A β-Mannanase from Bacillus subtilis B36: Purification, Properties, Sequencing, Gene Cloning and Expression in Escherichia coli

2006 ◽  
Vol 61 (11-12) ◽  
pp. 840-846 ◽  
Author(s):  
Ya Nan Li ◽  
Kun Meng ◽  
Ya Ru Wang ◽  
Bin Yao
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
Bacillus Subtilis ◽  
Specific Activity ◽  
Ph Value ◽  
High Specificity ◽  
Apparent Molecular Weight ◽  
Cloning And Expression ◽  
Gene Cloning And Expression ◽  
Reading Frame

Abstract MANB36, a secrete endo-β-1,4-D-mannanase produced by Bacillus subtilis B36, was puri­fied to homogeneity from a culture supernatant and characterized. The optimum pH value for the mannanase activity of MANB36 is 6.4 and the optimum temperature is 50 °C. The enzyme activity of MANB36 is remarkably thermostable at 60 °C and the specific activity of MANB36 is 927.84 U/mg. Metal cations (except Hg2+ and Ag+), EDTA and 2-mercaptoetha- nol (2-ME) have no effects on enzyme activity. This enzyme exhibits high specificity with the substituted galactomannan locust bean gum (LBG). The gene encoding for MANB36, manB36, was cloned by PCR and sequenced. manB36 contains a single open reading frame (ORF) consisting of 1104 bp that encodes a protein of 367 amino acids. The predicted mo­lecular weight of 38.13 kDa, calculated by the deduced protein of the gene manB36 without signal peptide, coincides with the apparent molecular weight of 38.0 kDa of the purified MANB36 estimated by SDS-PAGE. The mature protein of MANB36 has been expressed in Escherichia coli BL21 and the expressed mannanase has normal bioactivity.

scholarly journals A novel chitosanase from Bacillus sp. Strain K17: Gene cloning and expression in Escherichia coli

2002 ◽  
Vol 2 (1) ◽  
pp. 227-228 ◽  
Author(s):  
R. Yatsunami ◽  
Y. Sakihama ◽  
M. Suzuki ◽  
T. Fukazawa ◽  
S. Shimizu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gene Cloning ◽  
Cloning And Expression ◽  
Gene Cloning And Expression ◽  
Bacillus Sp ◽  
Expression In Escherichia Coli

scholarly journals Identification and Function of the pdxY Gene, Which Encodes a Novel Pyridoxal Kinase Involved in the Salvage Pathway of Pyridoxal 5′-Phosphate Biosynthesis in Escherichia coli K-12

1998 ◽  
Vol 180 (7) ◽  
pp. 1814-1821 ◽  
Author(s):  
Yong Yang ◽  
Ho-Ching Tiffany Tsui ◽  
Tsz-Kwong Man ◽  
Malcolm E. Winkler
Keyword(s):  
Escherichia Coli ◽  
De Novo ◽  
Specific Activity ◽  
Salvage Pathway ◽  
Pyridoxal Kinase ◽  
Triple Mutant ◽  
Reading Frame ◽  
E Coli ◽  
Specific Activities ◽  
K 12

ABSTRACT pdxK encodes a pyridoxine (PN)/pyridoxal (PL)/pyridoxamine (PM) kinase thought to function in the salvage pathway of pyridoxal 5′-phosphate (PLP) coenzyme biosynthesis. The observation that pdxK null mutants still contain PL kinase activity led to the hypothesis that Escherichia coli K-12 contains at least one other B6-vitamer kinase. Here we support this hypothesis by identifying the pdxY gene (formally, open reading frame f287b) at 36.92 min, which encodes a novel PL kinase. PdxY was first identified by its homology to PdxK in searches of the complete E. coli genome. Minimal clones of pdxY + overexpressed PL kinase specific activity about 10-fold. We inserted an omega cassette intopdxY and crossed the resultingpdxY::ΩKanr mutation into the bacterial chromosome of a pdxB mutant, in which de novo PLP biosynthesis is blocked. We then determined the growth characteristics and PL and PN kinase specific activities in extracts ofpdxK and pdxY single and double mutants. Significantly, the requirement of the pdxB pdxK pdxY triple mutant for PLP was not satisfied by PL and PN, and the triple mutant had negligible PL and PN kinase specific activities. Our combined results suggest that the PL kinase PdxY and the PN/PL/PM kinase PdxK are the only physiologically important B6vitamer kinases in E. coli and that their function is confined to the PLP salvage pathway. Last, we show thatpdxY is located downstream from pdxH (encoding PNP/PMP oxidase) and essential tyrS (encoding aminoacyl-tRNATyr synthetase) in a multifunctional operon.pdxY is completely cotranscribed with tyrS, but about 92% of tyrS transcripts terminate at a putative Rho-factor-dependent attenuator located in thetyrS-pdxY intercistronic region.

scholarly journals Escherichia coli S-adenosylhomocysteine/5′-methylthioadenosine nucleosidase. Purification, substrate specificity and mechanism of action

1985 ◽  
Vol 232 (2) ◽  
pp. 335-341 ◽  
Author(s):  
F Della Ragione ◽  
M Porcelli ◽  
M Cartenì-Farina ◽  
V Zappia ◽  
A E Pegg
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
Mechanism Of Action ◽  
Specific Activity ◽  
Maximal Rate ◽  
Purification Procedure ◽  
Protein Affinity ◽  
Naturally Occurring ◽  
Purine Ring ◽  
Ribose Moiety

