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1.
The gene encoding the cyclodextrin glucanotransferase of Paenibacillus pabuli US132 was connected to the amylase signal peptide of Bacillus stearothermophilus. This leads to an efficient secretion of the recombinant enzyme into the culture medium of Escherichia coli as an active form contrasting with the native construction leading to a periplasmic production. The optimum cultivation conditions
for the maximum expression were optimized, using a Box-Behnken design under the response surface methodology, and found to
be a post-induction temperature of 24°C, an induction-starting A600 nm of 0.85, an isopropyl-β-D-thiogalactopyranoside level of 0.045 mM and a post-induction time of 3.9 h. The screening of media components and their
concentration were achieved using a Plackett-Burman and a Box-Behnken designs sequentially. Under the optimized conditions
selected and in agreement with the predicted model, an activity of 6.03 U/mL was attained. This CGTase production was three-times
higher than that using the non-optimized culture conditions (2 U/mL). 相似文献
2.
Maldonado LM Hernández VE Rivero EM Barba de la Rosa AP Flores JL Acevedo LG De León Rodríguez A 《Biomolecular engineering》2007,24(2):217-222
A human interferon beta (hINF-beta) synthetic gene was optimized and expressed in Escherichia coli BL21-SI using a vector with the T7 promoter. To determine the best culture conditions such as culture medium, temperature, cell density and inducer concentration, we used the response surface methodology and a Box-Behnken design to get the highest hINF-beta production. The maximum hINF-beta production of 61 mg l(-1) was attained using minimum medium and the following predicted optimal conditions: temperature of 32.5 degrees C, cell density of 0.64, and inducer concentration of 0.30 M NaCl. This is the first report showing the successful performance of the BL21-SI system in a minimum medium. The response surface methodology is effective for the optimization of recombinant protein production using synthetic genes. 相似文献
3.
Bussari B Saudagar PS Shaligram NS Survase SA Singhal RS 《Journal of industrial microbiology & biotechnology》2008,35(1):49-58
Cephamycin C is an extracellular broad spectrum β-lactam antibiotic produced by Streptomyces clavuligerus, S. cattleya and Nocardia lactamdurans. In the present study, different substrates for solid-state fermentation were screened for maximum cephamycin C production
by S. clavuligerus NT4. The fermentation parameters such as substrate concentration, moisture content, potassium dihydrogen phosphate, inoculum
size and ammonium oxalate were optimized by response surface methodology (RSM). The optimized conditions yielded 21.68 ± 0.76 mg gds−1 of cephamycin C as compared to 10.50 ± 1.04 mg gds−1 before optimization. Effect of various amino acids on cephamycin C production was further studied by using RSM, which resulted
in increased yield of 27.41 ± 0.65 mg gds−1. 相似文献
4.
Najla Gasmi Franck Fudalej Héla Kallel Jean-Marc Nicaud 《Applied microbiology and biotechnology》2011,89(1):109-119
In this work, we investigated the effect of codon bias and consensus sequence (CACA) at the translation initiation site on
the expression level of heterologous proteins in Yarrowia lipolytica; human interferon alpha 2b (hIFN-α2b) was studied as an example. A codon optimized hIFN-α2b gene was synthesized according
to the frequency of codon usage in Y. lipolytica. Both wild-type (IFN-wt) and optimized hIFN-α2b (IFN-op) genes were expressed under the control of a strong inducible promoter
acyl-co-enzyme A oxidase (POX2). Protein secretion was directed by the targeting sequence of the extracellular lipase (LIP2):
pre–proLIP2. Codon optimization increased protein production by 11-fold, whereas the insertion of CACA sequence upstream of
the initiation codon of IFN-op construct resulted in 16.5-fold increase of the expression level; this indicates that translational
efficiency plays an important part in the increase of hIFN-α2b production level. The replacement of the pre–proLIP2 signal
secretion with the LIP2 pre-region sequence followed by the X-Ala/X-Pro stretch but without the pro-region also increased
the secretion of the target protein by twofold, suggesting therefore that the LIP2 pro-region is not necessary for extracellular
secretion of small heterologous proteins in Yarrowia lipolytica. 相似文献
5.
