Purification and properties of recombinant β-glucosidase of the hyperthermophilic bacterium Thermotoga maritima

A -glucosidase of the hyperthermophilic bacterium Thermotoga maritima has been purified from a recombinant Escherichia coli clone expressing the corresponding gene. The enzyme was found to be a dimer with an apparent molecular mass of approximately 95 kDa as determined by size exclusion chromatography. It was composed of two apparently identical subunits of about 47 kDa (determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis). The enzyme had a bbroadsubstrate specificity and attacked -glucoside, -galactoside, -fucoside, and, to a very small extent, also -xyloside substrates. -Glycosidic bonds were not hydrolysed. Kinetic measurement of the hydrolysis of o-nitrophenyl--d-glucopyranoside (oNPGlc) and o-nitrophenyl--d-galactopyranoside (oNPGal) in the concentration ranges 0.05–20 mm and 0.1–10 mm, respectively, at 75°C resulted in non-linear Lineweaver-Burk and Eadie-Hofstee 3lots whereas cellobiose and lactose did not induce this type of effect. Lactose caused substrate inhibition above 350 mm. The enzyme was optimally active at about pH 6.1. The T. maritima -glucosidase represents the most thermostable -glucosidase described to date. In 50 mm sodium phosphate buffer, pH 6.2, at an enzyme concentration of 50 g/ml, the pure enzyme without additives retained more than 60% of its initial activity after a 6-h incubation at 95°C. Correspondence to: W. Liebl     

Constitutive high-level expression of a codon-optimized β-fructosidase gene from the hyperthermophile Thermotoga maritima in Pichia pastoris

Enzymes for use in the sugar industry are preferred to be thermotolerant. In this study, a synthetic codon-optimized gene encoding a highly thermostable β-fructosidase (BfrA, EC 3.2.1.26) from the bacterium Thermotoga maritima was expressed in the yeast Pichia pastoris. The gradual increase of the transgene dosage from one to four copies under the control of the constitutive glyceraldehyde 3-phosphate dehydrogenase promoter had an additive effect on BfrA yield without causing cell toxicity. Maximal values of cell biomass (115 g/l, dry weight) and overall invertase activity (241 U/ml) were reached at 72 h in fed-batch fermentations using cane sugar as the main carbon source for growth. Secretion driven by the Saccharomyces cerevisiae α-factor signal peptide resulted in periplasmic retention (44 %) and extracellular release (56 %) of BfrA. The presence of N-linked oligosaccharides did not influence the optimal activity, thermal stability, kinetic properties, substrate specificity, and exo-type action mode of the yeast-secreted BfrA in comparison to the native unglycosylated enzyme. Complete inversion of cane sugar at initial concentration of 60 % (w/v) was achieved by periplasmic BfrA in undisrupted cells reacting at pH 5.5 and 70 °C, with average productivity of 4.4 g of substrate hydrolyzed per grams of biomass (wet weight) per hour. The high yield of fully active glycosylated BfrA here attained by recombinant P. pastoris in a low-cost fermentation process appears to be attractive for the large-scale production of this thermostable enzyme useful for the manufacture of inverted sugar syrup.     

High-level Expression of an α-l-arabinofuranosidase from Thermotoga maritima in Escherichia coli for the Production of Xylobiose from Xylan

To efficiently produce xylobiose from xylan, high-level expression of an α-l-arabinofuranosidase gene from Thermotoga maritima was carried out in Escherichia coli. A 1.5-kb DNA fragment, coding for an α-l-arabinofuranosidase of T. maritima, was inserted into plasmid pET-20b without the pelB signal sequence leader, and produced pET-20b-araA1 with 8 nt spacing between ATG and Shine–Dalgarno sequence. A maximum activity of 12 U mg⁻¹ was obtained from cellular extract of E. coli BL21-CodonPlus (DE3)-RIL harboring pET-20b-araA1. The over-expressed α-l-arabinofuranosidase was purified 13-fold with a 94% yield from the cellular extract of E. coli by a simple heat treatment. Production of xylooligosaccharides from corncob xylan by endoxylanase and α-l-arabinofuranosidase was examined by TLC and HPLC: xylobiose was the major product from xylan at 90 °C and its proportion in the xylan hydrolyzates increased with the reaction time. Hydrolysis with in the xylanase absence of α-l-arabinofuranosidase gave only half this yield. Revisions requested 27 October 2005; Revisions received 5 September 2005     

