Cloning and characterization of a thermostable and halo-tolerant endoglucanase from <Emphasis Type="Italic">Thermoanaerobacter tengcongensis</Emphasis> MB4

A β-1,4-endoglucanase (Cel5A) was cloned from the genomic DNA of saccharolytic thermophilic eubacterium Thermoanaerobacter tengcongensis MB4 and functionally expressed in Escherichia coli. Substrate specificity analysis revealed that Cel5A cleaves specifically the β-1,4-glycosidic linkage in cellulose with high activity (294 U mg⁻¹; carboxymethyl cellulose sodium (CMC)). On CMC, kinetics of Cel5A was determined (K _m 1.39 ± 0.12 g l⁻¹; k _cat/K _m 1.41 ± 0.13 g⁻¹ s⁻¹). Cel5A displays an activity optimum between 75 and 80 °C. Residues Glu187 and Glu289 were identified as key catalytic amino acids by sequence alignment. Interestingly, derived from a non-halophilic bacterium, Cel5A exhibits high residual activities in molar concentration of NaCl (3 M, 49.3%) and KCl (4 M, 48.6%). In 1 M NaCl, 82% of Cel5A activity is retained after 24 h incubation. Molecular Dynamics studies performed at 0 and 3 M NaCl, correlate the Cel5A stability to the formation of R-COO⁻···Na⁺ ···⁻OOC-R salt bridges within the Cel5A tertiary structure, while activity possibly relates to the number of Na⁺ ions trapped into the negatively charged active site, involving a competition mechanism between substrate and Na⁺. Additionally, Cel5A is remarkably resistant in ionic liquids 1-butyl-3-methyllimidazolium chloride (1 M, 54.4%) and 1-allyl-3-methylimidazolium chloride (1 M, 65.1%) which are promising solvents for cellulose degradation and making Cel5A an attractive candidate for industrial applications.     

Two highly thermostable paralogous single-stranded DNA-binding proteins from <Emphasis Type="Italic">Thermoanaerobacter tengcongensis</Emphasis>

The thermophilic bacterium Thermoanaerobacter tengcongensis has two single-stranded DNA-binding (SSB) proteins, designated TteSSB2 and TteSSB3. In a SSB complementation assay in Escherichia coli, only TteSSB3 took over the in vivo function of EcoSSB. We have cloned the ssb genes obtained by PCR and have developed E. coli overexpression systems. The TteSSB2 and TteSSB3 consist of 153 and 150 amino acids with a calculated molecular mass of 17.29 and 16.96 kDa, respectively. They are the smallest known bacterial SSB proteins. The homology between amino acid sequences of these proteins is 40% identity and 53% similarity. They are functional as homotetramers, with each monomer encoding one single-stranded DNA binding domain (OB-fold). In fluorescence titrations with poly(dT), both proteins bind single-stranded DNA with a binding site size of about 40 nt per homotetramer. Thermostability with half-life of about 30 s at 95 degrees C makes TteSSB3 similar to the known SSB of Thermus aquaticus (TaqSSB). The TteSSB2 was fully active even after 6 h incubation at 100 degrees C. Here, we show for the first time paralogous thermostable homotetrameric SSBs, which could be an attractive alternative for known homodimeric thermostable SSB proteins in their applications for molecular biology methods and analytical purposes.     

Cloning,expression, and characterization of thermostable Manganese superoxide dismutase from <Emphasis Type="Italic">Thermoascus aurantiacus</Emphasis> var. <Emphasis Type="Italic">levisporus</Emphasis>

A superoxide dismutase (SOD) gene of Thermoascus aurantiacus var. levisporus, a thermophilic fungus, was cloned, sequenced, and expressed in Pichia pastoris and its gene product was characterized. The coding sequence predicted a 231 residues protein with a unique 35 amino acids extension at the N-terminus indicating a mitochondrial-targeting sequence. The content of Mn was 2.46 μg/mg of protein and Fe was not detected in the purified enzyme. The enzyme was found to be inhibited by NaN₃, but not by KCN or H₂O₂. These results suggested that the SOD in Thermoascus aurantiacus var. levisporus was the manganese superoxide dismutase type. In comparison with other MnSODs, all manganese-binding sites were also conserved in the sequence (H88, H136, D222, H226). The molecular mass of a single band of the enzyme was estimated to be 21.7 kDa. The protein was expressed in tetramer form with molecular weight of 68.0 kDa. The activity of purified protein was 2,324 U/mg. The optimum temperature of the enzyme was 55°C and it exhibited maximal activity at pH 7.5. The enzyme was thermostable at 50 and 60°C and the half-life at 80°C was approximately 40 min.     

