Molecular cloning and expression of the pyranose 2-oxidase cDNA from<Emphasis Type="Italic"> Trametes ochracea</Emphasis> MB49 in<Emphasis Type="Italic"> Escherichia coli</Emphasis>

A cDNA of a structural gene encoding pyranose 2-oxidase (P2O) from Trametes ochracea strain MB49 was cloned into Escherichia coli strain BL21(DE3) on a multicopy plasmid under the control of the trc promoter. Synthesis of P2O was studied in batch cultures in LB or M9-based mineral medium at 28°C. While there was a low specific activity of P2O in LB medium, the enzyme was synthesised constitutively in mineral medium and represented 3% of the cell soluble protein (0.3 U mg^–1). The effect of isopropyl -d-thiogalactoside on the expression of P2O was studied in mineral medium at 25 and 28°C. The synthesis of P2O at 28°C corresponded to 39% of the cell soluble protein but the major portion of P2O (93%) was in the form of non-active inclusion bodies (activity of P2O equalled 0.19 U mg^–1). At 25°C, the amount of P2O represented 14% of the cell soluble protein and the activity of P2O was 1.1 U mg^–1. The soluble enzyme represented 70% of the total amount of P2O.     

Using native hydantoinase promoter to induce d-carbamoylase soluble expression in Escherichia coli

By use of PCR, the genes encoding d-carbamoylase from A. radiobacter TH572 were cloned in plasmid pET30a and transformed into Escherichia coli BL21 (DE3) to overexpress d-carbamoylase. However, almost all of the protein remained trapped in inclusion bodies. To improve the expression of the properly folded active enzyme, a constitutive plasmid of pGEMT-DCB was constructed using the native hydantoinase promoter (P_Hase) whose optimal length was confirmed to 209 bp. Furthermore, the RBS region in the downstream of P_Hase was optimized to increase the expression level, so the plasmid pGEMT-R-DCB was constructed and transformed into E. coli strain Top10F′. The enzyme activity of Top10F′/pGEMT-R-DCB grown at 37 °C was found to be 0.603 U/mg (dry cell weight, DCW) and increase 58-fold over cells of BL21 (DE3) harboring the plasmid pET-DCB grown at 28 °C.     

Heterologous expression of an extra-cellular lipase from the basidiomycete Pleurotus sapidus

The 1641 bp cDNA encoding an extra-cellular lipase of the basidiomycete Pleurotus sapidus (Lip2) was cloned from a cDNA library. Expression of the cDNA in Escherichia coli, with and without signal sequence, led to the production of recombinant Lip2, mainly as inclusion bodies with low catalytic activity. Refolding yielded catalytically active protein. A C-terminal His tag was used for purification and immunochemical detection. The recombinant lipase hydrolysed xanthophyll esters with high efficiency, and omitting the signal sequence did not alter the catalytic properties. The P. sapidus lipase represents the first enzyme of the lipase/esterase family from a basidiomycetous fungus characterised on the molecular level and expressed in a manageable host.     

Escherichia coli expression and in vitro activation of a unique ligninolytic peroxidase that has a catalytic tyrosine residue

Heterologous expression of Trametes cervina lignin peroxidase (LiP), the only basidiomycete peroxidase that has a catalytic tyrosine, was investigated. The mature LiP cDNA was cloned into the pET vector and used to transform Escherichia coli. Recombinant LiP protein accumulated in inclusion bodies as an inactive form. Refolding conditions for its in vitro activation—including incorporation of heme and structural Ca²⁺ ions, and formation of disulfide bridges—were optimized taking as a starting point those reported for other plant and fungal peroxidases. The absorption spectrum of the refolded enzyme was identical to that of wild LiP from T. cervina suggesting that it was properly folded. The enzyme was able to oxidize 1,4-dimethoxybenzene and ferrocytochrome c confirming its high redox potential and ability to oxidize large substrates. However, during oxidation of veratryl alcohol (VA), the physiological LiP substrate, an unexpected initial lag period was observed. Possible modification of the enzyme was investigated by incubating it with H₂O₂ and VA (for 30 min before dialysis). The pretreated enzyme showed normal kinetics traces for VA oxidation, without the initial lag previously observed. Steady-state kinetics of the pretreated LiP were almost the same as the recombinant enzyme before the pretreatment. Moreover, the catalytic constant (k_cat) for VA oxidation was comparable to that of wild LiP from T. cervina, although the Michaelis–Menten constant (K_m) was 8-fold higher. The present heterologous expression system provides a valuable tool to investigate structure–function relationships, and autocatalytic activation of the unique T. cervina LiP.     

