共查询到20条相似文献,搜索用时 78 毫秒
1.
K. Srinivasa Babu T. Muthukumaran Aju Antony S. D. Prem Singh Samuel M. Balamurali V. Murugan S. Meenakshisundaram 《Biotechnology letters》2009,31(5):659-664
Human granulocyte-macrophage colony stimulating factor (hGMCSF) is an important therapeutic cytokine. As a novel attempt to
purify hGMCSF protein, without the enzymatic cleavage of the affinity tag, an intein-based system was used. The gene was fused
by overlap extension PCR to the intein sequence at its N-terminal in pTYB11 vector. The hGMCSF was expressed as a fusion protein in E. coli BL21(DE3), and E. coli GJ1158. In the former, the protein was expressed as inclusion bodies and upon purification the yield was 7 mg/l with a specific
activity of 0.5 × 107 IU/mg. In salt-inducible E. coli GJ1158, hGMCSF was expressed in a soluble form at 20 mg/l and a specific activity of 0.9 × 107 IU/mg. The intein-hGMCSF was purified on a chitin affinity column by cleaving intein with 50 mM DTT resulting in a highly
pure 14.7 kDa hGMCSF. 相似文献
2.
The structural gene for sphingomyelinase (SMase) from Streptomyces
griseocarneus, was introduced into Streptomyces lividans using a shuttle vector, pUC702, for Escherichia coli/S. lividans. High-level secretory production of SMase was achieved using the promoter, signal sequence and terminator regions of phospholipase
D from Streptoverticillium cinnamoneum. The transformant constitutively expressed a high specific activity of SMase extracellularly during batch culture. Maximum
SMase activity (555 ± 114 U/mg protein) was with 1.75 M MgCl2 which was about 50-fold more than that with 10 mM MgCl2. 相似文献
3.
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
4.
Zhina Qiao Meijuan Xu Minglong Shao Youxi Zhao Mengfei Long Taowei Yang Xian Zhang Shangtian Yang Hideki Nakanishi Zhiming Rao 《Engineering in Life Science》2020,20(1-2):7-16
4‐Hydroxyisoleucine, a promising drug, has mainly been applied in the clinical treatment of type 2 diabetes in the pharmaceutical industry. l ‐Isoleucine hydroxylase specifically converts l‐ Ile to 4‐hydroxyisoleucine. However, due to its poor thermostability, the industrial production of 4‐hydroxyisoleucine has been largely restricted. In the present study, the disulfide bond in l ‐isoleucine hydroxylase protein was rationally designed to improve its thermostability to facilitate industrial application. The half‐life of variant T181C was 4.03 h at 50°C, 10.27‐fold the half‐life of wild type (0.39 h). The specific enzyme activity of mutant T181C was 2.42 ± 0.08 U/mg, which was 3.56‐fold the specific enzyme activity of wild type 0.68 ± 0.06 U/mg. In addition, molecular dynamics simulation was performed to determine the reason for the improvement of thermostability. Based on five repeated batches of whole‐cell biotransformation, Bacillus subtilis 168/pMA5‐idoT181C recombinant strain produced a cumulative yield of 856.91 mM (126.11 g/L) 4‐hydroxyisoleucine, which is the highest level of productivity reported based on a microbial process. The results could facilitate industrial scale production of 4‐hydroxyisoleucine. Rational design of disulfide bond improved l ‐isoleucine hydroxylase thermostability and may be suitable for protein engineering of other hydroxylases. 相似文献
5.
Ribitsch D Karl W Wehrschütz-Sigl E Tutz S Remler P Weber HJ Gruber K Stehr R Bessler C Hoven N Sauter K Maurer KH Schwab H 《Applied microbiology and biotechnology》2009,81(5):875-886
In the course of a microbial screening of soil samples for new oxidases, different enrichment strategies were carried out.