Characterization of a thermostable β-glucuronidase from Thermotoga maritima expressed in Arabidopsis thaliana

TmGUSI, a gene identical to that encoding a thermostable β-glucuronidase in the hyperthermophilic anaerobe Thermotoga maritima, has been synthesized using a PCR-based two-step DNA synthesis and codon optimization for plants, and expressed in both Escherichia coli and Arabidopsis thaliana. TmGUSI expressed in transformed E. coli cells exhibited maximum hydrolytic activity at 65?°C and pH 6.5 and retained more than 80% activity after incubation at 85?°C for 30?min. TmGUSI activity in transgenic A. thaliana plants containing TmGUSI was also stable over the temperature range 65-80?°C. Our data suggest that β-glucuronidase from T. maritima can serve as a useful thermostable marker in higher plants.     

Expression and characterization of a thermostable β-xylosidase from the hyperthermophile, Thermotoga maritima

A thermostable beta-xylosidase from a hyperthermophilic bacterium, Thermotoga maritima, was over-expressed in Escherichia coli using the T7 polymerase expression system. The expressed beta-xylosidase was purified in two steps, heat treatment and immobilized metal affinity chromatography, and gave a single band on SDS-PAGE. The maximum activity on p-nitrophenyl beta-D-xylopyranoside was at 90 degrees C and pH 6.1. The purified enzyme had a half-life of over 22-min at 95 degrees C, and retained over 57% of its activity after holding a pH ranging from 5.4 to 8.5 for 1 h at 80 degrees C. Among all tested substrates, the purified enzyme had specific activities of 275, 50 and 29 U mg(-1) on pNPX, pNPAF, and pNPG, respectively. The apparent Michaelis constant of the beta-xylosidase was 0.13 mM for p NPX with a V (max) of 280 U mg(-1). When the purified beta-xylosidase was added to xylanase, corncob xylan was hydrolized completely to xylose.     

Cloning and expression in Escherichia coli of a Thermoanaerobacter cellulolyticus gene coding for heat-stable β-glucanase

Summary A 4.8 kb HindIII fragment of Thermoanaerobacter cellulolyticus DNA cloned in Escherichia coli was shown to direct the synthesis of -glucanase. The enzyme produced by the transformant was extremely heat-stable and the optimum temperature for the enzyme reaction was 80°C. The cloned enzyme could hydrolyse carboxymethyl cellulose and lichenan, but could not digest laminarin, xylan and cellobiose. Although T. cellulolyticus secreted cellulase(s) into the medium, most of the cloned enzyme activity was detected only in cytoplasm in the recombinant clone.     

Expression of a β-galactosidase gene from Lactobacillus sake in Escherichia coli

Summary A -galactosidase gene from Lactobacillus sake coding for lactose hydrolysis was cloned and expressed in Escherichia coli. Chromosomal DNA from L. sake was partially digested with the restriction enzyme Sau3AI, and the 3–6 Kb fragment was ligated to the cloning vector pSP72 digested with BamHI. One E. coli transformant expressing -galactosidase was isolated on X-gal plates. It contained a plasmid with an insertion of approx. 4 Kb. The restriction map of the recombinant plasmid was constructed. The characteristics of the recombinant -galactosidase were compared with those of the wild type. The optima pH and temperature for both enzymes was 6.5 and 50°C, respectively. Stability of the enzymes at different temperatures and activity on lactose were determined.     

Coexpression of β-mannanase and xylanase genes in Escherichia coli

[目的]β-甘露聚糖酶和木聚糖酶都属于半纤维素酶,它们已经同时运用于工农业生产的许多领域.构建β-甘露聚糖酶和木聚糖酶共表达菌株并进行相关评价.[方法]通过设计一个共同的酶切位点,将菌株Bacillus subtilis BE-91中的β-甘露聚糖酶和木聚糖酶基因串联到表达载体pET28a(+)上,转化大肠杆菌构建了一株能够共表达β-甘露聚糖酶和木聚糖酶的菌株B.pET28a-man-xyl.[结果]菌株诱导21h后,发酵液中β-甘露聚糖酶和木聚糖酶的酶活分别为713.34 U/mL和1455.83 U/mL,是胞内酶活的11.8倍和2.53倍.[结论]SDS-PAGE分析、水解圈活性检测和胞外酶与胞内酶酶活检测表明:两个酶均以功能蛋白独立分泌到胞外.此外,与β-甘露聚糖酶和木聚糖酶单独酶解半纤维素相比,复合酶的酶解效果更好.菌株的成功构建为复合酶制剂(半纤维素酶制剂)的研究和生产奠定基础.     

