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1.
The amino acid l-theanine (γ-glutamylethylamide) has potential important applications in the food and pharmaceutical industries and increased
demand for this compound is expected. It is the major “umami” (good taste) component of tea and its favorable physiological
effects on mammals have been reported. An enzymatic method for the synthesis of l-theanine involving recombinant Escherichia coli γ-glutamyltranspeptidase (GGT) has been developed. We report here the application of small ubiquitin-related modifier (SUMO)
fusion technology to the expression and purification of recombinant Escherichia coli γ-GGT. In order to obtain γ-GGT with high theanine-forming activity, safety, and low cost for food and pharmaceutics industry,
M9 (consisting of glycerol and inorganic salts) and 0.1% (w/v) lactose were selected as culture medium and inducer, respectively.
The fusion protein was expressed in soluble form in E. coli, and expression was verified by SDS-PAGE and western blot analysis. The fusion protein was purified to 90% purity by nickel–nitrilotriacetic
acid (Ni–NTA) resin chromatography with a yield of 115 mg per liter fermentation culture. After the SUMO/γ-GGT fusion protein
was cleaved by the SUMO protease, the cleaved sample was reapplied to a Ni–NTA column. Finally, about 62 mg recombinant γ-GGT
was obtained from 1 l fermentation culture with no less than 95% purity. The recombinant γ-GGT showed great transpeptidase
activity, with 1500 U of purified recombinant γ-GGT in a 1-l reaction system, a biosynthesis yield of 41 g of l-theanine was detected by paper chromatography or high pressure liquid chromatography (HPLC). Thus, the application of SUMO
technology to the expression and purification of γ-GGT potentially could be employed for the industrial production of l-theanine. 相似文献
2.
Prabhu P Tiwari MK Jeya M Gunasekaran P Kim IW Lee JK 《Applied microbiology and biotechnology》2008,81(2):283-290
Based on analysis of the genome sequence of Bacillus licheniformis ATCC 14580, an isomerase-encoding gene (araA) was proposed as an l-arabinose isomerase (L-AI). The identified araA gene was cloned from B. licheniformis and overexpressed in Escherichia coli. DNA sequence analysis revealed an open reading frame of 1,422 bp, capable of encoding a polypeptide of 474 amino acid residues
with a calculated isoelectric point of pH 4.8 and a molecular mass of 53,500 Da. The gene was overexpressed in E. coli, and the protein was purified as an active soluble form using Ni–NTA chromatography. The molecular mass of the purified enzyme
was estimated to be ~53 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and 113 kDa by gel filtration chromatography,
suggesting that the enzyme is a homodimer. The enzyme required a divalent metal ion, either Mn2+or Co2+, for enzymatic activity. The enzyme had an optimal pH and temperature of 7.5 and 50°C, respectively, with a k
cat of 12,455 min−1 and a k
cat/K
m of 34 min−1 mM−1 for l-arabinose, respectively. Although L-AIs have been characterized from several other sources, B. licheniformis L-AI is distinguished from other L-AIs by its wide pH range, high substrate specificity, and catalytic efficiency for l-arabinose, making B. licheniformis L-AI the ideal choice for industrial applications, including enzymatic synthesis of l-ribulose. This work describes one of the most catalytically efficient L-AIs characterized thus far. 相似文献
3.
Xuefeng Wu Shaotong Jiang Mo Liu Lijun Pan Zhi Zheng Shuizhong Luo 《Journal of industrial microbiology & biotechnology》2011,38(4):565-571
Semicontinuous fermentation using pellets of Rhizopus oryzae has been recognized as a promising technology for l-lactic acid production. In this work, semicontinuous fermentation of R. oryzae AS 3.819 for l-lactic acid production has been developed with high l-lactic acid yield and volumetric productivity. The effects of factors such as inoculations, CaCO3 addition time, and temperature on l-lactic acid yield and R. oryzae morphology were researched in detail. The results showed that optimal fermentation conditions for the first cycle were: inoculation
with 4% spore suspension, CaCO3 added to the culture medium at the beginning of culture, and culture temperature of 32–34°C. In orthogonal experiments, high
l-lactic acid yield was achieved when the feeding medium was (g/l): glucose, 100; (NH4)2SO4, 2; KH2PO4, 0.1; ZnSO4·7H2O, 0.33; MgSO4·7H2O, 0.15; CaCO3, 50. Twenty cycles of semicontinuous fermentation were carried out in flask culture. l-lactic acid yield was 78.75% for the first cycle and 80–90% for the repeated cycles; the activities of lactate dehydrogenases
(LDH) were 7.2–9.2 U/mg; fermentation was completed in 24 h for each repeated cycle. In a 7-l magnetically stirred fermentor,
semicontinuous fermentation lasted for 25 cycles using pellets of R. oryzae AS 3.819 under the optimal conditions determined from flask cultures. The final l-lactic acid concentration (LLAC) reached 103.7 g/l, and the volumetric productivity was 2.16 g/(l·h) for the first cycle;
in the following 19 repeated cycles, the final LLAC reached 81–95 g/l, and the volumetric productivities were 3.40–3.85 g/(l·h). 相似文献
4.