With choline as the only carbon source, a microorganism was isolated and identified as Arthrobacter nicotianae. From this strain, a gene coding for a choline oxidase was isolated from chromosomal DNA. This gene named codA was cloned in Escherichia coli BL21-Gold and the protein (An_CodA) heterologously overexpressed as a soluble intracellular protein of 59.1 kDa. Basic biochemical characterization of
purified protein revealed a pH optimum of 7.4 and activity over a broad temperature range (15–70 °C). Specific activities
were determined toward choline chloride (4.70 ± 0.12 U/mg) and the synthetic analogs bis(2-hydroxyethyl)-dimethylammonium
chloride (0.05 ± 0.45 × 10–2 U/mg) and tris-(2-hydroxyethyl)-methylammonium methylsulfate (0.01 ± 0.12 × 10–2 U/mg). With increasing number of oxidizable groups, a significant decrease in activity was noted. Determination of kinetic
parameters in atmorspheric oxygen resulted in K
M = 1.51 ± 0.09 mM and V
max = 42.73 ± 0.42 mU/min for choline chloride and K
M = 4.77 ± 0.76 mM and V
max = 48.40 ± 2.88 mU/min for the reaction intermediate betaine aldehyde respectively. Nuclear magnetic resonance spectroscopic
analysis of the products formed during the enzyme reaction with choline chloride showed that in vitro the intermediate betaine
aldehyde exists also free in solution. 相似文献
6.
The extracellular inulinase structural gene was isolated from the genomic DNA of the marine yeast Pichia guilliermondii strain 1 by PCR. The gene had an open reading frame of 1,542 bp long encoding an inulinase. The coding region of the gene
was not interrupted by any intron. It encoded 514 amino acid residues of a protein with a putative signal peptide of 18 amino
acids and the calculated molecular mass of 58.04 kDa. The protein sequence deduced from the inulinase structural gene contained
the inulinase consensus sequences (WMNXPNGL) and (RDPKVF). It also had ten conserved putative N-glycosylation sites. The inulinase from P. guilliermondii strain 1 was found to be closely related to that from Kluyveromyces marxianus. The inulinase gene without the signal sequence was subcloned into pPICZαA expression vector and expressed in Pichia pastoris X-33. The expressed fusion protein was analyzed by SDS-PAGE and western blotting and a specific band with molecular mass
of about 60 kDa was found. Enzyme activity assay verified the recombinant protein as an inulinase. A maximum activity of 58.7 ± 0.12 U/ml
was obtained from the culture supernatant of P. pastoris X-33 harboring the inulinase gene. A large amount of monosaccharides, disaccharides and oligosaccharides were detected after
the hydrolysis of inulin with the crude recombinant inulinase. 相似文献
7.
Erika Nahomy Marino-Marmolejo Antonio De León-Rodríguez Ana Paulina Barba de la Rosa Leticia Santos 《Molecular biotechnology》2009,42(1):61-67
Analysis of the Thermoplasma acidophilum DSM 1728 genome identified two putative alcohol dehydrogenase (ADH) open reading frames showing 50.4% identity against each
other. The corresponding genes Ta0841 and Ta1316 encode proteins of 336 and 328 amino acids with molecular masses of 36.48 and 36.01 kDa, respectively. The genes were expressed
in Escherichia coli and the recombinant enzymes were functionally assessed for activity. Throughout the study only Ta1316 ADH resulted active
in the oxidative reaction in the pH range 2–8 (optimal pH 5.0) and temperatures from 25 to 90°C (optimal 75°C). This ADH catalyzes
the oxidation of several alcohols such as ethanol, methanol, 2-propanol, butanol, and pentanol during the reduction of the
cofactor NAD+. The highest activity was found in the presence of ethanol producing optically pure acetaldehyde. The specific enzyme activity
of the purified Ta1316 ADH with ethanol as a substrate in the optimal conditions was 628.7 U/mg. 相似文献
8.
A cellulase-free xylanase production by Thermomyces lanuginosus SSBP using bagasse pulp was examined under submerged (SmC) and solid-state cultivation (SSC). Higher level of xylanase activity
(19,320 ± 37 U g−1 dried carbon source) was obtained in SSC cultures than in SmC (1,772 ± 15 U g−1 dried carbon source) after 120 h with 10% inoculum. The biobleaching efficacy of crude xylanase was tested on bagasse pulp,
and the maximum brightness of 46.1 ± 0.06% was observed with 50 U of crude xylanase per gram of pulp, which was 3.8 points
higher than the brightness of untreated samples. Reducing sugars (26 ± 0.1 mg g−1) and UV-absorbing lignin-derived compounds in the pulp filtrates were observed as maximum in 50 U of crude xylanase-treated
samples. T. lanuginosus SSBP has potential applications due to its high productivity of xylanase and its efficiency in pulp bleaching. 相似文献
9.
