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1.
Isolation and characterization of a type II restriction endonuclease from Streptococcus thermophilus
The alpha-toxin (phospholipase C) of Clostridium perfringens has been reported to contain catalytically essential zinc ions. We report here that histidine residues are essential for the co-ordination of these ion(s). Incubation of alpha toxin with diethylpyrocarbonate, a histidine modifying reagent, did not result in the loss of phospholipase C activity unless the protein was first incubated with EDTA, suggesting that zinc ions normally protect the susceptible histidine residues. When the amino acid sequences of three phospholipase C's were aligned, essential zinc binding histidine residues in the non-toxic B. cereus phospholipase C were found in similar positions in the toxic C. perfringens enzyme and the weakly toxic C. bifermentans phospholipase C. 相似文献
2.
3.
The Escherichia coli DNA-dependent RNA polymerase (RPase) holoenzyme (alpha 2 beta beta' sigma) possesses 2 mol equiv of Zn: beta and beta' subunits each contain one Zn ion. An in vitro metal-substitution method developed earlier (method I) was used to remove the two intrinsic Zn ions and then to reconstitute other metal ions into the beta subunit of RPase. One Cd or Hg ion was successfully reconstituted into half-active enzymes (rec-Cd1- or rec-Hg1-RPase), while Mn or Ni ion was not incorporated. A new, simplified in vitro metal-substitution method (method II), which omitted the low-pH treatment and subsequent urea dialysis in method I, was devised in this study. Consequently, Zn or Cd could be incorporated into both the beta and beta' subunits, resulting in rec-Zn2- or rec-Cd2-RPase, respectively. However, only one Hg was incorporated, probably due to steric hindrance by the large size of the Hg ion, while Mn, Ni, or Cr was not bound by the reconstituted enzyme, which instead incorporated only one Zn. Analysis of the metal content of various reconstituted RPases indicated that without low-pH treatment Zn bound to both the beta and beta' subunits when Zn concentrations were higher than 2 X 10(-6)M, but it bound only to the beta' subunit at lower concentrations. Moreover, low-pH treatment destroys the metal binding site in the beta' subunit. The metal sites on the beta and beta' subunits did not have significant affinity for the transition metals such as Mn, Ni, and Cr.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
4.
George A. Somkuti Daniel K. Y. Solaiman Dennis H. Steinberg 《Biotechnology letters》1993,15(8):773-778
Summary The streptococcal cloning vector pIL253 (4.96-kbp, Emr) was used to introduce the Streptomyces antibioticus tyrosinase (mel) gene (1.56-kbp) into S. thermophilus, an important microbe in dairy fermentations. Electrotransformants of S. thermophilus ST128 contained 6.51-kbp recombinant plasmids which probed positively in Southern hybridizations with the biotin-labeled mel fragment. Western blots of cell extracts resolved by SDS-PAGE showed the presence of a ca. 31-kDa band thus confirming the synthesis of tyrosinase protein by genetic transformants. 相似文献
5.
A 63-base-pair synthetic promoter, sP1, was synthesized on the basis of the nucleotide sequence of a putative Streptococcus thermophilus promoter. When inserted upstream from the Streptomyces cho operon in a recombinant plasmid, pUCO195P-36, sP1 activated the expression of the cho genes in Escherichia coli, as shown by the production of cholesterol oxidase by the transformants. The sP1-driven cholesterol oxidase production in pUCO195P-36-transformed cells was estimated to be 40% of that produced by Plac-mediated cho expression in a pUCO193-containing host. The recombinant pUCO195P-36 appeared to be segregationally less stable in E. coli DH5 than in HB101. Its non-expressing counterpart, pUCO195P-1, was stable in both E. coli strains. The activity of sP1 was further demonstrated in E. coli by the expression of a Streptomyces melC operon. When placed upstream from the test operon in the pMCU22aPa construct, sP1 activated the melC expression as shown by the production of tyrosinase at (3.0±0.3) × 10–3 U/mg and (16.0±1.0) × 10–3 U/mg protein equivalent of cell extract in the absence and presence of isopropyl -d-thio-galactopyranoside, respectively. The presence of a counter-oriented Plac at the 3 end of the operon in the pMCU22bPa plasmid reduced the sP1-mediated tyrosinase production by about 85%.Mention of brand or firm names does not constitute an endoresement by the U.S. Department of Agriculture over others of a similar nature not mentioned 相似文献
6.
