scholarly journals A New Thermoactive Pullulanase from Desulfurococcus mucosus: Cloning, Sequencing, Purification, and Characterization of the Recombinant Enzyme after Expression in Bacillus subtilis

2000 ◽  
Vol 182 (22) ◽  
pp. 6331-6338 ◽  
Author(s):  
Fiona Duffner ◽  
Costanzo Bertoldo ◽  
Jens T. Andersen ◽  
Karen Wagner ◽  
Garabed Antranikian
Keyword(s):  
Bacillus Subtilis ◽  
Divalent Cations ◽  
Culture Fluid ◽  
Amino Acid Identity ◽  
Recombinant Enzyme ◽  
Gene Encoding ◽  
Amylolytic Enzymes ◽  
Chromatography Analysis ◽  
The Stability ◽  
Ph Range

ABSTRACT The gene encoding a thermoactive pullulanase from the hyperthermophilic anaerobic archaeon Desulfurococcus mucosus (apuA) was cloned in Escherichia coli and sequenced. apuA from D. mucosusshowed 45.4% pairwise amino acid identity with the pullulanase fromThermococcus aggregans and contained the four regions conserved among all amylolytic enzymes. apuA encodes a protein of 686 amino acids with a 28-residue signal peptide and has a predicted mass of 74 kDa after signal cleavage. The apuAgene was then expressed in Bacillus subtilis and secreted into the culture fluid. This is one of the first reports on the successful expression and purification of an archaeal amylopullulanase in a Bacillus strain. The purified recombinant enzyme (rapuDm) is composed of two subunits, each having an estimated molecular mass of 66 kDa. Optimal activity was measured at 85°C within a broad pH range from 3.5 to 8.5, with an optimum at pH 5.0. Divalent cations have no influence on the stability or activity of the enzyme. RapuDm was stable at 80°C for 4 h and exhibited a half-life of 50 min at 85°C. By high-pressure liquid chromatography analysis it was observed that rapuDm hydrolyzed α-1,6 glycosidic linkages of pullulan, producing maltotriose, and also α-1,4 glycosidic linkages in starch, amylose, amylopectin, and cyclodextrins, with maltotriose and maltose as the main products. Since the thermoactive pullulanases known so far from Archaeaare not active on cyclodextrins and are in fact inhibited by these cyclic oligosaccharides, the enzyme from D. mucosus should be considered an archaeal pullulanase type II with a wider substrate specificity.

scholarly journals Transcriptional Control of the Low-Temperature-Inducible des Gene, Encoding the Δ5 Desaturase of Bacillus subtilis

1999 ◽  
Vol 181 (22) ◽  
pp. 7028-7033 ◽  
Author(s):  
Pablo S. Aguilar ◽  
Paloma Lopez ◽  
Diego de Mendoza
Keyword(s):  
Bacillus Subtilis ◽  
Transcriptional Control ◽  
Unsaturated Fatty Acids ◽  
Functional Expression ◽  
Mrna Levels ◽  
Gene Encoding ◽  
Major Mechanism ◽  
Temperature Downshift ◽  
The Stability

ABSTRACT The Bacillus subtilis des gene encodes the cold-inducible Δ5 lipid desaturase involved in the formation of unsaturated fatty acids from saturated phospholipid precursors. Here, we describe the expression pattern of the des gene in response to a temperature downshift from 37 to 20°C. We found that the synthesis of des mRNA is undetectable at 37°C but dramatically induced upon the temperature downshift. Decay characteristics of the des transcript as well as the in vivo decay of B. subtilis bulk mRNA were investigated. The results showed that the stability of the des transcript as well as of bulk mRNA lasted substantially longer at 20°C than at 37°C. Functional expression of des at 37°C was achieved by exchanging its promoter with the non-cold shock spacpromoter. These data provide the first direct evidence that temperature-mediated control of transcription is the major mechanism regulating the mRNA levels of the B. subtilis desaturase. The present results also demonstrate that the only component of the desaturation system regulated by temperature is the desaturase enzyme.

Pullulanase Type I from Fervidobacterium pennavorans Ven5: Cloning, Sequencing, and Expression of the Gene and Biochemical Characterization of the Recombinant Enzyme

1999 ◽  
Vol 65 (5) ◽  
pp. 2084-2091 ◽  
Author(s):  
Costanzo Bertoldo ◽  
Fiona Duffner ◽  
Per L. Jorgensen ◽  
Garabed Antranikian
Keyword(s):  
Amino Acids ◽  
Biochemical Characterization ◽  
Signal Sequence ◽  
Thermotoga Maritima ◽  
Amino Acid Identity ◽  
Type I ◽  
Start Site ◽  
Gene Encoding ◽  
Amylolytic Enzymes ◽  
E Coli