Cloning and characterization of thermostable esterase from <Emphasis Type="Italic">Archaeoglobus fulgidus</Emphasis>

Thermostable esterase gene was cloned (Est-AF) from extremophilic microorganisms, Archaeoglobus fulgidus DSM 4304. The protein analysis result showed that Est-AF is monomer with total 247 amino acids and molecular weight of estimated 27.5 kDa. It also showed repeating units G-X-S-X-G (GHSLG) (residues 86 approximately 90) which is reported as active site of known esterases, and the putative catalytic triad composed of Ser88, Asp198 and His226. The esterase activity test with various acyl chain length of rho-nitrophenol resulted that Est-AF showed highest specific activity with rho-nitrophenylbutyrate (pNPC4) and rapidly decrease with rho-nitrophenyl ester contain more than 8 carbon chain. These results represent that cloned enzyme is verified as a carboxylesterase but not a lipase because esterase activity is decreased with rho-nitrophenyl ester contains more than 8 carbon chains but lipase activity does not affected with carbon chain length. Optimum temperature of esterase reaction with rho-nitrophenylbutyrate (pNPC4) was 80 degrees C. When ketoprofen ethyl ester was used as a substrate, activity of Est-AF showed the highest value at 70 degrees C, and 10% of activity still remains after 3 h of incubation at 90 degrees C. This result represents Est-AF has high thermostability with comparison of other esterases that have been reported. However, Est-AF showed low enantioselectivity with ketoprofen ethyl ester. Optimum pH of Est-AF is between pH 7.0 and pH 8.0. Km value of ketoprofen ethyl ester is 1.6 mM and, Vmax is 1.7 micromole/mg protein/min. Est-AF showed similar substrate affinity but slower reaction with ketoprofen ethyl ester compare with esterase from mesophilic strain P. fluorescens.     

High-level expression and characterization of a highly thermostable chitosanase from <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The sequence of an endo-chitosanase gene (CSN) from Aspergillus fumigatus was optimized based on the preferred codons of Pichia pastoris and synthesized in vitro through overlapping PCR (CSN-P). The gene was cloned into a yeast expression vector, pHBM905A, and secretorily expressed in Pichia pastoris GS115. The yield of CSN-P reached ~3 mg/ml with a high-density fermentation in a 14 l fermenter and the enzyme activity was ~25,000 U/ml. The enzyme had half-lives of 2.5 h at 80°C, 1 h at 90°C and 32 min at 100°C. It retained 70% activity after incubation with 10 M urea at room temperature for 30 min. This enzyme was used for a large-scale preparation of oligosaccharides: 3 g enzyme converted 200 kg chitosan into oligosaccharides in 24 h at 60°C.     

Cytoplasmic expression of a thermostable invertase from <Emphasis Type="Italic">Thermotoga maritima</Emphasis> in <Emphasis Type="Italic">Lactococcus lactis</Emphasis>

Objectives

To evaluate the secretory and cytoplasmic expression of a thermostable Thermogata maritima invertase in Lactococcus lactis.

Results

The thermostable invertase from T. maritima was cloned with and without the USP45 secretory peptide into the pNZ8148 vector for nisin-inducible expression in L. lactis. The introduction of an USP45 secretion peptide at the N-terminal of the enzyme led to a loss of protein solubility. Computational homology modeling and hydrophobicity studies indicated that the USP45 peptide exposes a stretch of hydrophobic amino acids on the protein surface resulting in lower solubility. Removal of the USP45 secretion peptide allowed a soluble and functional invertase to be expressed intracellularly in L. lactis. Immobilized metal affinity chromatography purification of the cell lysate with nickel-NTA gave a single protein band on SDS-PAGE, while E. coli-expressed invertase consistently co-purified with an additional band. The yields of the purified invertase from E. coli and L. lactis were 14.1 and 6.3 mg/l respectively.

Conclusions

Invertase can be expressed in L. lactis and purified in a functional form. L. lactis is a suitable host for the production of food-grade invertase for use in the food and biotechnology industries.