Baek-Rock Oh Jeong-Woo Seo Min Ho Choi Chul Ho Kim 《Biotechnology and Bioprocess Engineering》2008,13(6):666-670
To produce 1,3-propanediol (1,3-PD) from crude glycerol, cultivation conditions were optimized by response surface methodology
(RSM) based on a 25 factorial central composite design (CCD). RSM was adopted to derive a statistical model for the individual and interactive
effects of crude glycerol, (NH4)2SO4, pH, cultivation time and temperature on the production of 1,3-PD. Optimal conditions for maximum 1,3-PD production were
as follows: crude glycerol, 35 g/L; (NH4)2SO4, 8 g/L; pH, 7.37; cultivation time, 10.8 h; temperature, 36.88°C. Under these optimal conditions, the design expert presented
the maximal numerical solution with a predicted 1,3-PD production level of up to 13.74 g/L. The experimental production of
1,3-PD yielded 13.8 g/L, which was in close agreement with the model prediction. 相似文献
6.
Prodigiosin is a natural red pigment with algicidal activity against Cochlodinium polykrikoides, a major harmful red-tide microalga. To increase the yield of prodigiosin, a mutant of Hahella chejuenesis KCTC 2396, assigned M3349, was developed by an antibiotic mutagenesis using chloramphenicol. When cultured in Sucrose-based Marine Broth medium (SMB), M3349 could produce prodigiosin at 1.628+/-0.06 g/L, while wild type producing at 0.658+/-0.12 g/L under the same conditions. To increase the yield of prodigiosin production by M3349, significant medium components were determined using a two-level Plackett-Burman statistical design technique. Among fourteen components included in SMB medium, NaCl, Na2SiO3, MgCl2, H3BO3, Na2HPO4, Na2SO4, and CaCl2 were determined to be important for prodigiosin production. The medium formulation was finally optimized using a Box-Behnken design as follows: sucrose 10.0, peptone 8.0, yeast extract 2.0, NaCl 10.0, Na2SO4 12.0, CaCl2 1.8, MgCl2 0.7 g/L; and H3BO3 22.0, Na2HPO4 20.0, Na2SiO3 8.0 mg/L. The predicted maximum yield of prodigiosin in the optimized medium was 2.43 g/L by the Box-Behnken design, while the practical production was 2.60+/-0.176 g/L, which was 3.9 times higher than wild type with SMB Medium (0.658 g/L). 相似文献
7.
Periyasamy Ashok Kumar Muthu Manikandan Vijayaraghavan Kannan 《Biotechnology and Bioprocess Engineering》2010,15(4):641-650
Medium optimization for the nuclease (RNase) production by Bacillus firmus VKPACU-1 was studied using the one-factor-at-a-time method and Response Surface Methodology (RSM). One-factor-at-a-time methodology
was used to study the effects of carbon, nitrogen, phosphorus sources, and physical conditions such as pH and temperature,
on nuclease (RNase) production. After optimizing the carbon (glucose) and nitrogen (tryptone) sources in the culture medium
the physical conditions, pH (6.5) and temperature (35°C) were also optimized. Later these conditions were chosen as the main
factors and used in the experimental design. The central composite design (CCD) of the RSM was employed to evaluate the interactive
effects of these four variables. The optimized values obtained by the statistical analysis showed that glucose 5.95 g/L, tryptone
22.5 g/L, pH 6.5, and temperature 35°C affected maximum nuclease (RNase) production. When utilizing these proposed optimized
conditions, the model predicted nuclease (RNase) production of 43.6 U/mL and in the validation experiments, the nuclease production
obtained was 46.5 U/mL. The nuclease production in medium optimized by RSM was 26% higher, than in the non-optimized medium. 相似文献
8.
R. Preetha N. S. Jayaprakash Rosamma Philip I. S. Bright Singh 《Biotechnology and Bioprocess Engineering》2007,12(5):548-555
A marine isolate ofMicrococcus MCCB 104 has been identified as an aquaculture probiotic antagonistic toVibrio. In the present study different carbon and nitrogen sources and growth factors in a mineral base medium were optimized for
enhanced biomass production and antagonistic activity against the target pathogen,Vibrio harveyi, following response surface methodology (RSM). Accordingly the minimum and maximum limits of the selected variables were
determined and a set of fifty experiments programmed employing central composite design (CCD) of RSM for the final optimization.