Wan-Chi Liang Min-Guan Lin Meng-Chun Chi Hui-Yu Hu Huei-Fen Lo Hui-Ping Chang Long-Liu Lin 《Archives of microbiology》2009,191(7):583-593
Bacillus licheniformis DnaK (BlDnaK) is predicted to consist of a 45-kDa N-terminal ATPase domain and a 25-kDa C-terminal substrate-binding domain. In this
study, the full-length BlDnaK and its T86W and three C-terminally truncated mutants were constructed to evaluate the role of up to C-terminal 255 amino
acids of the protein. The steady-state ATPase activity for BlDnaK, T86W, T86W/ΔC120, T86W/ΔC249, and T86W/ΔC255 was 65.68, 53.21, 116.04, 321.38, and 90.59 nmol Pi/min per mg, respectively.
In vivo, BldnaK, T86W and T86W/ΔC120 genes allowed an E. coli
dnaK756-ts mutant to grow at 44°C. Except for T86W/ΔC255, simultaneous addition of B. licheniformis DnaJ and GrpE, and NR-peptide synergistically stimulated the ATPase activity of BlDnaK, T86W, T86W/ΔC120, and T86W/ΔC249 by 16.9-, 13.9-, 33.9-, 9.9-fold, respectively. Measurement of intrinsic tryptophan
fluorescence revealed significant alterations of microenvironment of aromatic amino acids in the C-terminally truncated mutants.
The temperature-dependent signal in the far-UV region for T86W was consistent with that of BlDnaK, but the C-terminally truncated mutant proteins showed a higher sensitivity toward temperature-induced denaturation.
These results suggest that C-terminal truncations alter the ATPase activity and thermal stability of BlDnaK and induce the conformation change of the ATPase domain.
Wan-Chi Liang and Min-Guan Lin contributed equally to this work. 相似文献
10.
Kameshnee Naidoo Manimaran Ayyachamy Kugen Permaul Suren Singh 《Bioprocess and biosystems engineering》2009,32(5):689-695
Xanthomonas campestris pv phaseoli produced an extracellular endoinulinase (9.24 ± 0.03 U mL−1) in an optimized medium comprising of 3% sucrose and 2.5% tryptone. X. campestris pv. phaseoli was further subjected to ethylmethanesulfonate mutagenesis and the resulting mutant, X. campestris pv. phaseoli KM 24 demonstrated inulinase production of 22.09 ± 0.03 U mL−1 after 18 h, which was 2.4-fold higher than that of the wild type. Inulinase production by this mutant was scaled up using
sucrose as a carbon source in a 5-L fermenter yielding maximum volumetric (21,865 U L−1 h−1) and specific (119,025 U g−1 h−1) productivities of inulinase after 18 h with an inulinase/invertase ratio of 2.6. A maximum FOS production of 11.9 g L−1 h−1 and specific productivity of 72 g g−1 h−1 FOS from inulin were observed in a fermenter, when the mutant was grown on medium containing 3% inulin and 2.5% tryptone.
The detection of mono- and oligosaccharides in inulin hydrolysates by TLC analysis indicated the presence of an endoinulinase.
This mutant has potential for large-scale production of inulinase and fructooligosaccharides. 相似文献
11.
Heterologous expression and characterization of a proxidomal ascorbate peroxidase from <Emphasis Type="Italic">Populus tomentosa</Emphasis> 总被引:1,自引:0,他引:1
The present study reported for the first time, cloning, expression and characteristics of a Proxidomal APX gene (PpAPX) from
Populus tomentosa. The PpAPX gene encodes a protein of 287 amino acid residues with a calculated molecular mass of 31.58 kDa. The over-expressed
recombinant PpAPX protein showed high activity towards the substrates ascorbate aicd (ASA) and H2O2. At fixed ASA concentrations, the K
m and V
max values were 0.12 ± 0.01 mM and 23.4 ± 4.2 mmol/min mg for H2O2. And at fixed H2O2 concentrations, the K
m and V
max values were 0.53 ± 0.04 mM and 20.0 ± 2.3 mmol/min mg for ASA. 相似文献
12.
