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1.
A bacterial strain isolated from spoiled coconut and identified as Bacillus cereus was found capable of producing alkaline thermostable extracellular lipase. Optimum temperature, time, and pH for enzyme substrate
reaction were found to be 60 °C, 10 min, and 8.0 respectively. Common surfactants except Triton X 100 and cetyltrimethylammonium
bromide have no or very little inhibitory effects on enzyme activity. The enzyme was found to be stable in presence of oxidizing
agents and protease enzyme. The maximum lipase production was achieved at 30–33 °C, pH 8.0 on 24 h of fermentation using 50 ml
medium in a 250-ml Erlenmeyer flask. The superior carbon and nitrogen sources for lipase production were starch (2%) and ammonium
sulfate (nitrogen level 21.2 mg/100 ml), peptone (nitrogen level 297 mg/100 ml), and urea (nitrogen level 46.62 mg/100 ml)
in combination, respectively. The maximum enzyme activity obtained was 33 ± 0.567 IU/ml. 相似文献
2.
Rushi Liu Qinlu Lin Yi Sun Xiangyang Lu Yilan Qiu Ye Li Xiangrong Guo 《Applied biochemistry and biotechnology》2009,158(2):432-444
Prevention of the prevalence of HB depends upon the development of efficient diagnostic reagent and preventive vaccine. Pichia pastoris offers many advantages over the other expression systems in the production of recombinant HBsAg. In this study, we reported
that the recombinant P. pastoris strains were cultured in shake flasks and then scaled up in a 5.0-l bioreactor: approximately 27 mg/l of the protein and
the maximal cell OD at 600 nm of 310 were achieved in the bioreactor. The recombinant HBsAg was purified by three steps of
purification procedures. SDS-PAGE showed that the purified recombinant HBsAg constituted only one homogeneous band of ~24 kDa.
CsCl density gradient ultracentrifugation assay indicated that the density of the HBsAg was 1.2 mg/ml, which was in agreement
with the natural HBsAg, the HBsAg expressed in Saccharomyces cerevisiae and in mammalian cells. Electron microscope observation revealed that the purified recombinant HBsAg was homogeneous 22-nm
particles, suggesting the HBsAg expressed in P. pastoris was self-assembled to virus-like structures. Competitive ELISA indicated that P. pastoris-derived HBsAg possessed the excellent immunoreaction with anti-HBsAg. Animal immunization showed that the immunogenicity
of P. pastoris-derived HBsAg was superior to that of S. cerevisiae-derived HBsAg. Together, our results demonstrated that the recombinant HBsAg expressed in P. pastoris could provide promising, inexpensive, and large-scale materials for the diagnostic reagent and vaccine to prevent HBV infection.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.
Rushi Liu and Qinlu Lin contributed equally to this work. 相似文献
3.
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
4.
Silva-Santisteban BO Converti A Filho FM 《Applied biochemistry and biotechnology》2009,152(2):249-261
Cultivations of Kluyveromyces marxianus var. bulgaricus ATCC 16045 were performed on both minimal and complex media using different carbon and nitrogen sources either in the presence
or absence of aeration. The results collected were worked out and compared so as to provide a useful contribution to the optimization
of inulinase production. Kinetics of extracellular inulinase release were similar on glucose, fructose, and sucrose. Inulinase
was detected at basal level since the beginning of batch runs on these three carbon sources and overproduced after their depletion.
The highest inulinase activity in minimal medium containing 10 g/l sucrose (6.4 IU/ml) was obtained at an initial (NH4)2SO4 concentration of 5 g/l, whereas it was reduced to about one fourth of this value and detected only at the beginning under
nitrogen-limited conditions. The best sucrose concentrations for the enzyme production were 30 and 20 g/l in minimal and complex
media, yielding 15.4 and 208 IU/ml, respectively. In general, the enzyme activity was much higher in complex than in minimal
medium under all conditions. O2-enriched air neither improved inulinase production nor prevented ethanol formation. 相似文献
5.