The response surface plots of biomass showed similar pattern with that of antagonistic activity, which indicated a strong
correlation between the biomass and antagonism. The optimum concentration of the carbon sources, nitrogen sources, and growth
factors for both biomass and antagonistic activity were glucose (17.4 g/L), lactose (17 g/L), sodium chloride (16.9 g/L).
ammonium chloride (3.3 g/L), and mineral salts solution (18.3 mL/L). 相似文献
9.
The optimization of process parameters for high inulinase production by the marine yeast strain Cryptococcus aureus G7a in solid-state fermentation (SSF) was carried out using central composite design (CCD), one of the response surface methodologies
(RSMs). We found that moisture, inoculation size, the amount ratio of wheat bran to rice husk, temperature and pH had great
influence on inulinase production by strain G7a. Therefore, the CCD was used to evaluate the influence of the five factors
on the inulinase production by strain G7a. Then, five levels of the five factors above were further optimized using the CCD.
Finally, the optimal parameters obtained with the RSM were the initial moisture 61.5%, inoculum 2.75%, the amount ratio of
wheat bran to rice husk 0.42, temperature 29 °C, pH 5.5. Under the optimized conditions, 420.9 U g−1 of dry substrate of inulinase activity was reached in the solid-state fermentation culture of strain G7a within 120 h whereas
the predicted maximum inulinase activity of 436.2 U g−1 of inulinase activity of 436.2 U g−1 of dry weight was derived from the RSM regression. This is the highest inulinase activity produced by the yeast strain reported
so far. A large amount of monosaccharides and oligosaccharides were detected after inulin hydrolysis by the crude inulinase. 相似文献
10.
Rosa María Camacho Juan Carlos Mateos Orfil González-Reynoso Lilia Arely Prado Jesús Córdova 《Journal of industrial microbiology & biotechnology》2009,36(7):901-909
The present study was conducted to investigate the capability of Haloarcula marismortui to synthesize esterases and lipases, and the effect of physicochemical conditions on the growth and the production of esterases
and lipases. Finally, the effect of NaCl concentration and temperature on esterase and lipase activities was studied using
intracellular crude extracts. In order to confirm the genomic prediction about the esterase and lipase synthesis, H. marismortui was cultured on a rich medium and the crude extracts (intra- or extracellular) obtained were assayed for both activities
using p-nitrophenyl esters and triacylglycerides as substrates. Studies on the kinetics of growth and production of esterase and
lipase of H. marismortui were performed, reaching a maximum growth rate of 0.053 h−1 and maximal productions of intracellular esterase and lipase of 2.094 and 0.722 U l−1 using p-nitrophenyl valerate and p-nitrophenyl laurate, respectively. Both enzymes were produced as growth-associated metabolites. The effects of temperature,
pH, and NaCl concentration on the growth rate and production of enzymes were studied by using a Box–Behnken response surface
design. The three response variables were significantly influenced by the physicochemical factors and an interaction effect
between temperature and NaCl concentration was also evidenced. The surface response method estimated the following maximal
values for growth rate and productions of esterase and lipase: 0.086 h−1 (at 42.5°C, pH 7.4, and 3.6 mol l−1 NaCl), 2.3 U l−1 (at 50°C, pH 7.5, and 4.3 mol l−1 NaCl), and 0.58 U l−1 (at 50°C, pH 7.6, and 4.5 mol l−1 NaCl), respectively. Esterases were active at different salt concentrations, showing two optimal activities (at 0.5 and 5 mol l−1 NaCl), which suggested the presence of two different esterases. Interestingly, in the absence of salt, esterase retained
50% residual activity. Esterases and lipase activities were maximal at 45°C and inactive at 75°C. This study represents the
first report evidencing the synthesis of esterase and lipase by H. marismortui. 相似文献
11.