ABSTRACT The gene encoding the type I pullulanase from the extremely thermophilic anaerobic bacterium Fervidobacterium pennavorans Ven5 was cloned and sequenced in Escherichia coli. The pulA gene from F. pennavoransVen5 had 50.1% pairwise amino acid identity with pulA from the anaerobic hyperthermophile Thermotoga maritima and contained the four regions conserved among all amylolytic enzymes. The pullulanase gene (pulA) encodes a protein of 849 amino acids with a 28-residue signal peptide. The pulA gene was subcloned without its signal sequence and overexpressed in E. coli under the control of the trc promoter. This clone, E. coli FD748, produced two proteins (93 and 83 kDa) with pullulanase activity. A second start site, identified 118 amino acids downstream from the ATG start site, with a Shine-Dalgarno-like sequence (GGAGG) and TTG translation initiation codon was mutated to produce only the 93-kDa protein. The recombinant purified pullulanases (rPulAs) were optimally active at pH 6 and 80°C and had a half-life of 2 h at 80°C. The rPulAs hydrolyzed α-1,6 glycosidic linkages of pullulan, starch, amylopectin, glycogen, α-β-limited dextrin. Interestingly, amylose, which contains only α-1,4 glycosidic linkages, was not hydrolyzed by rPulAs. According to these results, the enzyme is classified as a debranching enzyme, pullulanase type I. The extraordinary high substrate specificity of rPulA together with its thermal stability makes this enzyme a good candidate for biotechnological applications in the starch-processing industry.

Schiff base equilibria. II. Beryllium complexes of N-n-butylsalicylideneimine and the hydrolysis of the Be2+ ion

1965 ◽  
Vol 18 (5) ◽  
pp. 651 ◽  
Author(s):  
RW Green ◽  
PW Alexander
Keyword(s):  
Aqueous Solution ◽  
Schiff Base ◽  
Complex Formation ◽  
Distribution Coefficient ◽  
Dilute Aqueous Solution ◽  
The Stability ◽  
Ph Range ◽  
Hydrolysis Of ◽  
Beryllium Complexes ◽  
Equilibria In Aqueous Solution

The Schiff base, N-n-butylsalicylideneimine, extracts more than 99.8% beryllium into toluene from dilute aqueous solution. The distribution of beryllium has been studied in the pH range 5-13 and is discussed in terms of the several complex equilibria in aqueous solution. The stability constants of the complexes formed between beryllium and the Schiff base are log β1 11.1 and log β2 20.4, and the distribution coefficient of the bis complex is 550. Over most of the pH range, hydrolysis of the Be2+ ion competes with complex formation and provides a means of measuring the hydrolysis constants. They are for the reactions: Be(H2O)42+ ↔ 2H+ + Be(H2O)2(OH)2, log*β2 - 13.65; Be(H2O)42+ ↔ 3H+ + Be(H2O)(OH)3-, log*β3 -24.11.

scholarly journals Investigations of ternary complexes of Co(II) and Ni(II) with thiodiacetate anion and 1,10-phenanthroline or 2,2’-bipyridine in aqueous solutions

2014 ◽  
Vol 13 (1) ◽  
Author(s):  
Dariusz Wyrzykowski ◽  
Joanna Pranczk ◽  
Dagmara Jacewicz ◽  
Aleksandra Tesmar ◽  
Bogusław Pilarski ◽  
...  
Keyword(s):  
Aqueous Solutions ◽  
Substitution Reactions ◽  
Experimental Conditions ◽  
Kinetic Measurements ◽  
Hydroxo Complexes ◽  
Binary Complexes ◽  
Vis Spectroscopy ◽  
Potentiometric Titration Method ◽  
The Stability ◽  
Ph Range

AbstractA potentiometric titration method (PT) and a stopped-flow kinetic technique monitored by a UV−Vis spectroscopy have been used to characterize the stability of series of Co(II)- and Ni(II)-thiodiacetato complexes, M(TDA), in the presence of 1,10-phenanthroline (phen) or 2,2’-bipyridine (bipy) in aqueous solutions. The stability constants of the binary (1:1), ternary (1:1:1) as well as the resulting hydroxo complexes were evaluated and compared to the corresponding oxydiacetate complexes. Based on the species distribution as a function of pH the relative predominance of the species in the system over a pH range was discussed. Furthermore, the kinetic measurements of the substitution reactions of the aqua ligands to phen or bipy in the coordination sphere of the binary complexes M(TDA) were performed in the 288–303 K temperature range, at a constant concentration of phen or bipy and at seven different concentrations of the binary complexes (0.2–0.5 mM). The kinetic stability of the M(TDA) complexes was discussed in relation to the experimental conditions and the kind of the auxiliary ligands (phen/bipy). Moreover, the influence of the type of primary ligand (thiodiacetate/oxydiacetate) on the substitution rate of the auxiliary ligands was also compared.

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