Molecular cloning and expression of a Micromonospora chalcae β-glucosidase encoding gene in Escherichia coli

A Sau3A I genomic library from the actinomycete Micromonospora chalae was constructed in Escherichia coli using the expression vector pUC18. Using the chromogenic substrate 5-bromo-4-chloro-3-indolyl--glucoside (X-glu), a number of positive recombinant colonies were identified. One of those exhibiting the strongest phenotype contained a recombinant plasmid, pANNA1 which harboured a 4.2kb DNA insert. Using restriction endonuclease site mapping and subcloning strategies a 2.3kb DNA fragment encoding the -glucosidase activity was identified. Characterization of the strongly expressed recombinant enzyme demonstrated that it had a dramatically increased thermal stability at 50 °C. The Km values obtained for the recombinant enzyme and that from M. chalcae using the substrate p-nitrophenyl--D-glucoside were 0.19mM and 0.25mM, respectively.     

Identification of a halophilic α-amylase gene from Escherichia coli JM109 and characterization of the recombinant enzyme

A halophilic α-amylase (EAMY) gene from Escherichia coli JM109 was overexpressed in E. coli XL10-Gold and the recombinant protein was purified and characterized. The activity of the EAMY depended on the presence of both Na⁺ and Cl^?, and had maximum activity in 2 M NaCl at 55 °C and pH 7.0. When 2 % (w/v) soluble starch was used as substrate, the specific activity was about 1,090 U mg^?1 protein. This is the first report on identifying a halophilic α-amylase with high specific activity from non-halophilic bacteria.     

Epidemiology of Extended-Spectrum β-Lactamase Producing Escherichia coli in the Stools of Returning Japanese Travelers,and the Risk Factors for Colonization

Objective

Travel overseas has recently been considered a risk factor for colonization with drug-resistant bacteria. The purpose of this study was to establish the epidemiology and risk factors associated with the acquisition of drug-resistant bacteria by Japanese travelers.

Methods

Between October 2011 and September 2012, we screened the stools of 68 Japanese returning travelers for extended-spectrum β-lactamase (ESBL) producing Escherichia coli. All specimens were sampled for clinical reasons. Based on the results, the participants were divided into an ESBL-producing E. coli positive group (18 cases; 26%) and an ESBL-producing E. coli negative group (50 cases; 74%), and a case-control study was performed. Microbiological analyses of ESBL-producing strains, including susceptibility tests, screening tests for metallo-β-lactamase, polymerase chain reaction amplification and sequencing of bla _CTX-M genes, multilocus sequence typing, and whole genome sequencing, were also conducted.

Results

In a univariate comparison, travel to India was a risk factor (Odds Ratio 13.6, 95% Confidence Interval 3.0–75.0, p<0.0001). There were no statistical differences in the characteristics of the travel, such as backpacking, purpose of travel, interval between travel return and sampling stool, and duration of travel. Although 10 of 13 analyzed strains (77%) produced CTX-M-15, no ST131 clone was detected.

Conclusion

We must be aware of the possibilities of acquiring ESBL-producing E. coli during travel in order to prevent the spread of these bacteria not only in Japan but globally.     

Cloning,nucleotide sequence,and overexpression in Escherichia coli of the β-tyrosinase gene from an obligately symbiotic thermophile,Symbiobacterium thermophilum

Symbiobacterium thermophilum is an obligately symbiotic thermophile that can grow only in coculture with a specific Bacillus strain. The amino acid sequences of fragments obtained by cyanogen bromide decomposition of the thermostable -tyrosinase (tyrosine phenol-lyase, E.C. 4.1.99.2) from this organism resembled that of the tryptophanase produced by the same organism. DNA-probing with the tryptophanase gene as the hybridization probe led to cloning in Escherichia coli of the -tyrosinase (tpl) gene. The nucleotide sequence revealed that the -tyrosinase of 458 amino acids (relative molecular mass, 52269) showed significant similarity in amino acid sequence to the tryptophanase over the entire sequence. DNA manipulation of the cloned tpl gene in E. coli led to production of 375 times as much -tyrosinase as that produced by the original S. thermophilum strain.     