In the present study, the cultural conditions for exopolysaccharide (EPS) production from probiotic yeast Lipomyces starkeyi VIT-MN03 were optimized using response surface methodology (RSM) to maximize the yield of EPS. Interactions among the various factors viz. sucrose concentration (1–3 g%), NaCl concentration (2–4 g%), pH (3–5), temperature (20–30 °C), and incubation period (20–40 days) during EPS production were studied using Box-Behnken design (BBD). The EPS was purified and characterized using various instrumental analyses. The properties like adhesion, antioxidant, biosurfactant, cholesterol removal, and binding ability to mutagens were also tested for EPS produced. Sixfold increase in EPS production (4.87 g L−1) by L. starkeyi VIT-MN03 was noted under optimized condition. EPS showed a high viscosity (1.8 Pa S−1) and good shear-thinning properties. Instrumental analysis showed that EPS was heteropolysaccharide composed of glucan, mannan, and rhamnan. Lipomyces starkeyi VIT-MN03 exhibited good self-adhesion (95%) and co-aggregation ability (93%). Adhesion efficiency for yeast inoculum containing 5.5 × 107 CFU mL−1 per 9.2 cm2 of Caco-2 cell (colorectal adenocarcinoma) was noted. The probiotic EPS displayed strong antioxidant ability to scavenge hydroxyl radical and DPPH by 58% and 71% respectively. In addition, biosurfactant activity (86%) and cholesterol removal (90%) ability of probiotic EPS was also tested. EPS bound cells of L. starkeyi VIT-MN03 showed good binding ability to mutagens. These results support the effectiveness of using RSM for maximum EPS production. To the best of our knowledge, this is the first report on optimization of EPS production by probiotic yeast. 相似文献
12.
Juan Long Xiao Zhao Fei Liang Nan Liu Yuying Sun Yongzhi Xi 《Journal of biological engineering》2018,12(1):22
Background
Fermentation condition optimization and nutrients screening are of equal importance for efficient production of plasmid DNA vaccines. This directly affects the downstream purification and final quality and yield of plasmid DNA vaccines. The present study aimed to optimize the fermentation conditions for high-throughput production of therapeutic DNA vaccine pcDNA-CCOL2A1 by engineered Escherichia coli DH5α, using the response surface method (RSM).Results
We hypothesized that optimized fermentation conditions significantly increase the yield of pcDNA-CCOL2A1 therapeutic DNA vaccine, a novel DNA vaccine for treating rheumatoid arthritis (RA). Single-factor analysis was performed to evaluate the optimal basal culture medium from LB, 2?×?YT, TB, M9 (Glycerol) and M9 (Glucose), respectively. Thereafter, the Plackett-Burman design (PBD) was used to ascertain the three most significant factors affecting the vaccine yields, followed by the paths of steepest ascent to move to the nearest region of maximum response. Initial screening through the PBD revealed that the most key factors were peptone, mannitol, and inoculum concentration. Subsequent use of RSM was further optimized for the production of therapeutic DNA vaccine pcDNA-CCOL2A1 through Box-Behnken design (BBD). The final optimized fermentation conditions were as follows: peptone, 25.86 g/L; mannitol, 8.08 g/L; inoculum concentration, OD?=?0.36. Using this statistical experimental design, the yield of therapeutic DNA vaccine pcDNA-CCOL2A1 markedly increased from 223.37 mg/L to339.32 mg/L under optimal conditions, and a 51.9% increase was observed compared with the original medium.Conclusions
The present results provide a basis for further production of high-quality and high-yield therapeutic DNA vaccine pcDNA-CCOL2A1 in pilot-scale and even industrial-scale.13.
Derakhti S Shojaosadati SA Hashemi M Khajeh K 《Preparative biochemistry & biotechnology》2012,42(3):203-216
Production of α-amylase in a laboratory-scale packed-bed bioreactor by Bacillus sp. KR-8104 under solid-state fermentation (SSF) with possibility of temperature control and monitoring was studied using wheat bran (WB) as a solid substrate. The simultaneous effects of aeration rate, initial substrate moisture, and incubation temperature on α-amylase production were evaluated using response surface methodology (RSM) based on a Box-Behnken design. The optimum conditions for attaining the maximum production of α-amylase were 37°C, 72% (w/w) initial substrate moisture, and 0.15 L/min aeration. The average enzyme activity obtained under the optimized conditions was 473.8 U/g dry fermented substrate. In addition, it was observed that the production of enzyme decreased from the bottom of the bioreactor to the top. 相似文献
14.