     

Cloning and expression of the sucrose phosphorylase gene from <Emphasis Type="Italic">Leuconostoc </Emphasis><Emphasis Type="Italic">mesenteroides</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The gene encoding sucrose phosphorylase (742sp) in Leuconostoc mesenteroides NRRL B-742 was cloned and expressed in Escherichia coli. The nucleotide sequence of the transformed 742sp comprised an ORF of 1,458 bp giving a protein with calculated molecular mass of 55.3 kDa. 742SPase contains a C-terminal amino acid sequence that is significantly different from those of other Leu. mesenteroides SPases. The purified 742SPase had a specific activity of 1.8 U/mg with a K _m of 3 mM with sucrose as a substrate; optimum activity was at 37°C and pH 6.7. The purified 742SPase transferred the glucosyl moiety of sucrose to cytosine monophosphate (CMP). Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cloning and expression of <Emphasis Type="Italic">mel</Emphasis> gene from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Previous work from our laboratory has shown that most of Bacillus thuringiensis strains possess the ability to produce melanin in the presence of l-tyrosine at elevated temperatures (42 °C). Furthermore, it was shown that the melanin produced by B. thuringiensis was synthesized by the action of tyrosinase, which catalyzed the conversion of l-tyrosine, via l-DOPA, to melanin. In this study, the tyrosinase-encoding gene (mel) from B. thuringiensis 4D11 was cloned using PCR techniques and expressed in Escherichia coli DH5 . A DNA fragment with 1179 bp which contained the intact mel gene in the recombinant plasmid pGEM1179 imparted the ability to synthesize melanin to the E. coli recipient strain. The nucleotide sequence of this DNA fragment revealed an open reading frame of 744 bp, encoding a protein of 248 amino acids. The novel mel gene from B.thuringiensis expressed in E. coli DH5 conferred UV protection on the recipient strain.     

Importance of glucose-6-phosphate dehydrogenase in taxol biosynthesis in <Emphasis Type="Italic">Taxus chinensis</Emphasis> cultures

The roles of glucose-6-phosphate dehydrogenase (G6PDH) in paclitaxel production were investigated in cell suspension cultures of Taxus chinensis. In the normal cultures, the trend of G6PDH activity was similar to that of cell growth. Addition of glutamate increased G6PDH activity, while dehydroepiandrosterone (DHEA) decreased G6PDH activity. In elicitor-treated cultures, cell growth was depressed, while G6PDH activity and taxol production were enhanced compared with the control. Glutamate recovered the depression of cell growth, and resulted in further increase in G6PDH activity and taxol production. Contrarily, DHEA exacerbated the depression of cell growth, and decreased G6PDH activity and taxol production induced by fungal elicítor. The results indicated that G6PDH played a critic role of taxol production by affecting cell viability.     

Cloning,expression, and characterization of serine protease from thermophilic fungus <Emphasis Type="Italic">Thermoascus aurantiacus</Emphasis> var. <Emphasis Type="Italic">levisporus</Emphasis>

The serine protease gene from a thermophilic fungus Thermoascus aurantiacus var. levisporus, was cloned, sequenced, and expressed in Pichia pastoris and the recombinant protein was characterized. The full-length cDNA of 2,592 bp contains an ORF of 1,482 bp encoding 494 amino acids. Sequence analysis of the deduced amino acid sequence revealed high homology with subtilisin serine proteases. The putative enzyme contained catalytic domain with active sites formed by three residues of Aspl83, His215, and Ser384. The molecular mass of the recombinant enzyme was estimated to be 59.1 kDa after overexpression in P. pastoris. The activity of recombinant protein was 115.58 U/mg. The protease exhibited its maximal activity at 50°C and pH 8.0 and kept thermostable at 60°C, and retained 60% activity after 60 min at 70° C. The protease activity was found to be inhibited by PMSF, but not by DTT or EDTA. The enzyme has broad substrate specificity such as gelatin, casein and pure milk, and exhibiting highest activity towards casein.     