Methee Khamduang Kanoktip Packdibamrung Jarun Chutmanop Yusuf Chisti Penjit Srinophakun 《Journal of industrial microbiology & biotechnology》2009,36(10):1267-1274
The production of l-phenylalanine is conventionally carried out by fermentations that use glucose or sucrose as the carbon source. This work
reports on the use of glycerol as an inexpensive and abundant sole carbon source for producing l-phenylalanine using the genetically modified bacterium Escherichia coli BL21(DE3). Fermentations were carried out at 37°C, pH 7.4, using a defined medium in a stirred tank bioreactor at various
intensities of impeller agitation speeds (300–500 rpm corresponding to 0.97–1.62 m s−1 impeller tip speed) and aeration rates (2–8 L min−1, or 1–4 vvm). This highly aerobic fermentation required a good supply of oxygen, but intense agitation (impeller tip speed
~1.62 m s−1) reduced the biomass and l-phenylalanine productivity, possibly because of shear sensitivity of the recombinant bacterium. Production of l-phenylalanine was apparently strongly associated with growth. Under the best operating conditions (1.30 m s−1 impeller tip speed, 4 vvm aeration rate), the yield of l-phenylalanine on glycerol was 0.58 g g−1, or more than twice the best yield attainable on sucrose (0.25 g g−1). In the best case, the peak concentration of l-phenylalanine was 5.6 g L−1, or comparable to values attained in batch fermentations that use glucose or sucrose. The use of glycerol for the commercial
production of l-phenylalanine with E. coli BL21(DE3) has the potential to substantially reduce the cost of production compared to sucrose- and glucose-based fermentations. 相似文献
5.
Guanosine 5′-triphosphate (GTP) is the key substrate for biosynthesis of guanosine 5′-diphosphate (GDP)-l-fucose. In this study, improvement of GDP-l-fucose production was attempted by manipulating the biosynthetic pathway for guanosine nucleotides in recombinant Escherichia coli-producing GDP-l-fucose. The effects of overexpression of inosine 5′-monophosphate (IMP) dehydrogenase, guanosine 5′-monophosphate (GMP) synthetase
(GuaB and GuaA), GMP reductase (GuaC) and guanosine–inosine kinase (Gsk) on GDP-l-fucose production were investigated in a series of fed-batch fermentations. Among the enzymes tested, overexpression of Gsk
led to a significant improvement of GDP-l-fucose production. Maximum GDP-l-fucose concentration of 305.5 ± 5.3 mg l−1 was obtained in the pH-stat fed-batch fermentation of recombinant E. coli-overexpressing Gsk, which corresponds to a 58% enhancement in the GDP-l-fucose production compared with the control strain overexpressing GDP-l-fucose biosynthetic enzymes. Such an enhancement of GDP-l-fucose production could be due to the increase in the intracellular level of GMP. 相似文献
6.
