Heterologous expression, refolding and functional characterization of two antifreeze proteins from Fragilariopsis cylindrus (Bacillariophyceae)

Antifreeze proteins (AFPs) provide protection for organisms subjected to the presence of ice crystals. The psychrophilic diatom Fragilariopsis cylindrus which is frequently found in polar sea ice carries a multitude of AFP isoforms. In this study we report the heterologous expression of two antifreeze protein isoforms from F. cylindrus in Escherichia coli. Refolding from inclusion bodies produced proteins functionally active with respect to crystal deformation, recrystallization inhibition and thermal hysteresis. We observed a reduction of activity in the presence of the pelB leader peptide in comparison with the GS-linked SUMO-tag. Activity was positively correlated to protein concentration and buffer salinity. Thermal hysteresis and crystal deformation habit suggest the affiliation of the proteins to the hyperactive group of AFPs. One isoform, carrying a signal peptide for secretion, produced a thermal hysteresis up to 1.53 °C ± 0.53 °C and ice crystals of hexagonal bipyramidal shape. The second isoform, which has a long preceding N-terminal sequence of unknown function, produced thermal hysteresis of up to 2.34 °C ± 0.25 °C. Ice crystals grew in form of a hexagonal column in presence of this protein. The different sequences preceding the ice binding domain point to distinct localizations of the proteins inside or outside the cell. We thus propose that AFPs have different functions in vivo, also reflected in their specific TH capability.     

Heterologous expression and regulation of the lysA genes of Pseudomonas aeruginosa and Escherichia coli

Summary Chlorsulfuron-resistant mutants of Arabidopsis thaliana were isolated by screening for growth of seedlings in the presence of the herbicide. Both whole plants and derived tissue cultures were resistant to concentrations of the herbicide approximately 300-fold higher than that required to prevent growth of the wild-type. The resistance is due to a single dominant nuclear mutation at a locus designated csr which has been genetically mapped to chromosome-3. Acetohydroxy acid synthase activity in extracts from chlorsulfuron-resistant plants was much less-susceptible to inhibition by chlorsulfuron and a structurally related inhibitor than the activity in wild-type extracts. This suggests that the csr locus is the structural gene for acetohydroxy acid synthase.     

Heterologous expression,purification, and characterization of xylose reductase from <Emphasis Type="Italic">Candida shehatae</Emphasis>

The xylose reductase (XR) gene (xyl1) from Candida shehatae was cloned and expressed in Escherichia coli, and purified as a His₆-tagged fusion protein. The recombinant XR had K_m values for NADH than NADPH of 150 μM and 20 μM, respectively. The optimal reaction was at pH 6.5 and 35°C. The enzyme was specific for d-xylese.     

Purification and characterization of an extracellular proline iminopeptidase from Arthrobacter nicotianae 9458

An extracellular proline iminopeptidase, with a molecular mass of about 53 kDa, was purified from Arthrobacter nicotianae 9458 and characterized. The enzyme had temperature and pH optima of 37 degrees C and 8.0, respectively, was completely inactivated by heating for 1 min at 80 degrees C and showed highest activity on Pro-pNA. The proline iminopeptidase was characterized by activity at low temperature, NaCl concentrations up to 7.5% and by high sensitivity to pH values 6.0, serine enzyme inhibitor PMSF and divalent cations, Fe2+, Sn2+, Cu2+, Zn2+, Hg2+, Co2+ and Ni2+. The extracellular proline iminopeptidase from A. nicotianae 9458 was able to hydrolyze proline-containing peptides at the pH, temperature and NaCl concentration typical of the surface of smear-ripened cheese and may contribute to proteolysis of these cheeses during ripening.     