《Arabian Journal of Chemistry》2022,15(5):103799
BackgroundStreptokinase (SK) is a potent plasminogen activator naturally produced by beta-hemolytic streptococcus bacteria and used as a thrombolytic drug.ObjectivesOptimize high yield production of recombinant streptokinase (rSK) in Escherichia coli and evaluate its thrombolytic activity.MethodsSynthetic gene encoding mature SK protein with optimization for rare codons and mRNA secondary structure was cloned into the expression vector pET-3a and transformed into Escherichia coli BL21 (DE3). Seed banks were established for high rSK expression clones. The native rSK protein expression was optimized using IPTG induction. The nonsoluble rSK inclusion bodies were purified, denatured in 6 M guanidinium chloride, and refolded using the rapid dilution method. The refolded rSK protein was purified using anion exchange chromatography and evaluated with ELISA. The activity of rSK was evaluated using the casein digestion method and in vitro blood clot lysis assay with reference drug Sedonase as standard.ResultsSeed banks with high stable expression of native rSk (MW 47 kDa) were established. High rSK expression was optimized using 1 mM IPTG at bacterial OD600 0.6. The refolded rSK was prepared and purified successfully with high productivity (494 mg purified rsk/L culture). Using ELISA, the purified rSK molecular identity and conservation of native SK epitopes were confirmed. The enzymatic activity of the purified rSK was 1.945x106 IU/mg with 62.94 ± 2.3% clot lysis efficiency.ConclusionA high yield production of proper rSK protein with in vitro thrombolytic activity similar to commercial SK has been achieved, suggesting a more cost-effective industrial production of its biosimilar drug. 相似文献
6.
Keivan Majidzadeh-A Vahid Khalaj Davami Fatemeh Hemayatkar Mahdi Barkhordari Farzaneh Adeli Ahmad Fereidoun Mahboudi 《Applied biochemistry and biotechnology》2010,162(7):2037-2048
Human tissue plasminogen activator (t-PA) plays a pivotal role in the treatment of acute myocardial infarction, ischemic stroke,
and deep vein thrombosis. It has the benefit of generating no adverse effects such as fibrinogen depletion, systemic hemorrhage,
and immunologic reactions. Human t-PA is a serine-protease enzyme containing 527 amino acid residues in five structural domains.
The correct folding of t-PA requires the correct pairing of 17 disulfide bridges in the molecule. A gene encoding full-length
human t-PA was cloned into pPICZαA expression vector downstream of alcohol oxidase promoter and α-mating signal sequence from
Saccharomyces cerevisiae and flush with the kex2 cleavage site to express the protein with a native N terminus. The methylotrophic yeast, Pichia pastoris GS115 strain, was transformed with this cassette, and methanol utilizing (mut+) transformants were selected for production
and secretion of human t-PA into culture media. SDS–PAGE and Western blot analysis showed the expressed bands of t-PA protein.
Zymography test indicated suitable folding and proper function of the expressed recombinant human t-PA in conversion of plasminogen
to plasmin and gelatin lysis. Amidolytic activity test showed the amidolytic activity of 1,650 IU/ml. The results of this
study concluded that P. pastoris methylotrophic yeast can be a suitable alternative for mammalian and prokaryotic expression systems to produce t-PA. 相似文献
7.
Ramadhar Kumar S. Balaji T. S. Uma A. B. Mandal P. K. Sehgal 《Applied biochemistry and biotechnology》2010,160(1):30-39
A Bacillus subtilis (MTCC9102) isolate was shown to produce significant amount of keratinase under optimized conditions in solid-state fermentation
using Horn meal as a substrate. Optimized value for moisture, inoculum, and aeration were found to be 100% (v/w), 50% (v/w), and 150% (w/w), respectively, and the optimum nitrogen source was peptone and carbon source was dextrose. Maximum keratinolytic activity
was observed at 48 h after incubation, and the optimum age (24 h) of inoculum was significant. The influence of cultivation
temperature and initial pH of the medium on keratinase production revealed the optimum values for the temperature and pH as
37 °C and 7, respectively. Maximum keratinase activity of the crude extract was 15,972 U/mg/ml. These results indicate that
this bacterial strain shows a high biotechnological potential for keratinase production in solid-state fermentation, and use
of the horn meal as the substrate can be implemented for keratinous solid wastes management. 相似文献
8.
Christina Bohlin Leif J. Jönsson Robyn Roth Willem H. van Zyl 《Applied biochemistry and biotechnology》2006,129(1-3):195-214
Convenient expression systems for efficient heterologous production of different laccases are needed for their characterization
and application. The laccase cDNAs lcc1 and lcc2 from Trametes versicolor were expressed in Pichia pastoris and Aspergillus niger under control of their respective glyceraldehyde-3-phosphate dehydrogenase promoters and with the native secretion signal
directing catalytically active laccase to the medium. P. pastoris batch cultures in shake-flasks gave higher volumetric activity (1.3 U/L) and a better activity to biomass ratio with glucose
than with glycerol or maltose as carbon source. Preliminary experiments with fed-batch cultures of P. pastoris in bioreactors yielded higher activity (2.8 U/L) than the shake-flask experiments, although the levels remained moderate
and useful primarily for screening purposes. With A. niger, high levels of laccase (2700 U/L) were produced using a minimal medium containing sucrose and yeast extract. Recombinant
laccase from A. nigher harboring the lcc2 cDNA was purified to homogeneity and it was found to be a 70-kDa homogeneous enzyme with biochemical and catalytic properties
similar to those of native T. versicolor laccase A. 相似文献
9.