Biosynthesis of guanosine 5′-diphosphate-l-fucose (GDP-l-fucose) requires NADPH as a reducing cofactor. In this study, endogenous NADPH regenerating enzymes such as glucose-6-phosphate
dehydrogenase (G6PDH), isocitrate dehydrogenase (Icd), and NADP+-dependent malate dehydrogenase (MaeB) were overexpressed to increase GDP-l-fucose production in recombinant Escherichia coli. The effects of overexpression of each NADPH regenerating enzyme on GDP-l-fucose production were investigated in a series of batch and fed-batch fermentations. Batch fermentations showed that overexpression
of G6PDH was the most effective for GDP-l-fucose production. However, GDP-l-fucose production was not enhanced by overexpression of G6PDH in the glucose-limited fed-batch fermentation. Hence, a glucose
feeding strategy was optimized to enhance GDP-l-fucose production. Fed-batch fermentation with a pH-stat feeding mode for sufficient supply of glucose significantly enhanced
GDP-l-fucose production compared with glucose-limited fed-batch fermentation. A maximum GDP-l-fucose concentration of 235.2 ± 3.3 mg l−1, corresponding to a 21% enhancement in the GDP-l-fucose production compared with the control strain overexpressing GDP-l-fucose biosynthetic enzymes only, was achieved in the pH-stat fed-batch fermentation of the recombinant E. coli overexpressing G6PDH. It was concluded that sufficient glucose supply and efficient NADPH regeneration are crucial for NADPH-dependent
GDP-l-fucose production in recombinant E. coli. 相似文献
7.
Jiaping Song Chuanwen Zhou Rui Liu Xudong Wu Di Wu Xiaojian Hu Yu Ding 《Molecular biology reports》2010,37(4):1823-1829
The crystal structures of almost all the enzymes of arginine metabolism have been determined, but arginine decarboxylase’s
structure is not resolved yet. In order to characterize and crystallize arginine decarboxylase, we overexpressed biosynthetic
arginine decarboxylase (ADC; EC 4.1.1.19, encoded by the speA gene) from Escherichia coli in the T7 expression system as a cleavable poly-His-tagged fusion construct. The expressed recombinant His10-ADC (77.3 kDa) was first purified by Ni–NTA affinity chromatography, then proteolytically digested with Tobacco Etch Virus
(TEV) protease to remove the poly-His fusion tag, and finally purified by anion exchange chromatography. The His10 tag removed recombinant ADC (74.1 kDa)’s typical yield was 90 mg from 1 l of culture medium with purity above 98%. The recombinant
ADC was assayed for decarboxylase activity, showing decarboxylase activity of 2.8 U/mg, similar to the purified native E. coli ADC. The decarboxylase activity assay also showed that the purified recombinant ADC tolerated broad ranges of pH (pH 6–9)
and temperature (20–80°C). Our research may facilitate further studies of ADC structure and function, including the determination
of its crystal structure by X-ray diffraction. 相似文献
8.
Xylose reductase (XR) is a key enzyme in biological xylitol production, and most XRs have broad substrate specificities. During
xylitol production from biomass hydrolysate, non-specific XRs can reduce l-arabinose, which is the second-most abundant hemicellulosic sugar, to the undesirable byproduct arabitol, which interferes
with xylitol crystallization in downstream processing. To minimize the flux from l-arabinose to arabitol, the l-arabinose-preferring, endogenous XR was replaced by a d-xylose-preferring heterologous XR in Candida tropicalis. Then, Bacillus licheniformis
araA and Escherichia coli araB and araD were codon-optimized and expressed functionally in C. tropicalis for the efficient assimilation of l-arabinose. During xylitol fermentation, the control strains BSXDH-3 and KNV converted 9.9 g l-arabinose l−1 into 9.5 and 8.3 g arabitol l−1, respectively, whereas the recombinant strain JY consumed 10.5 g l-arabinose l−1 for cell growth without forming arabitol. Moreover, JY produced xylitol with 42 and 16% higher productivity than BSXDH-3
and KNV, respectively. 相似文献
9.
Jin Zhou Ju Chu Yong-Hong Wang Si-Liang Zhang Ying-Ping Zhuang Zhong-Yi Yuan 《World journal of microbiology & biotechnology》2008,24(6):789-796
An intracellular S-adenosylmethionine synthetase (SAM-s) was purified from the fermentation broth of Pichia pastoris GS115 by a sequence chromatography column. It was purified to apparent homogeneity by (NH4)2SO4 fractionation (30–60%), anion exchange, hydrophobic interaction, anion exchange and gel filtration chromatography. HPLC showed
the purity of purified SAM-s was 91.2%. The enzyme was purified up to 49.5-fold with a final yield of 20.3%. The molecular
weight of the homogeneous enzyme was 43.6 KDa, as determined by electro-spray ionization mass spectrometry (ESI-MS). Its isoelectric
point was approximately 4.7, indicating an acidic character. The optimum pH and temperature for the enzyme reaction were 8.5
and 35 °C, respectively. The enzyme was stable at pH 7.0–9.0 and was easy to inactivate in acid solution (pH ≤ 5.0). The temperature
stability was up to 45 °C. Metal ions, such as, Mn2+ and K+ at the concentration of 5 mM had a slight activation effect on the enzyme activity and the Mg2+ activated the enzyme significantly. The enzyme activity was strongly inhibited by heavy metal ions (Cu2+ and Ag2+) and EDTA. The purified enzyme from the transformed Pichia pastoris synthesized S-adenosylmethionine (SAM) from ATP and l-methionine in vitro with a K
m of 120 and 330 μM and V
max of 8.1 and 23.2 μmol/mg/min for l-methionine and ATP, respectively. 相似文献
10.
