-kDa thermostable family 1 Glycosyl Hydrolase (GH1) enzyme with β-glucosidase and β-galactosidase activities was purified
to homogeneity from the seeds of Putranjiva roxburghii belonging to Euphorbiaceae family. N-terminal and partial internal amino acid sequences showed significant resemblance to plant GH1 enzymes. Kinetic
studies showed that enzyme hydrolyzed p-nitrophenyl β-d-glucopyranoside (pNP-Glc) with higher efficiency (K
cat/K
m = 2.27 × 104 M−1 s−1) as compared to p-nitrophenyl β-d-galactopyranoside (pNP-Gal; K
cat/K
m = 1.15 × 104 M−1 s−1). The optimum pH for β-galactosidase activity was 4.8 and 4.4 in citrate phosphate and acetate buffers respectively, while
for β-glucosidase it was 4.6 in both buffers. The activation energy was found to be 10.6 kcal/mol in the temperature range
30–65 °C. The enzyme showed maximum activity at 65 °C with half life of ~40 min and first-order rate constant of 0.0172 min−1. Far-UV CD spectra of enzyme exhibited α, β pattern at room temperature at pH 8.0. This thermostable enzyme with dual specificity
and higher catalytic efficiency can be utilized for different commercial applications. 相似文献
13.
Yao Guo Fengxia Lu Haizhen Zhao Yanchong Tang Zhaoxin Lu 《Applied biochemistry and biotechnology》2010,162(2):498-509
A gene of glucose oxidase (GOD) from Aspergillus niger Z-25 was cloned and sequenced. The entire open reading frame (ORF) consisted of 1,818 bp and encoded a putative peptide of
605 amino acids. The gene was fused to the pPICZαA plasmid and overexpressed in Pichia pastoris SMD1168. The recombinant GOD (rGOD) was secreted into the culture using MF-α factor signal peptide under the control of the
AOX1 promoter. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that rGOD exhibited a single band at around
94 kDa. The maximal GOD activity of approximately 40 U/mL was achieved in shake flask by induction under optimal conditions
after 7 days. rGOD was purified by ammonium sulfate precipitate leading to a final specific activity of 153.46 U/mg. The optimum
temperature and pH of the purified enzyme were 40 °C and 6.0, respectively. Over 88% of maximum activity was maintained below
40 °C. And the recombinant enzyme displayed a favorable stability in the pH range from 4.0 to 8.0. The Lineweaver–Burk plotting
revealed that rGOD exhibited a K
m value of 16.95 mM and a K
cat value of 484.26 s−1. 相似文献
14.
Sequential acidic precipitation followed by a single chromatographic step (gel filtration) allowed the recovery of a collagenolytic
fraction containing several proteases from by-products of snow crab (Chionoecetes opilio). The partial purification was particularly efficient to recover tryptic (purification fold = 1,352.5; yield = 110%) but
also chymotryptic, elastolytic, and collagenolytic activities. A temperature of 40 °C and pH 8.0–8.5 were optimal for enzyme
activity, which was stable for 2 h under these conditions. Calcium was not required for stability and thus activity. The isoelectric
points of the protein components ranged from 3.7 to 4.6. Zymography revealed 29 and 48 kDa major components and others from
22 to 56 kDa. Enzymes were inhibited by PMSF and TLCK but were insensitive to TPCK. In view of these properties, the proteases
likely belong to the serine collagenase group. Inhibition by EDTA could be due to a mechanism other than Ca2+ chelation. Using a food system (ground fish), the fraction was more proteolytic than a commercial bacterial protease, suggesting
potential applications in enzymatic hydrolysis processes. 相似文献
15.
Farshad Sohbatzadeh Abasalt Hosseinzadeh Colagar Saeed Mirzanejhad Somayeh Mahmodi 《Applied biochemistry and biotechnology》2010,160(7):1978-1984
We developed and employed a new geometrical structure of dielectric barrier discharge in atmospheric pressure for bacterial
broad spectrum sterilization. We utilized a plasma source having an AC power supply at 50 HZ and 5,400 V (rms value). We prepared
suspensions of the Gram-negative bacteria species (Escherichia coli, Pseudomonas aeruginosa) and a Gram-positive of Bacillus cereus with Luria–Bertani broth media up to OD600 nm = 0.25 of McFarland standard. Afterglow of non-thermal atmospheric pressure plasma treated these suspensions. The influence
of the atmospheric plasma afterglow on the species was assayed in different time durations 5, 10, and 15 min. The spectroscopic
results of this investigation indicated that the survival reduction of the species can reach to 100% for P. aeruginosa in an exposure time of 10 min, E. coli and B. cereus in an exposure time of 15 min. 相似文献
16.
A feeding technology that was suitable for improving the nisin production by Lactococcus lactis subsp. lactis W28 was established. The effects of initial sucrose concentration (ISC) in the fermentation broth, feeding time, and feeding
rate on the fermentation were studied. It was observed that a fed-batch culture (ISC = 10 g l−1) with 100 ml sucrose solution (190 g l−1) being evenly fed (9–10 ml h−1) into the fermenter after 3-h fermentation gave the best performance in terms of biomass and nisin yield. Under these conditions,
the total biomass and the total nisin yield were approximately 23% and 51% higher than those in batch fermentation, respectively.