Purification and characterization of two extracellular proteinases from Arthrobacter nicotianae 9458

Two extracellular serine proteinases with molecular masses of about 53–55 and 70–72 kDa, were purified from Arthrobacter nicotianae 9458 and characterized. The enzymes differed with respect to temperature optimum, 55–60 and 37°C, respectively, tolerance to low values of pH and temperature, heat stability, sensitivity to EDTA and sulfhydryl blocking agents, and hydrophobicity. Both proteinases were optimally active in the pH range of 9.0 and 9.5, had considerable activity at pH 6.0 on α_s1- and β-caseins, and tolerated NaCl over 5%. Specificity on casein fractions was generally similar and β-casein was more susceptible to hydrolysis than α_s1-casein. The proteinases of Arthrobacter spp. may play a significant role in ripening of the smear surface-ripened cheeses.     

Enzymatic Characterization of an Amine Oxidase from Arthrobacter sp. Used to Measure Phosphatidylethanolamine

Ethanolamine oxidase was screened with the aim of using it to establish a novel enzymatic phosphatidylethanolamine assay. Ethanolamine oxidase activity was detected in the crude extract of Arthrobacter sp., and the enzyme was purified more than 15-fold in three steps with a 54% yield. SDS–PAGE revealed the presence of only one band, which migrated, with an apparent molecular mass of 70 kDa. Biochemical characterization of the enzyme showed phenylethylamine to be the preferred substrate, with the highest kcat/Km value. The primary structure, determined by sequencing the cloned gene, showed a high degree of identity to Cu-containing phenylethylamine oxidase (64%). When heterologously overexpressed in Escherichia coli, the enzyme exhibited only a trace of amine oxidase activity, but high levels of activity emerged after exposure to Cu²⁺, as is typical of recombinant copper amine oxidases. Preliminary application of this enzyme coupled with phospholipase D for determination of phosphatidylethanolamine is also described. This is the first enzymatic method for the measurement of phosphatidylethanolamine.     

Heterologous expression and functional characterization of two hybrid poplar cell-wall invertases

The expression of two hybrid poplar cell-wall invertases (EC 3.2.1.26; PaxgINV1 and PaxgINV2) were previously shown to be spatially and temporally regulated in the vegetative tissues. The expression of PaxgINV1 was linked to processes relating to dormancy, while PaxgINV2 expression was prominent in tissues undergoing growth and expansion. In an effort to further elucidate the physiological roles of these key cell wall enzymes, PaxgINV1 and PaxgINV2 were heterologously expressed in the methylotrophic yeast Pichia pastoris. Three-dimensional predictive models of the poplar invertases revealed a structural channel containing both the conserved β-fructofuranosidase and cell-wall invertase motifs, suggesting that this channel is the putative active site of these enzymes. Recombinant PaxgINV1 and PaxgINV2 had pH optima of 4.8 and 5.6 and temperature optima of 45 and 40°C, respectively. Functional characterization revealed the ability for both enzymes to hydrolyze the fructose residue of sucrose, raffinose, stachyose and verbascose, with PaxgINV2 having higher specific activity for each of the substrates tested. The K _m values of sucrose/raffinose/stachyose were 1.7/1.8/5.0 mM for PaxgINV1 and 1.6/1.7/1.9 mM for PaxgINV2, respectively. Activity analyses in the presence of various metal cations showed that PaxgINV2 was strongly inhibited by Cu²⁺, Zn²⁺ and Hg²⁺, while PaxgINV1 was only weakly inhibited by these cations. The results from this study, coupled with previous expression data, suggest that PaxgINV1 and PaxgINV2 have distinct roles with respect to the physiology and development of hybrid poplar, specifically phloem unloading and processes related to dormancy and bud break.     