Bacillus coagulans has been of great commercial interest over the past decade owing to its strong ability of producing optical pure l-lactic acid from both hexose and pentose sugars including l-arabinose with high yield, titer and productivity under thermophilic conditions. The l-arabinose isomerase (L-AI) from Bacillus coagulans was heterologously over-expressed in Escherichia coli. The open reading frame of the L-AI has 1,422 nucleotides encoding a protein with 474 amino acid residues. The recombinant
L-AI was purified to homogeneity by one-step His-tag affinity chromatography. The molecular mass of the enzyme was estimated
to be 56 kDa by SDS-PAGE. The enzyme was most active at 70°C and pH 7.0. The metal ion Mn2+ was shown to be the best activator for enzymatic activity and thermostability. The enzyme showed higher activity at acidic
pH than at alkaline pH. The kinetic studies showed that the K
m, V
max and k
cat/K
m for the conversion of l-arabinose were 106 mM, 84 U/mg and 34.5 mM−1min−1, respectively. The equilibrium ratio of l-arabinose to l-ribulose was 78:22 under optimal conditions. l-ribulose (97 g/L) was obtained from 500 g/l of l-arabinose catalyzed by the enzyme (8.3 U/mL) under the optimal conditions within 1.5 h, giving at a substrate conversion
of 19.4% and a production rate of 65 g L−1 h−1. 相似文献
11.
Yoshinori Takagi Teruhide Sugisawa Tatsuo Hoshino 《Applied microbiology and biotechnology》2009,82(6):1049-1056
A single-stage continuous fermentation process for the production of 2-keto-l-gulonic acid (2KGA) from l-sorbose using Ketogulonigenium vulgare DSM 4025 was developed. The chemostat culture with the dilution rate that was calculated based on the relationship between
the 2KGA production rate and the 2KGA concentration was feasible for production with high concentration of 2KGA. In this system,
112.2 g/L of 2KGA on the average was continuously produced from 114 g/L of l-sorbose. A steady state of the fermentation was maintained for the duration of more than 110 h. The dilution rate was kept
in the range of 0.035 and 0.043 h−1, and the 2KGA productivity was 3.90 to 4.80 g/L/h. The average molar conversion yield of 2KGA from l-sorbose was 91.3%. Under the optimal conditions, l-sorbose concentration was kept at 0 g/L. Meanwhile, the dissolved oxygen level was changing in response to the dilution rate
and 2KGA concentration. In the dissolved oxygen (DO) range of 16% to 58%, it was revealed that the relationship between DO
and D possessed high degree of positive correlation under the l-sorbose limiting condition (complete consumption of l-sorbose). Increasing D closer to the critical value for washing out point of the continuous fermentation, DO value tended to be gradually increased
up to 58%. In conclusion, an efficient and reproducible continuous fermentation process for 2KGA production by K. vulgare DSM 4025 could be developed using a medium containing baker’s yeast without using a second helper microorganism. 相似文献
12.