Habibollah Faraji Mohammad Ramezani Baratali Mashkani Hamid R. Sadeghnia Hamid M. Benhangi Saeed Hosseini Teshnizi Fatemeh Soltani 《Biotechnology progress》2019,35(4):e2819
Staphylokinase (SAK) is a promising thrombolytic agent for the treatment of patients suffering from blood-clotting disorders. To increase the potency of SAK and to minimize vessel reocclusion, a new construct bearing SAK motif fused to tsetse thrombin inhibitor (TTI) via a 20-amino acid linker with 2 RGD (2 × arginine-glycine-aspartic acid inhibiting platelet aggregation via attachment to integrin receptors of platelet) was codon optimized and expressed comparatively in Pichia pastoris GS115 as a Mut+ strain and KM71H as a Muts strain. Fusion protein was optimized in terms of best expression condition and fibrinolytic activity and compared with the rSAK. Expression level of the designed construct reached up to 175 mg/L of the culture medium after 72-hr stimulation with 2.5% methanol and remained steady for 3–4 days. The highest expression was obtained at the range of 2–3% methanol. The SAK-2RGD-TT (relative activity >82%) was more active at 25–37 °C than rSAK (relative activity of 93%). Further, it showed relative activity >80% at pH ranges of 7–9. Western blot analysis showed two bands of nearly 27 and 24 kDa at ratio of 5 to 3, respectively. The specific fibrinolytic activity of the SAK-2RGD-TTI was measured as 8,269 U/mg, and 19,616 U/mg for the nonpurified and purified proteins, respectively. Deglycosylation by using tunicamycin in culture medium resulted in higher fibrinolytic activity of SAK-2RGD-TTI (2.2 fold). Consequently, compared to the rSAK, at the same equimolar proportion, addition of RGD and TTI fragments could increase fibrinolytic activity. Also, P. pastoris can be considered as an efficient host for overexpression of the soluble SAK-2RGD-TTI with high activity without requiring a complicated purification procedure. 相似文献
13.
The extracellular lipase structural gene was isolated from cDNA of Aureobasidium pullulans HN2-3 by using SMARTTM RACE cDNA amplification kit. The gene had an open reading frame of 1245 bp long encoding a lipase. The coding region of the
gene was interrupted by only one intron (55 bp). It encodes 414 amino acid residues of a protein with a putative signal peptide
of 26 amino acids. The protein sequence deduced from the extracellular lipase structural gene contained the lipase consensus
sequence (G-X-S-X-G) and three conserved putative N-glycosylation sites. According to the phylogenetic tree of the lipases,
the lipase from A. pullulans was closely related to that from Aspergillus fumigatus (XP_750543) and Neosartorya fischeri (XP_001257768) and the identities were 50% and 52%, respectively. The mature peptide encoding cDNA was subcloned into pET-24a
(+) expression vector. The recombinant plasmid was expressed in Escherichia coli BL21(DE3). The expressed fusion protein was analyzed by SDS-PAGE and western blotting and a specific band with molecular
mass of about 47 kDa was found. Enzyme activity assay verified the recombinant protein as a lipase. A maximum activity of
0.96 U/mg was obtained from cellular extract of E. coli BL21(DE3) harboring pET-24a(+)LIP1. Optimal pH and temperature of the crude recombinant lipase were 8.0 and 35 °C, respectively and the crude recombinant lipase
had the highest hydrolytic activity towards peanut oil. 相似文献
14.
Shreenath Prasad Anurag Misra Ved Prakash Jangir Abhishek Awasthi Jog Raj Tek Chand Bhalla 《World journal of microbiology & biotechnology》2007,23(3):345-353
Rhodococcus sp. NDB 1165, a nitrile-transforming organism was isolated from temperate forest soil of Himalayas. The nitrilase (EC 3.5.5.2)
activity of this organism had higher substrate specificity toward aromatic nitriles (benzonitrile, 3-cyanopyridine and 4-cyanopyridine)
and unsaturated aliphatic nitrile (acrylonitrile) in comparison to saturated aliphatic nitriles (acetonitrile, propionitrile,
butyronitrile and isobutyronitrile) nitrile and arylacetonitrile (phenylacetonitrile and indole-3-acetonitrile). The nitrilase
of Rhodococcus sp. NDB 1165 was inducible in nature and propionitrile proved to be an efficient inducer. However, the salts of ferrous and
cobalt ions had an inhibitory effect. Under optimized reaction conditions (pH 8.0 and temperature 45°C) the nitrilase activity
of this organism was 2.39 ± 0.07 U/mg dry cell mass (dcm). The half-life of this enzyme was 150 min and 40 min at 45°C and
50°C respectively. However, it was quite stable at 40°C and around 58 % activity was retained even after 6 h at this temperature.