S-Adenosylhomocysteine/5′-methylthioadenosine nucleosidase (EC 3.2.2.9) was purified to homogeneity from Escherichia coli to a final specific activity of 373 mumol of 5′-methylthioadenosine cleaved/min per mg of protein. Affinity chromatography on S-formycinylhomocysteine-Sepharose is the key step of the purification procedure. The enzyme, responsible for the cleavage of the glycosidic bond of both S-adenosylhomocysteine and 5′-methylthioadenosine, was partially characterized. The apparent Km for 5′-methylthioadenosine is 0.4 microM, and that for S-adenosylhomocysteine is 4.3 microM. The maximal rate of cleavage of S-adenosylhomocysteine is approx. 40% of that of 5′-methylthioadenosine. Some 25 analogues of the two naturally occurring thioethers were studied as potential substrates or inhibitors of the enzyme. Except for the analogues modified in the 5′-position of the ribose moiety or the 2-position of the purine ring, none of the compounds tested was effective as a substrate. Moreover, 5′-methylthioformycin, 5′-chloroformycin, S-formycinylhomocysteine, 5′-methylthiotubercidin and S-tubercidinylhomocysteine were powerful inhibitors of the enzyme activity. The results obtained allow the hypothesis of a mechanism of enzymic catalysis requiring as a key step the protonation of N-7 of the purine ring.

scholarly journals Properties of human testis-specific lactate dehydrogenase expressed from Escherichia coli

1991 ◽  
Vol 273 (3) ◽  
pp. 587-592 ◽  
Author(s):  
K M LeVan ◽  
E Goldberg
Keyword(s):  
Escherichia Coli ◽  
Lactate Dehydrogenase ◽  
Prokaryotic Expression ◽  
Specific Activity ◽  
Human Testis ◽  
Reading Frame ◽  
E Coli ◽  
Heat Stable ◽  
Prokaryotic Expression Vector ◽  
Tac Promoter

The cDNA encoding the C4 isoenzyme of lactate dehydrogenase (LDH-C4) was engineered for expression in Escherichia coli. The Ldh-c open reading frame was constructed as a cassette for production of the native protein. The modified Ldh-c cDNA was subcloned into the prokaryotic expression vector pKK223-3. Transformed E. coli cells were grown to mid-exponential phase, and induced with isopropyl beta-D-thiogalactopyranoside for positive regulation of the tac promoter. Induced cells expressed the 35 kDa subunit, which spontaneously formed the enzymically active 140 kDa tetramer. Human LDH-C4 was purified over 200-fold from litre cultures of cells by AMP and oxamate affinity chromatography to a specific activity of 106 units/mg. The enzyme was inhibited by pyruvate concentrations above 0.3 mM, had a Km for pyruvate of 0.03 mM, a turnover number (nmol of NADH oxidized/mol of LDH-C4 per min at 25 degrees C) of 14,000 and was heat-stable.

scholarly journals Bacillus subtilis phenylalanyl-tRNA synthetase genes: cloning and expression in Escherichia coli and B. subtilis.

1989 ◽  
Vol 171 (2) ◽  
pp. 1228-1232 ◽  
Author(s):  
A A Brakhage ◽  
H Putzer ◽  
K Shazand ◽  
R J Röschenthaler ◽  
M Grunberg-Manago
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Trna Synthetase ◽  
Cloning And Expression ◽  
Expression In Escherichia Coli

Gene Cloning and Expression of a Bacteroides Gingivalis-Specific Protein Antigen in Escherichia Coli

1988 ◽  
Vol 2 (2) ◽  
pp. 310-314 ◽  
Author(s):  
Y. Abiko ◽  
M. Hayakawa ◽  
H. Aoki ◽  
H. Takiguchi
Keyword(s):  
Escherichia Coli ◽  
Specific Protein ◽  
Phage Clone ◽  
Cloning And Expression ◽  
Protein Antigen ◽  
Periodontal Pathogen ◽  
Gene Cloning And Expression ◽  
Chromosomal Dna ◽  
Cell Extracts ◽  
Bacteroides Gingivalis

Gene banks of chromosomal DNA from Bacteroides gingival is 381 were constructed utilizing the bacteriophage replacement vector λCharon4A. A clone encoding a protein antigen from B. gingivalis was identified by Western-blot screening, with use of antiserum induced to extracts of B. gingivalis cells. DNA fragments from the phage clone were subcloned into the plasmid vector pACYC184 to yield an immunoreactive clone. Cell extracts from the subclone reacted with antiserum against B. gingivalis, but did not react with antisera to B. asaccharolyticus, B. intermedius, or B. melaninogenicus. The antiserum against the purified clone products reacted with N-lauryl sarcosine extracts from B. gingivalis cells, but did not react with those of other Bacteroides cells. In addition, human serum from periodontitis patients reacted with the clone product by Western electrophoretic transfer and immunoblotting analysis. These data suggest that the gene coding for a B. gingivalis-specific protein antigen was successfully cloned and functionally expressed in Escherichia coli. This clone product may prove useful for further studies of B. gingival is as a periodontal pathogen.

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