Deutch CE 《Antonie van Leeuwenhoek》2011,99(4):781-793
Staphylococcus saprophyticus strains ATCC 15305, ATCC 35552, and ATCC 49907 were found to require l-proline but not l-arginine for growth in a defined culture medium. All three strains could utilize l-ornithine as a proline source and contained l-ornithine aminotransferase and Δ1-pyrroline-5-carboxylate reductase activities; strains ATCC 35552 and ATCC 49907 could use l-arginine as a proline source and had l-arginase activity. The proline requirement also could be met by l-prolinamide, l-proline methyl ester, and the dipeptides l-alanyl-l-proline and l-leucyl-l-proline. The bacteria exhibited l-proline degradative activity as measured by the formation of Δ1-pyrroline-5-carboxylate. The specific activity of proline degradation was not affected by addition of l-proline or NaCl but was highest in strain ATCC 49907 after growth in Mueller–Hinton broth. A membrane fraction from this
strain had l-proline dehydrogenase activity as detected both by reaction of Δ1-pyrroline-5-carboxylate with 2-aminobenzaldehyde (0.79 nmol min−1 mg−1) and by the proline-dependent reduction of p-iodonitrotetrazolium (20.1 nmol min−1 mg−1). A soluble fraction from this strain had Δ1-pyrroline-5-carboxylate dehydrogenase activity (88.8 nmol min−1 mg−1) as determined by the NAD+-dependent oxidation of dl-Δ1-pyrroline-5-carboxylate. Addition of l-proline to several culture media did not increase the growth rate or final yield of bacteria but did stimulate growth during
osmotic stress. When grown with l-ornithine as the proline source, S. saprophyticus was most susceptible to the proline analogues L-azetidine-2-carboylate, 3,4-dehydro-dl-proline, dl-thiazolidine-2-carboxylate, and l-thiazolidine-4-carboxylate. These results indicate that proline uptake and metabolism may be a potential target of antimicrobial
therapy for this organism. 相似文献
13.
Jian Feng Li Jie Zhang Zhen Zhang Hong Wei Ma Jia Xin Zhang Shuang Quan Zhang 《The protein journal》2010,29(5):314-319
The human beta defensins-4 (hBD4) exhibit a broad range of antimicrobial properties and are thought to be ideal therapeutic
agents because of their potential ability to circumvent the problems of acquired resistance often observed with other antimicrobial
therapies. We report here the application of small ubiquitin-related modifier (SUMO) fusion technology to the expression and
purification of cationic antibacterial peptide hBD4. The fusion protein expressed in a soluble form was purified to a purity
of 90% by Ni-IDA chromatography and 637 mg protein of interest was obtained per liter of fermentation culture. After the SUMO-hBD4
fusion protein was cleaved by the SUMO protease at 30 °C for 1 h, the cleaved sample was re-applied to a Ni-IDA. Finally,
about 166 mg recombinant hBD4 was obtained from 1 L fermentation culture with no less than 96% purity and the recombinant
hBD4 had similar antimicrobial properties to the synthetic hBD4. Thus, the SUMO-mediated peptide expression and purification
system potentially could be employed for the production of recombinant cytotoxic peptides. 相似文献
14.
Yaser Hassan Dewir Nisha Singh Shakira Shaik Ashley Nicholas 《In vitro cellular & developmental biology. Plant》2010,46(1):41-46
The present study reports a simple protocol for indirect shoot organogenesis and plant regeneration of Sutherlandia using rachis and stem segments. Different concentrations (0.0–68.08 μmol l−1) of thidiazuron (TDZ) were used for callus induction and shoot organogenesis. The highest percentage of callus formation
(97.5%) and the highest percentage of explants forming shoots (88.8%) were obtained from rachis explants cultured onto Murashige
and Skoog (MS) medium (Murashige and Skoog, Physiol. Plant. 15:473–495, 1962) supplemented with 45.41 μmol l−1 TDZ. Scanning electron microscopy demonstrated the early development of adventitious shoots derived from callus cultures.
Shoot clusters were further developed and grown in MS hormone-free medium. The presence of l-canavanine was determined by thin-layer chromatography and confirmed after column fractionation using silica gel and nuclear
magnetic resonance spectroscopy. Individual shoots were rooted on different concentrations and combinations of MS salt strength
and IBA. Half-strength MS salt medium supplemented with 24.6 μmol l−1 IBA was optimal for root induction in which 78% of shoots were rooted. The in vitro plants were successfully acclimatized in a growth chamber with a 90% survival rate. 相似文献
15.