The V
max and K
m value of this nitrilase were 1.67 μmol/ml min and 0.1 M respectively using 3-cyanopyridine as substrate. However, the decrease
in V
max and K
m values (0.56 μmol/ml min and 0.02 M, respectively) were ␣observed at >0.05 M 3-cyanopyridine which revealed that this enzyme
experienced uncompetitive inhibition at higher substrate concentrations. Under optimized reaction conditions, 1.6 M 3-cyanopyridine
was successfully converted in to nicotinic acid using 2.0 mg resting cells (dcm)/ml reaction mixture in 11 h. This is the
highest production of nicotinic acid i.e. 8.95 mg/mg resting cells (dcm)/h as compared to nitrilase systems reported hitherto. 相似文献
15.
Ning Guo Fang Gong Zhenming Chi Jun Sheng Jing Li 《Journal of industrial microbiology & biotechnology》2009,36(4):499-507
In order to isolate inulinase overproducers of the marine yeast Pichia guilliermondii, strain 1, cells were mutated by using UV light and LiCl2. One mutant (M-30) with enhanced inulinase production was obtained. Response surface methodology (RSM) was used to optimize
the medium compositions and cultivation conditions for inulinase production by the mutant in solid-state fermentation. The
initial moisture, inoculum, the amount ratio of wheat bran to rice bran, temperature, pH for the maximum inulinase production
by the mutant M-30 were found to be 60.5%, 2.5%, 0.42, 30°C and 6.50, respectively. Under the optimized conditions, 455.9 U/grams
of dry substrate (gds) of inulinase activity was reached in the solid state fermentation culture of the mutant M-30 whereas
the predicted maximum inulinase activity of 459.2 U/gds was derived from RSM regression. Under the same conditions, its parent
strain only produced 291.0 U/gds of inulinase activity. This is the highest inulinase activity produced by the yeast strains
reported so far. 相似文献
16.
Recombinant truncated human heme oxygenase-1 (hHO-1) expressed in Escherichia coli was efficiently separated and purified from feedstock by DEAE-ion exchange expanded bed adsorption. Protocol optimization
of hHO-1 on DEAE adsorbent resulted in adsorption in 0 M NaCl and elution in 150 mM NaCl at a pH of 8.5. The active enzyme
fractions separated from the expanded bed column were further purified by a Superdex 75 gel filtration step. The specific
hHO-1 activity increased from 0.82 ± 0.05 to 24.8 ± 1.8 U/mg during the whole purification steps. The recovery and purification
factor of truncated hHO-1 of the whole purification were 72.7 ± 4.7 and 30.2 ± 2.3%, respectively. This purification process
can decrease the demand on the preparation of feedstock and simplify the purification process. 相似文献
17.
Riadh Ben Salah Ali Gargouri Robert Verger Youssef Gargouri Hafedh Mejdoub 《World journal of microbiology & biotechnology》2009,25(8):1375-1384
The sequence corresponding to the mature lipase of Rhizopus oryzae WPG (ROLw) was subcloned in the pPIC9K expression vector, with a strong AOX1 promoter, to construct a recombinant lipase protein containing six histidine residues at the N-terminal. The His-tagged lipase
was expressed in Pichia Pastoris X33 and purified to homogeneity by a simple, one-step purification protocol using immobilized metal affinity chromatography (Ni-NTA
resin). High level expression of the lipase by Pichia Pastoris X33 cells harbouring the lipase gene containing expression vector was observed upon induction with 2.5 g/l methanol at 28°C;
the specific activity of the purified His6-ROLw was 1,500 or 760 U/mg using olive oil emulsion or tributyrin as substrates, respectively. To check the importance of
Asn 134 His substitution in the affinity and substrate selectivity of ROLw, the mutant His6-ROLw-N134H was overexpressed in Pichia Pastoris X33 and purified with the same nickel metal affinity column. The specific activity of the purified His-tagged ROLw-N134H
was 5,900 and 35 U/mg using olive oil emulsion or tributyrin as substrate. A comparative study of the wild type (His6-ROLw) and the mutant (His6-ROLw-N134H) proteins was carried out. A 3D structure model of ROLw was built using the RNL structure as template. We have
concluded that a slight increase in the exposed hydrophilic residues on the surface of ROLw as compared to RNL (ROLwN134H)
could be responsible for a higher selectivity of ROlw for long and short chain triacylglycerols at the lipid/water interface
and then explaining the importance of Asn 134 for the chain length specificity of ROLw. This property is quite rare among
Rhizopus lipases and gives this new lipase great potential for use in the field of biocatalysis. 相似文献
18.