Pradnya Deshpande Sajitha Nair Shubhangi Khedkar 《Applied biochemistry and biotechnology》2009,158(3):552-560
Water hyacinth (Eichhornia crassipes), an aquatic weed common to the subtropic/tropical regions, was utilized as an inexpensive lignocellulosic substrate for
production of cellulase by Trichoderma reesei. The effects of process parameters like substrate pretreatment, substrate concentration, initial medium pH, mode of inoculation,
and incubation temperature on cellulase production were investigated. Under optimal conditions, a maximal cellulase activity
of 0.22 ± 0.04 IU/ml (approximately 73.3 IU/g cellulose) was recorded at the end of 15-day incubation period. Specific activity
of the enzyme was 6.25 IU/mg protein. Hydrolysis of 1% substrate (water hyacinth) using crude enzyme dosage of 1.2 IU/g water
hyacinth showed 28.7% saccharification in 1 h. The observations in present study indicate that saccharification of cellulose
from water hyacinth was significantly higher by laboratory-produced cellulase than the commercial blend. 相似文献
10.
Production of Cellulolytic Enzymes by <Emphasis Type="Italic">Aspergillus phoenicis</Emphasis> in Grape Waste using Response Surface Methodology 总被引:1,自引:0,他引:1
Dedavid e Silva LA Lopes FC Silveira ST Brandelli A 《Applied biochemistry and biotechnology》2009,152(2):295-305
The production of cellulolytic enzymes by the fungus Aspergillus phoenicis was investigated. Grape waste from the winemaking industry was chosen as the growth substrate among several agro-industrial
byproducts. A 2 × 2 central composite design was performed, utilizing the amount of grape waste and peptone as independent
variables. The fungus was cultivated in submerged fermentation at 30 °C and 120 rpm for 120 h, and the activities of total
cellulases, endoglucanases, and β-glucosidases were measured. Total cellulases were positively influenced by the linear increase
of peptone concentration and decrease at axial concentrations of grape waste and peptone. Maximum activity of endoglucanase
was observed by a linear increase of both grape waste and peptone concentrations. Concentrations of grape waste between 5
and 15 g/L had a positive effect on the production of β-glucosidase; peptone had no significant effects. The optimum production
of the three cellulolytic activities was observed at values near the central point. A. phoenicis has the potential for the production of cellulases utilizing grape waste as the growth substrate. 相似文献
11.
Characterization of Thermo-stable Endoinulinase from a New Strain <Emphasis Type="Italic">Bacillus Smithii</Emphasis> T7 总被引:1,自引:0,他引:1
A new thermophilic inulinase-producing strain, which grows optimally at 60 °C, was isolated from soil samples with medium
containing inulin as a sole carbon source. It was identified as a Bacillus smithii by analysis of 16s rDNA. Maximum inulinase yield of 135.2 IU/ml was achieved with medium pH7.0, containing inulin 2.0%, (NH4)H2PO4 0.5%, yeast extract 0.5%, at 50 °C 200 rpm shaker for 72-h incubation. The purified inulinase from the extracellular extract
of B. smithii T7 shows endoinulinolytic activity. The optimum pH for this endoinulinase is 4.5 and stable at pH range of 4.0–8.0. The optimum
temperature for enzyme activity was 70 °C, the half life of the endoinulinase is 9 h and 2.5 h at 70 °C and 80 °C respectively.
Comparatively lower Michaelis–Menten constant (4.17 mM) and higher maximum reaction velocity (833 IU/mg protein) demonstrate
the endoinulinase’s greater affinity for inulin substrate. These findings are significant for its potential industrial application. 相似文献
12.