M. Helanto K. Kiviharju T. Granström M. Leisola A. Nyyssölä 《Applied microbiology and biotechnology》2009,83(1):77-83
l-Ribose is a rare and expensive sugar that can be used as a precursor for the production of l-nucleoside analogues, which are used as antiviral drugs. In this work, we describe a novel way of producing l-ribose from the readily available raw material l-arabinose. This was achieved by introducing l-ribose isomerase activity into l-ribulokinase-deficient Escherichia coli UP1110 and Lactobacillus plantarum BPT197 strains. The process for l-ribose production by resting cells was investigated. The initial l-ribose production rates at 39°C and pH 8 were 0.46 ± 0.01 g g−1 h−1 (1.84 ± 0.03 g l−1 h−1) and 0.27 ± 0.01 g g−1 h−1 (1.91 ± 0.1 g l−1 h−1) for E. coli and for L. plantarum, respectively. Conversions were around 20% at their highest in the experiments. Also partially purified protein precipitates
having both l-arabinose isomerase and l-ribose isomerase activity were successfully used for converting l-arabinose to l-ribose. 相似文献
16.
Xin Zhou Zhengping Zhang Xiaohe Jia Yifan Wu Lan Luo Zhimin Yin 《World journal of microbiology & biotechnology》2008,24(8):1267-1272
Bacillus subtilis glutamine synthetase (GS) was highly expressed (about 86% of total protein) as soluble protein in Escherichia coli BL21(DE3) containing pET28a-glnA, which was induced by 0.4 mM IPTG in LB medium, and maximal theanine-forming activity of
the recombinant GS induced in LB is 6.4 U/mg at a series concentration (0–100 mM) of Mn2+ at optimal pH 7.5. In order to get GS with high theanine-forming activity, safety, and low cost for food and pharmaceutics
industry, M9-A (details are described in “Materials and methods”) and 0.1% (w/v) lactose were selected as culture medium and
inducer respectively. Recombinant GS was also highly expressed (84% of total protein) and totally soluble in M9-A and the
specific activity of the recombinant GS is 6.2 U/mg which is approximate to that (6.4 U/mg) induced in LB in the presence
of 10 mM Mn2+ at optimal pH 7.5. The activity is markedly higher activated by Mn2+ than that by other nine bivalent cations. Furthermore, M9-B (5 μM Mn2+ was added into M9-A) was used to culture the recombinant strain and theanine-forming activity of the recombinant GS induced
in M9-B was improved 20% (up to 7.6 U/mg). Finally, theanine production experiment coupled with yeast fermentation system
was carried out in a 1.0 ml reaction system with 0.1 mg crude GS from M9-B or M9-A, and the yield of theanine were 15.3 and
13.1 g/L by paper chromatography and HPLC, respectively. 相似文献
17.
Satomura T Zhang XD Hara Y Doi K Sakuraba H Ohshima T 《Applied microbiology and biotechnology》2011,89(4):1075-1082
The activity of a dye-linked l-proline dehydrogenase (dye-l-proDH) was found in the crude extract of an aerobic hyperthermophilic archaeon, Pyrobaculum calidifontis JCM 11548, and was purified 163-fold through four sequential chromatography steps. The enzyme has a molecular mass of about
108 kDa and is a homodimer with a subunit molecular mass of about 46 kDa. The enzyme retained more than 90% of its activity
after incubation at 100 °C for 120 min (pH 7.5) or after incubation at pHs 4.5–9.0 for 30 min at 50 °C. The enzyme catalyzed
l-proline dehydrogenation to Δ1-pyroline-5-carboxylate using 2,6-dichloroindophenol (DCIP) as the electron acceptor and the Michaelis constants for l-proline and DCIP were 1.67 and 0.026 mM, respectively. The prosthetic group on the enzyme was identified as flavin adenine
dinucleotide by high-performance liquid chromatography. The subunit N-terminal amino acid sequence was MYDYVVVGAG. Using that
sequence and previously reported genome information, the gene encoding the enzyme (Pcal_1655) was identified. The gene was
then cloned and expressed in Escherichia coli and found to encode a polypeptide of 415 amino acids with a calculated molecular weight of 46,259. The dye-l-proDH gene cluster in P. calidifontis inherently differs from those in the other hyperthermophiles reported so far. 相似文献
18.