Heterologous expression of hexaheme fragments of a multidomain cytochrome from Geobacter sulfurreducens representing a novel class of cytochromes c

Multiheme cytochromes c are of great interest for researchers for a variety of reasons but difficult to obtain in quantities sufficient for the majority of studies. The genome of delta-proteobacterium Geobacter sulfurreducens contains more than a hundred genes coding for c-type cytochromes. Three of them represent a new class of multiheme cytochromes characterized by a mixed type of heme coordination and multidomain organization. We cloned and expressed in Escherichia coli three hexaheme fragments corresponding to two-domain fragments of one such protein containing 12 heme binding motifs and believed to consist of four triheme domains. Despite high sequence similarity among the fragments, expression levels varied significantly. Expression was optimized either by host strain variation or by reducing the rate of apoprotein synthesis. All three fragments were purified by cation exchange followed by gel filtration and were shown to contain six covalently attached hemes as confirmed by mass spectrometry. Their visible spectra are typical of c-type cytochromes. One of the fragments was crystallized and its preliminary X-ray structure shows two separate domains.     

厌氧菌Sunxiuqinia sp. SH-52外切型海藻酸裂解酶的异源表达及酶学特征

【背景】目前已报道的海藻酸分解菌多数为好氧菌,未见有关厌氧菌的报道。从分离的海藻酸分解菌中表征的海藻酸裂解酶大多为内切型海藻酸裂解酶,外切型较少。【目的】研究来自厌氧海藻酸分解菌的海藻酸裂解酶基因,表征新型海藻酸裂解酶并阐明其酶学性质,为海藻酸裂解酶的多样性和微生物降解海藻酸机制提供理论依据。【方法】对来自厌氧海藻酸分解菌Sunxiuqinia sp. SH-52的海藻酸裂解酶SHA-4编码基因进行克隆,分析基因序列,构建重组质粒PGEX-4T-1-SHA-4并在大肠杆菌中实现异源表达,经纯化后对其酶学性质及降解特征进行研究。【结果】该酶在28°C、用0.1 mmol/L IPTG (异丙基-β-D-硫代半乳糖苷)条件下诱导6 h达到最大表达量,纯化后酶的比活力达到21 U/mg。酶学性质分析表明SHA-4的最适温度为37°C;最适pH为7.5;对PolyMG (杂聚古罗糖醛酸-甘露糖醛酸嵌段)具有底物偏好性;Na+对该酶的活性具有抑制作用,而金属离子Cu~(2+)具有明显促进作用,使活性提高了约168%;SHA-4催化海藻酸的Km值为2.5 mg/mL,Vmax为8.7 mg/(mL·min);SHA-4为外切型海藻酸裂解酶,降解海藻酸终产物为单糖。【结论】异源表达了来自一株厌氧海藻酸分解菌Sunxiuqiniasp.SH-52的海藻酸裂解酶SHA-4,该酶是PL6家族中第一个对PolyMG有底物偏好性的外切型海藻酸裂解酶,而且活性较高,作为工具酶有很好的应用前景,为海藻酸降解机制的探索提供了新的线索。     

Heterologous expression and characterization of glycoside hydrolase with its potential applications in hyperthermic environment

With the progressive focus on renewable energy via biofuels production from lignocellulosic biomass, cellulases are the key enzymes that play a fundamental role in this regard. This study aims to unravel the characteristics of Thermotoga maritima MSB8 (Tma) (a hyperthermophile from hot springs) thermostable glycoside hydrolase enzyme. Here, a glycoside hydrolase gene of Thermotoga maritima (Tma) was heterologously expressed and characterized. The gene was placed in the pQE-30 expression vector under the T5 promotor, and the construct pQE-30-Gh was then successfully integrated into Escherichia coli BL21 (DH5α) genome by transformation. Sequence of the glycoside hydrolase contained an open reading frame of 2.124 kbp, encoded a polypeptide of 721 amino acid residues. The molecular weight of the recombinant protein estimated was 79 kDa. The glycoside hydrolase was purified by Ni⁺²-NTA affinity chromatography and its enzymatic activity was investigated. The recombinant enzyme is highly stable within an extreme pH range (2.0–7.0) and highly thermostable at 80 °C for 72 h indicating its viability in hyperthermic environment and acidic nature. Moreover, the Ca²⁺ and Mn²⁺ introduction stimulated the residual activity of recombinant enzyme. Conclusively, the thermostable glycoside hydrolase possesses potential to be exploited for industrial applications at hyperthermic environment.     