Heterologous expression in Escherichia coli of Neurospora crassa neutral trehalase as an active enzyme

Neutral trehalase from Neurospora crassa was expressed in Escherichia coli as a polypeptide of 84 kDa in agreement with the theoretical size calculated from the corresponding cDNA. The recombinant neutral trehalase, purified by affinity chromatography exhibited a specific activity of 80–150 mU/mg protein. Optima of pH and temperature were 7.0 and 30 °C, respectively. The enzyme was absolutely specific for trehalose, and was quite sensitive to incubation at 40 °C. The recombinant enzyme was totally dependent on calcium, and was inhibited by ATP, copper, silver, aluminium and cobalt. K_M was 42 mM, and V_max was 30.6 nmol of glucose/min. The recombinant protein was phosphorylated by cAMP-dependent protein kinase, but not significantly activated. Immunoblotting with polyclonal antiserum prepared against the recombinant protein showed that neutral trehalase protein levels increased during exponential phase of N. crassa growth and dropped at the stationary phase. This is the first report of a neutral trehalase produced in E. coli with similar biochemical properties described for fungi native neutral trehalases, including calcium-dependence.     

Cloning and High-Level Expression of the Enzymatic Region of Phytase in E. coli

Phytase is an important enzyme poses great nutritional significance in humans and monogastric animals diets. The phytase production yield using wild sources, including micro-organisms, plants, and animals is sorely low. Thus, recombinant expression of phytase has received increasing interest for achieving production rate. Escherichia coli is the most preferred host for expression of heterologous proteins but overexpression of recombinant phytase in E. coli, met with limited success due to the sequestration of the enzyme into inclusion bodies. In the present study, artificial phytases gene with excellent thermostability and activity were designed by detecting the enzymatic region of the E. coli phytase gene by employing bioinformatics tools. Then, the PCR amplified recombinant gene was expressed in E. coli and the active enzyme was recovered from inclusion bodies. Employing cysteine amino acid in the dialysis buffer succeed to the superior activity of the enzyme with a specific activity of 73.8 U/mg. The optimum temperature and pH for enzyme activity were determined at 60 °C and 4, respectively. The novel recombinant enzyme illustrated perfect thermostability up to 70 °C with maintenance 75% of its activity. The enzyme was stable at pH range of 2–10. Moreover, the effects of ions and chemical compounds on enzyme stability and activity were assessed.

     

Characterization of a extreme thermostable fructose-1,6-bisphosphate aldolase from hyperthermophilic bacterium Aquifex aeolicus

Fructose-1,6-bisphosphate (FBP) aldolase, is a glycolytic enzyme that catalyzes the reversible condensation reaction of FBP to dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P). The aldolase gene from Aquifex aeolicus was subcloned, overexpressed in E. coli and purified to 95% homogeneity. The purified enzyme was activated by high concentrations of NH₄⁺ and low concentrations of Co²⁺. The native molecular weight of the purified FBP aldolase was identified as 67 kDa (dimer) by gel filtration chromatography. The enzyme exhibits optimum pH at 6.5 and temperature at 90 °C. Based on the kinetic characterizations, the apparent K_m was calculated to be 4.4 ± 0.07 mM, while V_max was found to be 100 ± 0.02 μM min⁻¹ mg protein⁻¹. The recombinant protein showed extreme heat stability; no activity loss was observed even at 100 °C for 2 h. In addition, the thermophilic enzyme also showed higher stability against several organic solvents viz. acetonitrile, 1,4-dioxane, and methanol. With higher stability against both heat and organic solvents than any other class II aldolase, the A. aeolicus FBP aldolase is an attractive enzyme for use as a biocatalyst for industrial applications.     