Zhou H Liao X Liu L Wang T Du G Chen J 《Journal of industrial microbiology & biotechnology》2011,38(9):1219-1227
The l-phenylalanine (l-Phe) production by Escherichia
coli WSH-Z06 (pAP-B03) was frequently prevented by bacteriophage BP-1 infestation. To cope with the bacteriophage BP-1 problem
for an improved l-Phe production, one bacteriophage BP-1-resistant mutant, E. coli BR-42, was obtained from 416 mutant colonies of E. coli WSH-Z06 after N-methyl-N’-nitro-N-nitrosoguanidine (NTG) mutagenesis by selection for resistance to bacteriophage BP-1. The recombinant E. coli BR-42-carrying plasmid pAP-B03 had a high capacity in l-Phe production and a remarkable tolerance to 1 × 1010 pfu (plaque-forming unit)/ml bacteriophage stock. For an enhanced l-Phe production by E. coli BR-42 (pAP-B03), the effects of different feeding strategies including pH–stat, constant rate feeding, linear decreasing
rate feeding, and exponential feeding on l-Phe production were investigated; and a two-stage feeding strategy, namely exponential feeding at μ
set = 0.18 h−1 in the first 20 h and a following linear varying rate feeding with F = (−0.55 × t + 18.6) ml/h, was developed to improve l-Phe production. With this two-stage feeding approach, a maximum l-Phe titer of 57.63 g/l with a high l-Phe productivity (1.15 g/l/h) was achieved, which was 15% higher than the highest level (50 g/l) reported so far according
to our knowledge. The recombinant E. coli BR-42 (pAP-B03) is a potential l-Phe over-producer in substantial prevention of bacteriophage BP-1 infestation compared to its parent strain WSH-Z06 (pAP-B03). 相似文献
19.
Lessertia frutescens L., commonly known as cancer-bush, is a medicinally reputed plant species indigenous to southern Africa. Field leaf extracts
of this species are known to exhibit many curative properties. However, little is known about the bioactive compounds that
are present in in vitro leaf extracts and seed extracts. The objective of this study was to verify the presence of and quantify
l-canavanine, gamma amino butyric acid (GABA), arginine and d-pinitol in the seeds, field leaves and in vitro leaves of L. frutescens using gas and liquid chromatography. Methanolic extracts of in vitro leaves, field leaves and seeds were used. MRM chromatograms
were recorded for l-canavanine and arginine using tandem mass spectrometry. GC chromatograms were recorded for GABA and d-pinitol using gas chromatography. d-Pinitol was found to be most abundant and was 14.75 and 18.17 mg/g in in vitro and field leaf extracts respectively, followed
by GABA (7.29 and 3.48 mg/g), arginine (7.08 and 0.35 mg/g) and l-canavanine (0.55 and 0.08 mg/g). In the seed extracts, GABA content was found to be the highest (1.69 mg/g) followed by l-canavanine (0.37 mg/g), then d-pinitol (0.25 mg/g), and arginine (0.02 mg/g). In vitro leaves had higher quantities of all compounds, except for d-pinitol. This study therefore highlights the potential of bulking in vitro leaves for the extraction of the medicinal compounds,
l-canavanine and GABA. 相似文献
20.
Mark S. Ou Lonnie O. Ingram K. T. Shanmugam 《Journal of industrial microbiology & biotechnology》2011,38(5):599-605
Lactic acid is used as an additive in foods, pharmaceuticals, and cosmetics, and is also an industrial chemical. Optically
pure lactic acid is increasingly used as a renewable bio-based product to replace petroleum-based plastics. However, current
production of lactic acid depends on carbohydrate feedstocks that have alternate uses as foods. The use of non-food feedstocks
by current commercial biocatalysts is limited by inefficient pathways for pentose utilization. B. coagulans strain 36D1 is a thermotolerant bacterium that can grow and efficiently ferment pentoses using the pentose-phosphate pathway
and all other sugar constituents of lignocellulosic biomass at 50°C and pH 5.0, conditions that also favor simultaneous enzymatic
saccharification and fermentation (SSF) of cellulose. Using this bacterial biocatalyst, high levels (150–180 g l−1) of lactic acid were produced from xylose and glucose with minimal by-products in mineral salts medium. In a fed-batch SSF
of crystalline cellulose with fungal enzymes and B. coagulans, lactic acid titer was 80 g l−1 and the yield was close to 80%. These results demonstrate that B. coagulans can effectively ferment non-food carbohydrates from lignocellulose to l(+)-lactic acid at sufficient concentrations for commercial application. The high temperature fermentation of pentoses and
hexoses to lactic acid by B. coagulans has these additional advantages: reduction in cellulase loading in SSF of cellulose with a decrease in enzyme cost in the
process and a reduction in contamination of large-scale fermentations. 相似文献