Zhao J Wang Y Chu J Zhang S Zhuang Y Yuan Z 《Journal of industrial microbiology & biotechnology》2008,35(4):257-262
Pyruvate oxidase (PyOD) is a very useful enzyme for clinical diagnostic applications and environmental monitor. Optimization
of the fermentation medium for maximization of PyOD constitutively, production by Escherichia coli DH5α/pSMLPyOD was carried out. Response surface methodology (RSM) was used to optimize the medium constituents. A 26–2 fractional factorial design (first order model) was carried out to identify the significant effect of medium components towards
PyOD production. Statistical analysis of results shows that yeast extract, ammonium sulfate and composite phosphate were significant
factors on PyOD production. The optimized values of these three factors were obtained by RSM based on the result of a 23 central composite rotatable design. Under these proposed optimized medium, the model predicted a PyOD activity of 610 U/L
and via experimental rechecking the model, an activity of 670 U/L was attained. 相似文献
15.
Recombinant human interferon-gamma (rhIFN-γ) is a protein of great potential for clinical therapy due to its multiple biological
activities. However, overexpressing rhIFN-γ in Escherichia coli was found to accumulate as cytoplasmic inclusion bodies. In this work, a system for soluble and active expression of rhIFN-γ
was constructed by coexpressing chaperonin GroEL/GroES in E. coli. The rhIFN-γ gene was fused to a pET-28a expression vector, and rhIFN-γ was partially expressed as the soluble form following
coexpression with a second vector producing chaperonin GroEL/GroES. The fermentation of recombinant E. coli harboring rhIFN-γ and GroEL/GroES plasmids was investigated, and the optimized conditions were as follows: culture temperature
of 25°C, incubation time of 8 h, isopropyl-β-d-thio-galactoside concentration of 0.2 mM, and l-arabinose concentration of 0.5 g/L. As a result, the expression level of rhIFN-γ was improved accordingly by 2.2-fold than
the control, while a significantly positive correlation was also found between the ratio of supernatant to precipitate of
rhIFN-γ and the amount of chaperonin. Circular dichroism spectra, fluorescence spectra, size exclusion chromatography, and
chemical cross-linking method were applied to characterize rhIFN-γ, indicating that the three-dimensional structure of rhIFN-γ
was identical to that of the native rhIFN-γ. The enzyme-linked immunosorbent assay for active rhIFN-γ quantification showed
that coexpression yielded 72.91 mg rhIFN-γ per liter fermentation broth. Finally, protein–protein interactions between rhIFN-γ
and chaperonin were analyzed using the yeast two-hybrid system, which provided the direct evidence that chaperonin GroEL/GroES
interacted with rhIFN-γ to increase the soluble expression and presented the potential in producing efficiently recombinant
proteins. 相似文献
16.
Meng-Hao Cai Xiang-Shan Zhou Xue-Qian Sun Ke-Jing Tao Yuan-Xing Zhang 《Journal of industrial microbiology & biotechnology》2009,36(3):381-389
Statistical methodologies were employed to optimize submerged culture medium for the production of a novel antineoplastic
compound aspergiolide A by a marine-derived fungus Aspergillus glaucus HB1-19 for the first time. Orthogonal design was preformed to determine the initial composition. Then Plackett–Burman design
was applied to evaluate the influence of related nutrients, and yeast extract paste, soybean powder and sodium glutamate were
confirmed as critical factors in the medium. Response surface methodology (RSM) was finally taken as an effective approach
to optimize the combination of the obtained three significant factors. The predicted maximal aspergiolide A production of
62.4 mg/L appeared at the region where the concentrations of sodium glutamate, soybean powder, and yeast extract paste were
2, 1, and 1.07 g/L, respectively. Under the proposed optimized conditions, the experimental aspergiolide A production reached
71.2 mg/L. The correlation between predicted value and measured value of these experiments proved the validity of the response
model. After optimization, aspergiolide A production increased 4.22 times compared to that of the original medium. Elemental
analysis was finally taken into consideration, and carbon–nitrogen ratio in the medium increased from 20.1:1 to 86.6:1. This
great difference was inferred as the most important reason for production enhancement by metabolic pathway analysis. 相似文献
17.