Effect of feeding sun-dried seaweed (Ascophyllum nodosum) on fecal shedding of Escherichia coli O157:H7 by feedlot cattle and on growth performance of lambs

Thirty-two steers orally inoculated with a four-strain mixture (10¹⁰ CFU) of nalidixic acid-resistant Escherichia coli O157:H7 had sun-dried Ascophyllum nodosum seaweed (Tasco-14™) added to their barley-based diet (860 g/kg barley grain and 90 g/kg whole crop barley silage, dry matter basis) to assess its effectiveness in reducing fecal shedding of the pathogen. Steers were housed in four groups of eight and received Tasco-14™ in the diet, in place of barley, at levels (as fed) of 10 g/kg for 14 days (T1-14), 20 g/kg for 7 days (T2-7), 20 g/kg for 14 days (T2-14), or not at all (i.e., control, CON). The dietary treatments commenced 7 days after E. coli O157:H7 inoculation and fecal shedding patterns were examined over 14 weeks. Water, water–trough interface, feed and fecal pat samples were also collected weekly and cultured for E. coli O157:H7. Detection of the pathogen in fecal samples was less frequent (P<0.05) in T1-14 (99/168) and T2-7 (84/168) versus CON (135/168) and T2-14 (115/168), and the concentrations of E. coli O157:H7 recovered in feces from T1-14 and T2-7 steers were lower (P<0.005) than from CON or T2-14 steers. Rates of decline in shedding of E. coli O157:H7 were similar among treatments, but final numbers of E. coli O157:H7 were lower (P<0.05) in T1-14 and T2-7 as compared to T2-14 and CON. Fecal volatile fatty acid concentrations and pH were similar among treatments, suggesting no fecal alterations that were antagonistic to survival. E. coli O157:H7 was present in 1 (from T2-7) of 56 cattle drinking water samples, 2 of 56 (T1-14, CON) feed samples and 32 of 56 fecal pats. A second experiment investigated effects of the dietary treatment on growth performance of non-inoculated sheep. Tasco-14™ was administered to 40 individually fed Canadian Arcott lambs beginning at day 56 of a 105-day finishing period. The lambs received Tasco-14™ at 0 g/kg (control, CON), at 10 g/kg for 14 days (T1-14), 20 g/kg for 14 days (T2-14), 10 g/kg for 28 days (T1-28) or at 20 g/kg for 7 days (T2-7) as a top-dress on their pelleted, barley grain-based diet (n = 8). E. coli O157:H7 was not isolated from fecal samples collected at 4-week intervals, but generic E. coli populations were lower (P<0.05) in T1-28 lambs than in other treatments. Average daily gain, feed intake, feed efficiency and carcass traits did not differ among treatments. Our challenge study supports past studies showing that Tasco-14™ decreases shedding of E. coli O157:H7 by cattle. The lamb study shows that this additive did not directly affect feed intake or animal growth.     

Expression, purification and characterization of aprotinin and a human analogue of aprotinin

Aprotinin is a Kunitz-type inhibitor with a relatively broad specificity. It has been shown to be clinically useful for the management of hemorrhagic complications. In this report, small ubiquitin-related modifier (SUMO) linked with a hexa-histidine tag was used as a fusion partner for the production of recombinant aprotinin and a human aprotinin analogue (cloned form human cDNA library). Both fusion proteins were overexpressed mainly as inclusion bodies in Escherichia coli and accounted for approximately 28% of the total cell proteins. After purification by Ni-Sepharose affinity chromatography and renaturation, the fusion proteins were cleaved with SUMO protease 1. Aprotinin and its analogue were separated from the fusion partner by the subtractive chromatography using Ni-Sepharose and then further purified with CM-cellulose. Kinetic studies demonstrated that the amidolytic activity of plasmin was competitively inhibited by recombinant aprotinin with a K_i of 8.6 ± 2.4 nM, which was similar to the K_i (7.5 ± 2.7 nM) of natural aprotinin. The K_i of human aprotinin analogue was 22.7 ± 6.5 nM. The expression strategy described in this study allows convenient high yield and easy purification of small recombinant protease inhibitors with complete native sequences.     