Bo Guan Fengxiang Chen Jianyong Lei Yunhua Li Zuoying Duan Ruiyu Zhu Yun Chen Huazhong Li Jian Jin 《Applied biochemistry and biotechnology》2013,171(7):1792-1804
A constitutive expression vector for rhIL-2-HSA fusion protein production in yeast Pichia pastoris was constructed. The coding gene was placed in frame with the Saccharomyces cerevisiae α-factor secretion signal sequence under the control of the GAP promoter. The recombinant plasmid pGAPZαA-rhIL-2-HSA was integrated into the genome of the P. pastoris GS115. The effect of different carbon sources on rhIL-2-HSA fusion protein expression was evaluated in shaking flask cultures. We found that recombinant P. pastoris grew well and efficiently secreted rhIL-2-HSA fusion protein into the medium when using glucose as carbon source. To achieve higher production, the influence of initial pH and culture temperature was also evaluated. Fed-batch fermentation strategy using glucose as carbon source for constitutive expression of rhIL-2-HSA fusion protein was investigated in 5-L bioreactor and the expression level of rhIL-2-HSA could reach about 250 mg/L after 60-h fermentation. The rhIL-2-HSA fusion protein produced by this constitutive expression system was purified and exhibited a specific bioactivity of 1.040?×?106 IU/mg in vitro. This study described constitutive expression of rhIL-2-HSA fusion protein by P. pastoris and development of a simple high-cell density fermentation strategy for biologically active rhIL-2-HSA fusion protein using glucose as sole carbon source. 相似文献
13.
A fungal strain, marked as ECU0913, producing high activities of both cellulase and xylanase was newly isolated from soil
sample collected near decaying straw and identified as Penicillium sp. based on internal transcribed spacer sequence homology. The cultivation of this fungus produced both cellulase (2.40 FPU/ml)
and xylanase (241 IU/ml) on a stepwisely optimized medium at 30 °C for 144 h. The cellulase and xylanase from Penicillium sp. ECU0913 was stable at an ambient temperature with half-lives of 28 and 12 days, respectively. Addition of 3 M sorbitol
greatly improved the thermostability of the two enzymes, with half-lives increased by 2.3 and 188-folds, respectively. Catalytic
performance of the Penicillium cellulase and xylanase was evaluated by the hydrolysis of corn stover pretreated by steam explosion. With an enzyme dosage
of 50 FPU/g dry substrate, the conversions of cellulose and hemicellulose reached 77.2% and 47.5%, respectively, without adding
any accessory enzyme. 相似文献
14.
Recombinant clones of X-33 strain Pichia pastoris containing the marker gene yEGFP were prepared. The optimal methanol concentration in the medium for induction of heterologous expression was determined in the recombinant clones.__________Translated from Khimiya Prirodnykh Soedinenii, No. 1, pp. 60–62, January–February, 2005. 相似文献
15.
Tang XS Tang ZR Wang SP Feng ZM Zhou D Li TJ Yin YL 《Applied biochemistry and biotechnology》2012,166(3):640-651
Bovine lactoferrampin (LFA) and bovine lactoferricin (LFC) are two antimicrobial peptides located in the N1 domain of bovine lactoferrin. The bactericidal activity of the fused peptide LFA–LFC is stronger than that of either LFA
or LFC. The high cost of peptide production from either native digestion or chemical synthesis limits the clinical application
of antimicrobial peptides. The expression of recombinant peptides in yeast may be an effective alternative. In the current
study, the expression, purification, and antibacterial activity of LFA–LFC using the Pichia pastoris expression system are reported. The linearized expression vector pPICZaA–LFA–LFC was transformed into P. pastoris KM71 by electroporation, and positive colonies harboring the target genes were screened out and used for fermentation. The
recombinant LFA–LFC peptide was purified via two-step column chromatography and identified by tricine–sodium dodecyl sulfate–polyacrylamide
gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The results indicate
that P. pastoris is a suitable system for secreting LFA–LFC. The fermentation supernate and the purified LFA–LFC show high antimicrobial activities.
The current study is the first to report on the expression and purification of LFA–LFC in P. pastoris and may have potential practical applications in microbial peptide production. 相似文献
16.
Ching-Chuan Chang Cheon-Seok Park Dewey D. Y. Ryu 《Applied biochemistry and biotechnology》1998,74(3):173-189
The effect of polypeptide fractions of proteose peptone on the induction of cloned gene expression of rice α-amylase in recombinantYarrowia lipolytica which is under the control of itsXPR2 promoter, was studied. Gel-filtration chromatography with Sephacryl S-100 and Sephadex G-25 (coarse) gels was used to fractionate
the active polypeptide fractions from the proteose peptone. The polypeptide size fractions that were effective for the induction
of cloned gene expression ranged between mol wt of 1.0 and 6.0 kDa. The fed-batch culture experiments with active polypeptide
fractions were performed in a 6-L fermenter. The specific productivity of α-amylase and the enzyme yield based on nitrogen
source increased from 25.7 to 33.0 U/g cell·h and 4.96 to 6.73 U/(mg nitrogen consumed), respectively, when proteose peptone
was replaced by active polypeptide fractions in production medium. The specific productivity of α-amylase and the enzyme yield
further improved to 36.2 U/g cell·h and 8.14 U/(mg nitrogen consumed), respectively, when the glutamic acid-enriched active
polypeptide fractions in the production medium was used. The specific productivity of α-amylase and the enzyme yield were
improved by 41 and 64%, respectively, as compared with the results obtained with the medium containing proteose peptone. Through
medium design, a bioprocess strategy for heterologous protein production was developed and a significant productivity improvement
achieved. 相似文献
17.