Cloning of two cellobiohydrolase genes from <Emphasis Type="Italic">Trichoderma</Emphasis><Emphasis Type="Italic">viride</Emphasis> and heterogenous expression in yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Cellobiohydrolase genes cbhI and cbhII were isolated from Trichoderma viride AS3.3711 and T. viride CICC 13038, respectively, using RT-PCR technique. The cbhI gene from T. viride AS3.3711 contains 1,542 nucleotides and encodes a 514-amino acid protein with a molecular weight of approximately 53.96 kDa. The cbhII gene from T. viride CICC 13038 was 1,413 bp in length encoding 471 amino acid residues with a molecular weight of approximately 49.55 kDa. The CBHI protein showed high homology with enzymes belonging to glycoside hydrolase family 7 and CBHII is a member of Glycoside hydrolase family 6. CBHI and CBHII play a role in the conversion of cellulose to glucose by cutting the disaccharide cellobiose from the non-reducing end of the cellulose polymer chain. The two cellobiohydrolase (CBHI, CBHII) genes were successfully expressed in Saccharomyces cerevisiae H158. Maximal activities of transformants Sc-cbhI and Sc-cbhII were 0.03 and 0.089 units ml⁻¹ under galactose induction, respectively. The optimal temperatures of the recombinant enzymes (CBHI, CBHII) were 60 and 70°C, respectively. The optimal pHs of recombinant enzymes CBHI and CBHII were at pH 5.8 and 5.0, respectively.     

Cloning of a novel lipase gene, <Emphasis Type="Italic">lipJ08,</Emphasis> from <Emphasis Type="Italic">Candida rugosa</Emphasis> and expression in <Emphasis Type="Italic">Pichia pastoris</Emphasis> by codon optimization

A novel lipase gene, lipJ08, was cloned from Candida rugosa ATCC14830, along with the already reported five lipase genes (lip1–lip5). Nucleotide sequencing indicated that the lipJ08 gene contains a 1650 bp open reading frame (ORF) without introns. The deduced amino acid sequence corresponds to 534 amino acid residues, including a putative signal sequence of 15 amino acid residues. Seventeen of the non-universal serine codons (CTG) of lipJ08 were converted into universal serine codons (TCT) by PCR-based mutagenesis. The native and codon-optimized lipJ08 genes were expressed in Pichia pastoris. The hydrolytic activity of the recombinant LIPJ08 was 4.7 U/ml, whereas the activity of the recombinant wild-type lipase could not be detected.     

Purification and characterization of cytochrome <Emphasis Type="Italic">c</Emphasis><Subscript><Emphasis Type="Italic">6</Emphasis></Subscript> from <Emphasis Type="Italic">Acaryochloris marina</Emphasis>

Cytochrome c ₆ , (cyt c ₆) a soluble monoheme electron transport protein, was isolated and characterized from the chlorophyll d-containing cyanobacterium Acaryochoris marina, the type strain MBIC11017. The protein was purified using ammonium sulfate precipitation, ion exchange and gel filtration column chromatography, and fast performance liquid chromatography. Its molecular mass and pI have been determined to be 8.87 kDa and less than 4.2, respectively, by mass spectrometry and isoelectrofocusing (IEF). The protein has an alpha helical structure as indicated by CD (circular dichroism) spectroscopy and a reduction midpoint potential (E _m) of +327 mV versus the normal hydrogen electrode (NHE) as determined by redox potentiometry. Its potential role in electron transfer processes is discussed.     

Molecular characterization and expression analysis of <Emphasis Type="Italic">pathogenesis related</Emphasis> protein 6 from <Emphasis Type="Italic">Panax ginseng</Emphasis>

Panax ginseng Meyer is one of the important medicinal plants in the world, particularly in Asian countries. Ginseng encounters many stress exposure during its long cultivation period. However, the molecular mechanism of stress resistance is still poorly understood in spite of its importance. In this study, pathogenesis-related protein 6 (PR6), also called proteinase inhibitor (PI), was isolated from ginseng embryogenic callus, named PgPR6. The small size of PR6, containing an open reading frame of 219 bp encoding 72 amino acids, the typical characteristic of PR6 protein, shares the highest sequence similarity to PR6 of Theobroma cacao (69% identity). Sequence and structural analysis indicated that PgPR6 belongs to class Kunitz-type PI family. This is the first report pertaining to the identification of PR6 gene from the P. ginseng genome. The high-level expression of PgPR6 was observed in root as revealed by quantitative real-time PCR. The temporal expression analysis demonstrated that PgPR6 expression was highly up-regulated by signaling molecules, heavy metals, mechanical wounding, chilling, salt, sucrose, and mannitol stress, indicating that PgPR6 may play an important role in the molecular defense response of ginseng to a various range of environmental stresses.