Heterologous expression of a novel psychrophilic Cu/Zn superoxide dismutase from Deschampsia antarctica

Superoxide dismutase (SOD) catalyzes the conversion of the superoxide radical (O₂⁻) into oxygen and hydrogen peroxide. Deschampsia antarctica is a plant that grows in Antarctica and survives to extreme low temperature and high UV radiation, thus it is an ideal model to study novel antioxidants. A cDNA Cu/Zn-SOD gene from D. antarctica was cloned into a pET vector and expressed in Escherichia coli BL21-SI. 112 mg/L of recombinant Cu/Zn-SOD was attained in batch cultures in bioreactor. Using Ni-affinity gel chromatography, the recombinant Cu/Zn-SOD was recovered with a purity of 90% and a specific enzyme activity of 749 at 25 °C. However, zymogram test showed that the enzyme has more activity at 4 °C. This D. antarctica SOD could be used to reduce the oxidation of refrigerated and frozen foods.     

Biological characteristics and thermal requirements of a Brazilian strain of the parasitoid Trichogramma pretiosum reared on eggs of Pseudoplusia includens and Anticarsia gemmatalis

A new strain of the parasitoid Trichogramma pretiosum, was collected in Rio Verde County, State of Goiás, Central Brazil, and designated as T. pretiosum RV. This strain was then found to be the most effective one among several different strains of T. pretiosum tested in a parasitoid selection assay. Therefore, its biological characteristics and thermal requirements were studied, aiming at allowing its multiplication under controlled environmental conditions in the laboratory. The parasitoid was reared on eggs of Pseudoplusia includens and Anticarsia gemmatalis at different constant temperatures within an 18–32 °C temperature range. The number of annual generations of the parasitoid was also estimated at those temperatures. Results have shown that T. pretiosum RV developmental time, from egg to adult, was influenced by all temperatures tested within the range, varying from 6.8 to 20.3 days and 6.0 to 17.0 days on eggs of P. includens and A. gemmatalis, respectively. The emergence of T. pretiosum RV from eggs of A. gemmatalis was higher than 94% at all temperatures tested. When this variable was evaluated on eggs of P. includens, however, the figures were higher than that within the 18–30 °C range (more than 98%), and were also statistically higher than the emergence observed at 32 °C (90.2%). The sex ratio of the parasitoids emerged from eggs of A. gemmatalis decreased from 0.55 to 0.29 at 18–32 °C, respectively. However, for those emerged from eggs of P. includens, the sex ratio was similar (0.73, 0.72 and 0.71) at 20, 28 and 32 °C, respectively. The lower temperature threshold (Tb) and thermal constant (K) were 10.65 °C and 151.25 degree-days when the parasitoid was reared on eggs of P. includens; and 11.64 °C and 127.60 degree-days when reared on eggs of A. gemmatalis. The number of generations per month increased from 1.45 to 4.23 and from 1.49 to 4.79 when the parasitoid was reared on eggs of P. includens and A. gemmatalis, respectively, following the increases in the temperature.     

Influence of process temperature on recombinant enzyme activity in Escherichia coli fed-batch cultures

The influence of proteolysis over recombinant protein quality has been studied using rhamnulose 1-phosphate aldolase (RhuA) production as case example. Progressive induction by means of continuous isopropyl-β-d-thiogalactopyranoside (IPTG) dosage in Escherichia coli fed-batch cultures led to high specific levels of recombinant protein. However, the specific activity profile did not correlate to the specific protein content when the process was run at 37 °C and there was a decrease of the enzyme activity along the induction phase. Specific activity loss depending on the presence of an energy source was observed at short term, but protein degradation due to the action of energy-independent metalloproteases occurred after a longer time period. The effects of lowering the temperature were analysed on both mechanisms, and a reduction of the specific activity loss was observed when the process temperature was decreased to 28 °C. Lower plasmid copy number and specific production rates probably alleviated the metabolic load on host cell during recombinant protein overexpression, and a high increase of the enzyme activity was achieved in high cell density fed-batch cultures under these conditions.     