Methylotrophic yeast Pichia pastoris is convenient for the expression of eukaryotic foreign proteins owing to its potential for posttranslational modifications,
protein folding, and facile culturing. In this work, human interleukin (hIL)-2 was successfully produced as a secreted fusion
form in recombinant P. pastoris. By employing green fluorescent protein (GFP) as a monitoring fusion partner, clear identification of fusion protein expression
and quantification of intracellular hIL-2 were possible even though there was no correlation between culture supernatant fluorescence
and secreted hIL-2 owing to high media interference. Importantly, by the addition of casamino acids in basal medium, we were
able to enhance threefold amount of secreted hIL-2, which was present both as a fusion and as a clipped fragment. 相似文献
18.
Expression of Recombinant Proteins in <Emphasis Type="Italic">Pichia Pastoris</Emphasis> 总被引:5,自引:0,他引:5
Li P Anumanthan A Gao XG Ilangovan K Suzara VV Düzgüneş N Renugopalakrishnan V 《Applied biochemistry and biotechnology》2007,142(2):105-124
Pichia pastoris has been used extensively and successfully to express recombinant proteins. In this review, we summarize the elements required
for expressing heterologous proteins, and discuss various factors in applying this system for protein expression. These elements
include vectors, host strains, heterologous gene integration into the genome, secretion factors, and the glycosylation profile.
In particular, we discuss and evaluate the recent progress in optimizing the fermentation process to improve the yield and
stability of expressed proteins. Optimization can be achieved by controlling the medium composition, pH, temperature, and
dissolved oxygen, as well as by methanol induction and feed mode. 相似文献
19.
Verônica Ferreira Patricia C. Nolasco Aline M. Castro Juliana N. C. Silva Alexandre S. Santos Mônica C. T. Damaso Nei Pereira Jr. 《Applied biochemistry and biotechnology》2006,129(1-3):226-233
This work aims to evaluate cell recycle of a recombinant strain of Pichia pastoris GS115 on the Xylanase A (XynA) production of Thermomyces lanuginosus IOC-4145 in submerged fermentation. Fed-batch processes were carried out with methanol feeding at each 12h and recycling
cell at 24, 48, and 72 h. Additionally, the influence of the initial cell concentration was investigated. XynA production
was not decreased with the recycling time, during four cell recycles, using an initial cell concentration of 2.5 g/L. The
maximum activity was 14,050 U/L obtained in 24h of expression. However, when the initial cell concentration of 0.25 g/L was
investigated, the enzymatic activity was reduced by 30 and 75% after the third and fourth cycles, respectively. Finally, it
could be concluded that the initial cell concentration influenced the process performance and the interval of cell recycle
affected enzymatic production. 相似文献
20.
Kumar A Singh S Poddar P Prabhune A Pundle A 《Applied biochemistry and biotechnology》2009,157(3):463-472
Penicillin V acylase (PVA) is a pharmaceutically important enzyme as it plays a vital role in the manufacture of semi-synthetic
β-lactam antibiotics. Rhodotorula aurantiaca (NCIM 3425) produced high levels of intracellular penicillin V acylase after 18 h at pH 8.0 and temperature 27 °C. Fructose
was the best carbon source for PVA production, whereas tryptone was the best nitrogen source to produce the enzyme up to 170
and 1,088 IU/l of culture, respectively. Additionally, the cell-bound PVA activity was enhanced on treatment with cationic
detergent. Whole-cell activity was found to be doubled (204%) on treatment of 0.01 g dry weight of cells with 50 μg/ml solution
of N-cetyl-N,N,N-trimethylammoniumbromide at pH 8.0 for 1 h at room temperature. Atomic force microscopy images of permeabilized cells show
perturbation in the cell wall and offer first-ever visual illustration of surface structure modifications that occur during
permeabilization of R. aurantiaca cells leading to enhancement in activity of intracellular enzyme. 相似文献