Purification and characterization of arsenite oxidase from Arthrobacter sp.

The chemolithoautotroph, Arthrobacter sp.15b oxidizes arsenite to arsenate using a membrane bound arsenite oxidase. The enzyme arsenite oxidase is purified to its homogeneity and identified using MALDI-TOF MS analysis. Upon further characterization, it was observed that the enzyme is a heterodimer showing native molecular mass as ~100 kDa and appeared as two subunits of ~85 kDa LSU and 14 kDa SSU on SDS–PAGE. The V _max and K _m values of the enzyme was found to be 2.45 μM (AsIII)/min/mg) and 26 μM, respectively. The purified enzyme could withstand wide range of pH and temperature changes. The enzyme, however, gets deactivated in the presence of 1 mM of DEPC suggesting the involvement of histidine at the binding site of the enzyme. The peptide analysis of large sub unit of the enzyme showed close match with the arsenite oxidases of Burkholderia sp. YI019A and arsenite oxidase, Mo-pterin containing subunit of Alcaligenes faecalis. The small subunit, however, differed from other arsenite oxidases and matched only with 2Fe–2S binding protein of Anaplasma phagocytophilum. This indicates that Rieske subunits containing the iron–sulfur clusters present in the large as well as small subunits of the enzyme are integral part of the protein.     

Gene cloning,expression, and characterization of a novel trehalose synthase from <Emphasis Type="Italic">Arthrobacter aurescens</Emphasis>

Trehalose synthase (TreS) is an intramolecular transglycosylase. It specially catalyzes the conversion of maltose and trehalose. In this study, a novel treS gene, which had a length of 1,797 bp and encoded 598 amino acids, was cloned from Arthrobacter aurescens CGMCC 1.1892 and expressed in Escherichia coli. Thin layer chromatography results indicated that it could catalyze the conversion between maltose and trehalose in one step. However, the ion chromatography results showed that, as a byproduct, about 13% glucose was also produced. The purified recombinant enzyme had a molecular weight of 68 kDa and showed its optimal activity at 35 °C and pH 6.5. This enzyme was not thermostable, and its activity was increased by 1 mM Mg²⁺, Mn²⁺, and Ca²⁺ while strongly inhibited by 5 mM Cu²⁺ and SDS.     

Heterologous expression of functionally active enterolysin A, class III bacteriocin from Enterococcus faecalis, in Escherichia coli

The heterologous expression of enterolysin A (EnlA), heat-labile class III bacteriocin from Enterococcus faecalis II/1 with anti-listerial activity, was studied in Escherichia coli. The PCR amplified products of enterolysin A structural gene, N-terminal part of EnlA with endopeptidase-like activity and C-terminal part of EnlA similar to a lysis gene of bacteriophage, were cloned in prelinearized pQE-30UA expression vector. The expression of EnlA structural gene led to the synthesis and secretion of functional-active His-tagged enterolysin A protein, which was purified to homogeneity using His-Select™ Cartridge and was shown to be fully active against the indicator strain. The expression of N-terminal or C-terminal part of EnlA and deletion of last 58 amino acids from C-terminal domain of EnlA led to the synthesis of biologically non-active proteins.     