Cloning and expression of Citrobacter freundii β-isopropylmalate dehydrogenase gene in both Escherichia coli and Bacillus subtilis

Summary -Isopropylmalate (IPM) dehydrogenase gene of Citrobacter freundii was cloned in both Escherichia coli and Bacillus subtilis. Plasmid pCBL 1 containing C. freundii -IPM dehydrogenase gene was isolated using E. coli (leuB) as a host, pBR 322 as a vector and Hind III as an enzyme. The molecular weight (mol.wt.) of pCBL 1 was 7.7 megadalton (Md) and the plasmid was restricted at two sites by Hind III or Sal I, at three sites by BamH I and at four sites by Pst I. The second hybrid plasmid pCBL 2 containing -IPM dehydrogenase gene was reconstructed from 2.1 Md Pst I fragment of pCBL 1 and pBR 322. -IPM dehydrogenase activities of E. coli transformants with pCBL 1 or pCBL 2 were 2–7-fold higher than those of the present strains. The -IPM dehydrogenase gene was transferred from pBR 322 to pLS 353, a shuttle vector between E. coli and B. subtilis. The third plasmid, pCBL 3 (mol.wt. 5.6Md), was cloned in B. subtilis (leuC) and expressed the enzyme activity which complemented the Leucharacter. The enzyme activities of B. subtilis transformants with pCBL 3 were about 5-fold higher than those of present strains. Thus, the C. freundii gene was effectively expressed in both E. coli and B. subtilis.     

Cloning, analysis and expression of the gene for β-glycosidase of Thermus caldophilus GK24 and properties of the enzyme

The gene (bglT) encoding Thermus caldophilus GK24 -glycosidase (Tca -glycosidase) was cloned and sequenced. The gene contains an open reading frame encoding 431 amino acids with a M _r of 48 658 Da. The bglT gene was expressed under the control of tac promoter on a high-copy plasmid in E. coli. The recombinant Tca -glycosidase was purified 41.5-fold with a 59% yield and a specific activity of 83 U mg^–1 protein.     

Expression in Escherichia coli of a cloned β-glucanase gene from Bacillus amyloliquefaciens

Summary The recombinant phage G1 has been identified by screening 700 plaques of a Charon 4A library, containing DNA of Bacillus amyloliquefaciens, for phage clones directing the hydrolysis of lichenan in Escherichia coli, as indicated by haloes surrounding plaques on lichenan agar. The gene coding for an endo--1.3–1.4-glucanase was recloned within a 3.6 kb EcoRI fragment into the EcoRI site of plasmid pBR322, in both orientations.The location and extent of the bgl gene on the 3.6 kb Bacillus DNA insert was estimated by insertion mutagenesis with transposon Tn5 and restriction mapping of Tn5 insertions within or near to the bgl gene.The -glucanase synthesized by E. coli harbouring plasmids pEG1 or pEG2 was shown to accumulate mainly in the periplasmic space but -glucanase activities were also detected extracellulary and in the cytoplasm. The molecular weight of the enzyme synthesized in E. coli harbouring pEG1 was estimated by SDS-polyacrylamide gel electrophoresis to be about 24000. It was shown that the level of bgl gene expression in E. coli varies about 10-fold, depending on the orientation of the 3.6 kb DNA-fragment cloned within the EcoRI site of pBR322. After insertion of HindIII-DNA fragments from phage into the HindIII site of the -glucanase-high-expression plasmid pEG1, we obtained clones also showing an approximately 10-fold reduction in -glucanase activites. It was thus concluded that on plasmid pEG1 the leftward acting Ap^r (PI) promotor of plasmid pBR322 strongly increases the expression in E. coli of the cloned B. amyloliquefaciens bgl gene.Abbreviations Ap ampicillin, Km, kanamycin - kd kilodalton - kb kilobase pairs - moi multiplicity of infection - pfu plaque forming units - SDS sodium dodecylsulphate - Tc tetracycline