Over-expression in E. coli and purification of the human OCTN1 transport protein

The hOCTN1 amplified from skin fibroblast RNA was cloned in pET-28a(+) or in pH6EX3 plasmid. The encoded recombinant hOCTN1 resulted in a 6-His tagged fusion protein with a 34 or 21 amino acid extra N-terminal sequence in the pET-28a(+)-hOCTN1 or in the pH6EX3-hOCTN1 constructs, respectively. Both constructs were used to express the hOCTN1 in Escherichia coli Rosetta(DE3)pLysS. The best over-expression was obtained with the pH6EX3-hOCTN1 after 6 h of induction with IPTG at 28 °C. The expressed protein with an apparent molecular mass of 54 kDa, was collected in the insoluble fraction of the cell lysate. Further improvement was obtained using the E. coli RosettaGami2(DE3)pLysS strain to express the protein encoded by pH6EX3-hOCTN1. After 6 h of induction with IPTG at 28 °C, hOCTN1 accounted for 30% of the total protein in the insoluble pellet. This protein fraction was washed with Triton X-100 and deoxycholate, solubilized with a buffer containing 0.8% Sarkosyl, 3 M urea and applied to a Ni²⁺-chelating chromatography column. The homogeneously purified hOCTN1 was eluted with a buffer containing 50 mM imidazole, 0.1% Triton X-100 and 50 mM 2-mercaptoethanol. A yield of about 3 mg purified protein per liter of cell culture was obtained.     

Optimization for high-level expression of the Pichia guilliermondii recombinant inulinase in Pichia pastoris and characterization of the recombinant inulinase

The inulinase gene cloned from the marine-derived yeast Pichia guilliermondii strain 1 was expressed in Pichia pastoris X-33 and the conditions for overexpression of the inulinase were optimized. After the optimization of the conditions for production of the recombinant inulinase, 286.8 ± 5.4 U/ml and 8873 ± 55.3 U/mg of the recombinanat inulinase in the supernatant of the culture of 2-l fermentor were attained at 120 h of the fermentation and fermentation efficiency was 13.04 μg ± 0.4 of protein/ml/d. The recombinant inulinase was purified and characterized. The molecular weight of the purified recombinant inulinase was 57.6 kDa, which was higher than that of the native iunlinase. The optimal pH and temperature of the purified recombinant inulinase were 6.0 and 60 °C, respectively. Other biochemical characteristics of the purified recombinant inulinase were the same as those of the native inulinase produced by the marine-derived P. guilliermondii strain 1. The purified recombinant inulinase also had high exoinulinase activity. Therefore, the recombinant inulinase may have highly potential applications in food and pharmaceutical industies.     

Expression, purification, refolding and characterization of a putative lysophospholipase from Pyrococcus furiosus: retention of structure and lipase/esterase activity in the presence of water-miscible organic solvents at high temperatures

A putative lysophospholipase (PF0480) encoded by the Pyrococcus furiosus genome has previously been cloned and expressed in Escherichia coli. Studies involving crude extracts established the enzyme to be an esterase; however, owing presumably to its tendency to precipitate into inclusion bodies, purification and characterization have thus far not been reported. Here, we report the overexpression and successful recovery and refolding of the enzyme from inclusion bodies. Dynamic light scattering suggests that the enzyme is a dimer, or trimer, in aqueous solution. Circular dichroism and fluorescence spectroscopy show, respectively, that it has mixed beta/alpha structure and well-buried tryptophan residues. Conformational changes are negligible over the temperature range of 30–80 °C, and over the concentration range of 0–50% (v/v) of water mixtures with organic solvents such as methanol, ethanol and acetonitrile. The enzyme is confirmed to be an esterase (hydrolyzing p-NP-acetate and p-NP-butyrate) and also shown to be a lipase (hydrolyzing p-NP-palmitate), with lipolytic activity being overall about 18- to 20-fold lower than esterase activity. Against p-NP-palmitate the enzyme displays optimally activity at pH 7.0 and 70 °C. Remarkably, over 50% activity is retained at 70 °C in the presence of 25% acetonitrile. The high organic solvent stability and thermal stability suggest that this enzyme may have useful biodiesel-related applications, or applications in the pharmaceutical industry, once yields are optimized.     