Regulation of expression by divalent cations of a light-inducible gene in Arthrobacter photogonimos

Expression of the light-inducible (lipA) gene in Arthrobacter photogonimos is repressed by Ca²⁺ at a concentration greater than 0.1 M. Expression of lipA was induced by relatively high concentrations of Zn²⁺ Ni²⁺ or Co²⁺ in cell suspensions, an effect that was blocked by an increase in the concentration of Ca²⁺ in the medium. Zn²⁺ and other metals apparently overcame repression by Ca²⁺ by competing for a cellular binding site. Expression of lipA was also induced when the amount of free Ca²⁺ was lowered with ethylene-bis (oxyethylenenitrilo)tetraacetic acid (EGTA). Our results show that the lipA gene does not require Zn²⁺ or other divalent cation for expression and that it is regulated negatively by Ca²⁺.Accumulation of the mature product of this gene (light-inducible protein, LIP) was minimal in the presence of EGTA. Accumulation increased 10-to 20-fold when divalent cations such as Ca²⁺, Mn²⁺, Cu²⁺ or Zn²⁺ were added to cell suspensions treated with chelator. These divalent cations, which allowed the protein to achieve a protease-resistant form on the cell surface, could be substituted by protease inhibitors such as antipain, leupeptin or 1,10-phenanthroline. Our data can be explained by a biparous mechanism in which divalent cations regulate both expression of the lipA gene and accumulation of the gene product.Abbreviations LIP light-inducible protein - BAPTA 1,2-bis(o-aminophenoxy)ethanc-N,N,N,N-tetraacetic acid     

Heterologous expression, purification and characterisation of the extracellular domain of trypanosome invariant surface glycoprotein ISG75

The invariant surface glycoprotein ISG75 is a transmembrane glycoprotein occurring on the surface of the bloodstream-form Trypanozoon. This study describes the expression and purification of the N-terminal extracellular domain of ISG75, a novel target for development of diagnostic tests for trypanosomosis. To facilitate disulfide formation in the cytoplasm, a 1287-bp cDNA fragment encoding ISG75 from Trypanosoma brucei gambiense was expressed in a thioredoxin reductase, glutathione oxidoreductase double mutant Escherichia coli strain. An accessory plasmid pRIL, providing the argI, ileY, and leuW tRNAs, was necessary for efficient heterologous translation of the ISG75 mRNA. The recombinant double-tagged (streptavidine and histidine) ISG75 was purified by two-step affinity chromatography. Addition of L-glutamic acid and L-arginine in the buffer solutions was crucial to stabilise the protein during purification. The purified soluble protein was characterised by circular dichroism spectroscopy, reverse-phase high pressure liquid chromatography and mass spectrometry. It has an alpha-helical folded conformation, is homogeneous and pure (99%). Furthermore, sera of Trypanosoma brucei-infected animals specifically recognise this recombinant ISG75; and rabbit antiserum raised against the recombinant ISG75 detects all species of the Trypanozoon subgenus in parasite preparations.     

Heterologous expression and characterization of man gene from Bacillus Subtilis in Pichia Pastoris

β-Mannanase catalyzes endo-wise hydrolysis of the backbone of mannan and heteromannan, which are abundant in the cell wall structure of ungerminated leguminous seeds. The mature β-mannanase originated from Bacillus subtilis was expressed in Pichia pastoris, a methylotrophic yeast, using the leader peptide sequence of Saccharomyces cerevisiae α-factor. The cultivation of β-mannanase expressing Pichia pastoris yields up to 1.8 g/L protein. In the supernatant the activity of the 40 kDa—total mannanase attained a level of 1102.0 IU/mL. The properties of the β-mannanase were characterized. Optimum pH and temperature for the recombinant enzyme were 5.5 and 50°C respectively. The enzyme was stable at pH 5.0–10.0 and maintained over 30% original activity after incubating at 70°C for 30 min. __________ Translated from China Biotechnology, 2005, 26(7): 52–56 [译自: 中国生物工程杂志]     

Isolation and characterization of Arthrobacter sp. strain MCM B-436, an atrazine-degrading bacterium, from rhizospheric soil