When the sucrose concentration was controlled at 5–10 g l−1 in variable volume intermittent fed-batch fermentation (VVIF) with ISC = 10 g l−1, the total biomass and the total nisin yield were 29% and 60% above those in batch fermentation, respectively. The VVIF proved
to be effective to eliminate the substrate inhibition by maintaining sucrose at appropriate levels. It is also easy to be
scaled up, since various parameters involved in industrial production were taken into account. 相似文献
17.
Glucofructans from Saussurea lappa (Asteraceae) roots were studied. It was found that free fructose and oligomeric glucofructans (saccharose, 1-kestose, nystose,
1F-β-fructofuranosylnystose, and 1F-β-fructofuranosyl-1F-β-fructofuranosylnystose) were present. The dominant polymer Sl-GF (MW 51.4 kDa), which was a linear inulin-type glucofructan
consisting of β-(2 → 1)-bonded fructofuranose units, was isolated and characterized. The total content of glucofructans in Saussurea lappa roots was 476.97–578.27 mg/g. 相似文献
18.
F. Ceyda Dudak İsmail H. Boyacı Agnese Jurkevica Mahmud Hossain Zoraida Aquilar H. Brian Halsall Carl J. Seliskar William R. Heineman 《Analytical and bioanalytical chemistry》2009,393(3):949-956
A rapid and convenient assay system was developed to detect viable Escherichia coli in water. The target bacteria were recovered from solution by immunomagnetic separation and incubated in tryptic soy broth
with isopropyl-β-d-thiogalactopyranoside, which induces formation of β-galactosidase in viable bacteria. Lysozyme was used to lyse E. coli cells and release the β-galactosidase. β-Galactosidase converted 4-methylumbelliferyl-β-d-galactoside to 4-methylumbelliferone (4-MU), which was measured by fluorescence spectrophotometry using excitation and emission
wavelengths of 355 and 460 nm, respectively. Calibration graphs of 4-MU fluorescence intensity versus E. coli concentration showed a detection range between 8 × 104 and 1.6 × 107 cfu mL−1, with a total analysis time of less than 3 h. The advantage of this method is that it detects viable cells because it is
based on the activity of the enzyme intrinsic to live E. coli. 相似文献
19.
Xu Z Li S Fu F Li G Feng X Xu H Ouyang P 《Applied biochemistry and biotechnology》2012,166(4):961-973
d-tagatose is a ketohexose that can be used as a novel functional sweetener in foods, beverages, and dietary supplements. This
study was aimed at developing a high-yielding d-tagatose production process using alginate immobilized Lactobacillus fermentum CGMCC2921 cells. For the isomerization from d-galactose into d-tagatose, the immobilized cells showed optimum temperature and pH at 65 °C and 6.5, respectively. The alginate beads exhibited
a good stability after glutaraldehyde treatment and retained 90% of the enzyme activity after eight cycles (192 h at 65 °C)
of batch conversion. The addition of borate with a molar ratio of 1.0 to d-galactose led to a significant enhancement in the d-tagatose yield. Using commercial β-galactosidase and immobilized L. fermentum cells, d-tagatose was successfully obtained from lactose after a two-step biotransformation. The relatively high conversion rate and
productivity from d-galactose to d-tagatose of 60% and 11.1 g l−1 h−1 were achieved in a packed-bed bioreactor. Moreover, lactobacilli have been approved as generally recognized as safe organisms,
which makes this L. fermentum strain an attracting substitute for recombinant Escherichia coli cells among d-tagatose production progresses. 相似文献
20.
Xiaofeng Wang Yongchuan Sun Feng Ke Heyun Zhao Tao Liu Li Xu Yun Liu Yunjun Yan 《Applied biochemistry and biotechnology》2012,166(5):1355-1367
A gene encoding Yarrowia lipolytica lipase LIP2 (YlLIP2) was cloned into a constitutive expression vector pGAPZαA and electrotransformed into the Pichia pastoris X-33 strain. The high-yield clones obtained by high copy and enzyme activity screening were chosen as the host strains for
shaking flask and fermentor culture. The results showed that glucose was the optimum carbon source for YlLIP2 production,
and the maximum hydrolytic activity of recombinant YlLIP2 reached 1,315 U/ml under the flask culture at 28 °C, pH 7.0, for
48 h. The fed-batch fermentation was carried out in 3- and 10-l bioreactors by continuously feeding glucose into the growing
medium for achieving high cell density and YlLIP2 yields. The maximum hydrolytic activity of YlLIP2 and cell density obtained
in the 3-l bioreactor were 10,300 U/ml and 116 g dry cell weight (DCW)/l, respectively. The peak hydrolytic activity of YlLIP2
and cell density were further improved in the 10-l fermentor where the values respectively attained were 13,500 U/ml and 120 g
DCW/l. The total protein concentration in the supernatant reached 3.3 g/l and the cell viability remained approximately 99%
after 80 h of culture. Furthermore, the recombinant YlLIP2 produced in P. pastoris pGAP and pAOX1 systems have similar content of sugar (about 12%) and biochemical characteristics. The above results suggest
that the GAP promoter-derived expression system of P. pastoris is effective for the expression of YlLIP2 by high cell density culture and is probably an alternative to the conventional
AOX1 promoter expression system in large-scale production of industrial lipases. 相似文献