Q Wang LL Zhao JY Sun JX Liu XY Weng 《World journal of microbiology & biotechnology》2012,28(3):929-935
A modified error-prone PCR and high-throughout screening system based on 96-well plate were employed to improve catalytic
activity of a hybrid xylanase (ATx). The mutant (FSI-A124) with enhanced activity was further heterologously expressed in
Pichia pastoris under the control of GAP promoter. The recombinant xylanase driven by the Saccharomyces cerevisiae α-mating factor was secreted into culture medium. After growth in YPD medium for 96 h, xylanase activity in the culture supernatant
reached 66.1 U ml−1, which was 2.92 times as that of its parent. 6 × His-tagged purification increased the specific activity to 1557.61 U mg−1. The optimum temperature and pH of recombinant xylanase were 55°C and 6.0, respectively. A single amino acid substitution
(L49P) was observed within sequence of the mutant. Insight of the three dimensional structure revealed that proline possibly
produced weaker hydrogen bond, van der Waals force and hydrophobic interaction with other residues nearby than leucine, especially
for V174, contributing to the flexibility of catalytic residue E177. In this study, FSI-A124 exhibited higher xylanase activity
but poorer thermostability than its parent, indicating that activity and stability might be negatively correlated. 相似文献
19.
The methylotrophic yeast Pichia pastoris has been used for the expression of many proteins. However, limitations such as protein degradation and aggregation became
obvious when secreting heterologous protein-recombinant human consensus interferon-α mutant. Here, we investigate the effect
of induction temperature on the yield and stability of interferon mutant expressed by P. patoris with buffered complex medium. The best results in terms of interferon mutant bioactivity and specific bioactivity were obtained
when the microorganism was induced at 15°C, which were 2.91 × 108 ± 0.3 × 108 and 2.26 × 108 ± 0.23 × 108 IU mg−1, respectively. At the same time, the cells grew fast owing to high AOX1-specific activity, and interferon mutant expression
level reached 1.23 g l−1, which was almost 30 times higher than that in the flask. Also, the proteolytic degradation of interferon mutant was inhibited
completely because of lower protease bioactivity probably due to a reduced cell death rate at lower temperatures as well as
protection of yeast extract and peptone in complex medium. In addition, interferon mutant aggregation was repressed significantly
by the addition of Tween-80, and a specific bioactivity of 7.35 × 108 ± 0.56 × 108 IU mg−1 was obtained. These results should be applicable to other low-stability recombinant proteins expressed in P. pastoris. 相似文献
20.
<Emphasis Type="Italic">Thermotoga maritima</Emphasis> TM0298 is a highly thermostable mannitol dehydrogenase 总被引:1,自引:1,他引:0
Song SH Ahluwalia N Leduc Y Delbaere LT Vieille C 《Applied microbiology and biotechnology》2008,81(3):485-495
Thermotoga maritima TM0298 is annotated as an alcohol dehydrogenase, yet it shows high identity and similarity to mesophilic mannitol dehydrogenases.
To investigate this enzyme further, its gene was cloned and expressed in Escherichia coli. The purified recombinant enzyme was most active on fructose and mannitol, making it the first known hyperthermophilic mannitol
dehydrogenase. T. maritima mannitol dehydrogenase (TmMtDH) is optimally active between 90 and 100 °C and retains 63% of its activity at 120 °C but shows
no detectable activity at room temperature. Its kinetic inactivation follows a first-order mechanism, with half-lives of 57 min
at 80 °C and 6 min at 95 °C. Although TmMtDH has a higher V
max with NADPH than with NADH, its catalytic efficiency is 2.2 times higher with NADH than with NADPH and 33 times higher with
NAD+ than with NADP+. This cofactor specificity can be explained by the high density of negatively charged residues (Glu193, Asp195, and Glu196)
downstream of the NAD(P) interaction site, the glycine motif. We demonstrate that TmMtDH contains a single catalytic zinc
per subunit. Finally, we provide the first proof of concept that mannitol can be produced directly from glucose in a two-step
enzymatic process, using a Thermotoga neapolitana xylose isomerase mutant and TmMtDH at 60 °C. 相似文献