Atrazine is one of the most environmentally prevalent s-triazine-ring herbicides. The widespread use of atrazine and its toxicity necessitates search for remediation technology. As atrazine is still used in India as a major herbicide, exploration of atrazine-degrading bacterial community is of immense importance. Considering lack of reports on well characterized atrazine-degrading bacterial cultures from India and wide diversity and density of microorganisms in rhizosphere, soil sample from rhizosphere of atrazine-resistant plant was studied. Arthrobacter sp. strain isolated in this investigation utilizes atrazine as the sole nitrogen source. In addition, the bacterium degrades other triazines such as ametryn, cyanizine, propazine and simazine. PCR analysis confirms the presence of atzBCD and triazine hydrolase (trzN) genes on chromosomal DNA. Sequencing of the trzN gene reveals high sequence similarity with trzN from Nocardioides sp. C190. An inducible and intracellular atrazine chlorohydrolase enzyme was isolated and partially purified from this isolate. This study confirms the presence of atrazine-degrading microbial population in Indian soils and could be used efficiently for remediation of contaminated soils. Presence of trzN gene indicates possible presence of bacterial community with more efficient and novel enzymatic capabilities. Comparison of enzyme and gene structure of this isolate with other geographically distinct atrazine-degrading strains will help us in the better understanding of gene transfer and evolution.     

Heterologous expression and characterization of recombinant purple acid phosphatase from red kidney bean

Purple acid phosphatases (PAPs) are dinuclear metallohydrolases of widespread occurrence. In a first step to understand structure-function relationship of PAP from red kidney bean (kbPAP), we cloned its cDNA and functionally expressed the enzyme in insect cells. kbPAP cDNA encodes a protein of 459 amino acids with 99% identity to the published primary structure (T. Klabunde et al., Eur. J. Biochem. 226 (1994) 369-375). N-terminally the cDNA encodes 27 amino acids with characteristics for a signal directing the nascent protein to the endoplasmic reticulum. A baculovirus vector was constructed containing cDNAs of kbPAP and green fluorescent protein, the latter to serve as transfection and infection marker. Heterologous expression in High Five insect cells afforded a dimeric, disulfide-linked phosphatase of 110 kDa, identical to the mass of native kbPAP. Purification in three steps yielded 1.5 mg recombinant protein per liter of culture medium with a specific activity of 266 units/mg, slightly exceeding that of native kbPAP. The recombinant protein was functionally indistinguishable from native kbPAP, despite differences in glycosylation and sensitivity to redox reagents.     

Heterologous expression of cDNAs encoding monodehydroascorbate reductases from the moss, <Emphasis Type="Italic">Physcomitrella patens</Emphasis> and characterization of the expressed enzymes

Monodehydroascorbate reductase (MDHAR; EC 1.6.5.4) catalyses the reduction of the monodehydroascorbate (MDHA) radical to ascorbate, using NADH or NADPH as an electron donor, and is believed to be involved in maintaining the reactive oxygen scavenging capability of plant cells. This key enzyme in the ascorbate-glutathione cycle has been studied here in the moss Physcomitrella patens, which is tolerant to a range of abiotic stresses and is increasingly used as a model plant. In the present study, three cDNAs encoding different MDHAR isoforms of 47 kDa were identified in P. patens, and found to exhibit enzymic characteristics similar to MDHARs in vascular plants despite low-sequence identity and a distant evolutionary relationship between the species. The three cDNAs for the P. patens MDHAR enzymes were expressed in Escherichia coli and the active enzymes were purified and characterized. Each recombinant protein displayed an absorbance spectrum typical of flavoenzymes and contained a single non-covalently bound FAD coenzyme molecule. The K _m and k _cat values for the heterologously expressed PpMDHAR enzymes ranged from 8 to 18 μM and 120–130 s⁻¹, respectively, using NADH as the electron donor. The K _m values were at least an order of magnitude higher for NADPH. The K _m values for the MDHA radical were ∼0.5–1.0 μM for each of the purified enzymes, and further kinetic analyses indicated that PpMDHARs follow a ‘ping–pong’ kinetic mechanism. In contrast to previously published data, site-directed mutagenesis indicated that the conserved cysteine residue is not directly involved in the reduction of MDHA.