Purification, characterization and crystallization of pyrroline-5-carboxylate reductase from the hyperthermophilic archeon Sulfolobus Solfataricus

The gene SSO0495 (proC), which encodes pyrroline-5-carboxylate reductase (P5CR) from the thermoacidophilic archeon Sulfolobus solfataricus P2 (Ss-P5CR), was cloned and expressed. The purified recombinant enzyme catalyzes the thioproline dehydrogenase with concomitant oxidation of NAD(P)H to NAD(P)⁺. This archeal enzyme has an optimal alkaline pH in this reversible reaction and is thermostable with a half-life of approximately 30 min at 80 °C. At pH 9.0, the reverse activation rate is nearly 3-fold higher than at pH 7.0. The homopolymer was characterized by cross-linking and size exclusion gel filtration chromatography. Ss-P5CR was crystallized by the hanging-drop vapor-diffusion method at 37 °C. Diffraction data were obtained to a resolution of 3.5 Å and were suitable for X-ray structure determination.     

Cloning and Functional Expression of the Mucrosobin Protein, a β-Fibrinogenase of Trimeresurus mucrosquamatus (Taiwan Habu)

A venom-specific cDNA encoding for a thrombin-like enzyme designated as mucrosobin has been cloned and sequenced from the cDNA library of the venomous gland of Trimeresurus mucrosquamatus. The full-length cDNA of mucrosobin was assembled by oligonucleotide screening and 5′-rapid amplification of cDNA ends. The amino acid sequence deduced from the cDNA consists of 257 amino acid residues with a putative signal peptide of 24 residues. It is highly homologous to the other thrombin-like enzymes (batroxobin, mucofirase, and calobin), suggesting that it is a serine proteinase with a conserved catalytic triad of His⁴¹, Asp⁸⁴ and Ser¹⁷⁹ in the deduced form of mucrosobin protein. Northern blot analysis revealed that the mucrosobin gene encodes an mRNA of 1.5 kb and suggested a tissue-specific expression in the venomous gland. In an effort to study the biological property of mocrosobin, we have expressed the 28-kDa protein as inclusion bodies in Escherichia coli. For analyzing enzymatic activity, the inclusion bodies were solubilized and the recombinant protein was refolded with a two-step dialysis protocol. The refolded recombinant protein exhibited a specific β-fibrinogenolytic activity. This study offers a possibility of using genetic engineering to acquirie a functional snake venom protein with therapeutic potential.     

Expression, Purification, and Initial Characterization of the Recombinant Storage Protein Precursor ofTheobroma cacao

The gene encoding the 67-kDa cocoa storage protein precursor has been cloned fromTheobroma cacaoand expressed inEscherichia coliusing the pET expression system. The recombinant storage protein has been renatured from inclusion bodies at 30°C using 20 m glycine–NaOH buffer, pH 10.0, containing 1 m oxidized glutathione and 0.1% Brij. The renatured protein was purified and demonstrated to adopt a stable native conformation by optical spectroscopy. Secondary structure analysis from circular dichroism indicated the protein to be 23% α-helix and 38% β-sheet, in close agreement with values obtained using a secondary structure prediction program.     

Refolding and Purification of Zymomonas mobilis Levansucrase Produced as Inclusion Bodies in Fed-Batch Culture of Recombinant Escherichia coli

Zymomonas mobilis levansucrase was overproduced by the fed-batch culture of recombinant Escherichia coli harboring a novel expression system that is constitutively expressed by the promoter from the Rahnella aquatilis levansucrase gene. Most of the levansucrase was produced as inclusion bodies in the bacterial cytoplasm, accounting for approximately 20% of the total cellular protein. Refolding after complete denaturation by high concentrations of urea or guanidine hydrochloride was not successful, resulting in large amounts of insoluble aggregates. During the development of the refolding method, it was found that direct solubilization of the inclusion bodies with Triton X-100 reactivated the enzyme, with a considerable refolding efficiency. About 65% of inclusion body levansucrase was refolded into active levansucrase in the renaturation buffer containing 4% (v/v) Triton X-100. The in vitro refolded enzyme was purified to 95% purity by single-step DEAE–Sepharose ion exchange chromatography. Triton X-100 was removed by this ion exchange chromatography.