Bhalchandra K. Vaidya Snehal R. Mutalik Renuka M. Joshi Sanjay N. Nene Bhaskar D. Kulkarni 《Journal of industrial microbiology & biotechnology》2009,36(5):671-678
In the present work, statistical experimental methodology was used to enhance the production of amidase from Rhodococcus erythropolis MTCC 1526. R.
erythropolis MTCC 1526 was selected through screening of seven strains of Rhodococcus species. The Placket–Burman screening experiments suggested that sorbitol as carbon source, yeast extract and meat peptone
as nitrogen sources, and acetamide as amidase inducer are the most influential media components. The concentrations of these
four media components were optimised using a face-centred design of response surface methodology (RSM). The optimum medium
composition for amidase production was found to contain sorbitol (5 g/L), yeast extract (4 g/L), meat peptone (2.5 g/L), and
acetamide (12.25 mM). Amidase activities before and after optimisation were 157.85 units/g dry cells and 1,086.57 units/g
dry cells, respectively. Thus, use of RSM increased production of amidase from R.
erythropolis MTCC 1526 by 6.88-fold. 相似文献
18.
Acid phosphatase production by recombinant Arxula adeninivorans was carried out in submerged fermentation. Using the Plackett–Burman design, three fermentation variables (pH, sucrose concentration,
and peptone concentration) were identified to significantly affect acid phosphatase and biomass production, and these were
optimized using response surface methodology of central composite design. The highest enzyme yields were attained in the medium
with 3.9% sucrose and 1.6% peptone at pH 3.8. Because of optimization, 3.86- and 4.19-fold enhancement in enzyme production
was achieved in shake flasks (17,054 U g−1 DYB) and laboratory fermenter (18,465 U g−1 DYB), respectively. 相似文献
19.
You-Jung Lee Hye-Jin Kim Wa Gao Chung-Han Chung Jin-Woo Lee 《Biotechnology and Bioprocess Engineering》2012,17(2):227-235
The optimal conditions for production of carboxymethylcellulase (CMCase) of Bacillus amyloliquefaciens DL-3 by a recombinant Escherichia coli JM109/DL-3 were established at a flask scale using the response surface method (RSM). The optimal conditions of rice bran,
tryptone, and initial pH of the medium for cell growth extracted by Design Expert Software were 66.1 g/L, 6.2 g/L, and 7.2,
respectively, whereas those for production of CMCase were 58.0 g/L, 5.0 g/L, and 7.1. The analysis of variance (ANOVA) of
results from central composite design (CCD) indicated that significant factor (“probe > F” less than 0.0500) for cell growth was rice bran, whereas those for production of CMCase were rice bran and initial pH of
the medium. The optimal temperatures for cell growth and the production of CMCase by E. coli JM109/DL-3 were found to be 37°C. The optimal agitation speed and aeration rate of 7 L bioreactors for cell growth were 498
rpm and 1.4 vvm, whereas those for production of CMCase were 395 rpm and 1.1 vvm. The ANOVA of results indicated that the
aeration rate was more significant factor (“probe > F” less than 0.0001) than the agitation speed for cell growth and production of CMCase. The optimal inner pressure for cell
growth was 0.08 MPa, whereas that for the production of CMCase was 0.06 MPa. The maximal production of CMCase by E. coli JM109/DL-3 under optimized conditions was 871.0 U/mL, which was 3.0 times higher than the initial production of CMCase before
optimization. 相似文献
20.
Zhong-Ce Hu Wen-Jun Li Shu-Ping Zou Kun Niu 《Preparative biochemistry & biotechnology》2020,50(8):745-752
AbstractEchinocandin B, a kind of antimycotic with cyclic lipo-hexapeptides, was produced by fermentation with Aspergillus nidulans using fructose as main carbon source. The objective of this study was to screen a high-yield mutant capable of using cheap starch as main carbon source by atmospheric and room temperature plasma (ARTP) treatment in order to decrease the production cost of echinocandin B. A stable mutant A. nidulans ZJB19033, which can use starch as optimal carbon source instead of expensive fructose, was selected from two thousands isolates after several cycles of ARTP mutagenesis. To further increase the production of echinocandin B, the optimization of fermentation medium was performed by response surface methodology (RSM), employing Plackett-Burman design (PBD) followed by Box-Behnken design (BBD). The optimized fermentation medium provided the optimal yield of echinocandin B, 2425.9?±?43.8?mg/L, 1.3-fold compared to unoptimized medium. The results indicated that the mutant could achieve high echinocandin B production using cheap starch as main carbon source, and the cost of carbon sources in fermentation medium reduced dramatically by about 45%. 相似文献