Daniel K. Y. Solaiman George A. Somkuti 《Journal of industrial microbiology & biotechnology》1991,8(4):253-258
Summary A streptomycete gene coding for extracellular cholesterol oxidase (choA) was subcloned and expressed inEscherichia coli. The pUCO series recombinants were obtained by inserting thechoA gene into the uniqueKpnI site of pUC19 vector. Expression was observed with pUCO192A and pUCO193 constructs in which the cloned gene(s) were aligned with the upstreamlacZ promoter. Isopropyl -d-thioglucopyranoside (IPTG) enhanced this expression up to 2.5-fold. Specific Cho activity in the cell extracts of the stable pUCO193 transformant were 0.004 U and 0.007 U per mg protein without and with IPTG induction, respectively. Cho activity was detected in the spent medium of this culture, suggesting possible secretion of the enzyme. 相似文献
7.
Summary Bifunctional shuttle vector (pBN183) and recombinant plasmids (pDCO2 and pDCO3) carrying aStreptomyces cholesterol oxidase gene (choA) were deleted to varying degrees inStreptococcus thermophilus. Restriction mapping of the deleted plasmids led to the identification of deletion prone regions in the transforming DNAs. Sequence analysis revealed that direct repeats and hairpin structures occurred in these regions, suggesting that they are deleterious to the stability of plasmids inS. thermophilus. 相似文献
8.
Pseudomonas resinovorans phaC1
Pre
and phaC2
Pre
genes coding for poly(hydroxyalkanoate) (PHA) synthases were cloned by PCR and expressed in E. coli LS1298 (fadB). Repeat-unit composition analysis showed that -hydroxydecanoate (67–75 mol%) and -hydroxyoctanoate (25–33 mol%) are the major monomers of the PHA produced in cells grown on decanoate. Sequence analysis showed that the gene products of phaC1
Pre
and phaC2
Pre
had 61% identical (75% positive) amino-acid sequence matches, and both sequences contained a conserved /-hydrolase fold in the carboxy-terminal portion of the proteins. Switching the /-hydrolase folds of phaC1
Pre
and phaC2
Pre
yielded chimeric pha7 and pha8 genes that afforded PHA synthesis in E. coli LS1298. The repeat-unit compositions of PHA in cells containing pha7 and pha8 were similar to those found in transformants containing the parental genes. Deletion mutants of phaC1
Pre
and phaC2
Pre
that resulted in potential translational fusions also supported PHA synthesis with similar repeat-unit compositions. Chimeric genes obtained from the switching of fragments containing the /-hydrolase folds of phaC1
Pre
and Ralstonia eutropha phbC did not direct the synthesis of PHA in transformed cells. 相似文献
9.
Daniel K.Y. Solaiman 《Biotechnology Techniques》1998,12(11):829-832
An electroporation procedure for the transformation of Pseudomonas oleovorans was developed using a model plasmid, pCN51. The optimal electrotransformation was achieved with cells harvested at 45 to 60 min of growth and concentrated to a cell density of 5 OD600nm, plasmid concentration of 6 g per 100 l of cell suspension, and a 0.1-cm gap-width cuvette. Electroporation was performed at the settings of 250 , 25F and 2.5 kV. Transformation yields in the order of 103 colony-forming-unit per electroporation sample were obtained. This is a first report of the electroporation of the commercially valuable bacterium Ps. oleovorans. © Rapid Science Ltd. 1998 相似文献
10.
High‐titer production and strong antimicrobial activity of sophorolipids from Rhodotorula bogoriensis
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Rhodotorula bogoriensis produces sophorolipids (SLs) that contain 13‐hydroxydocosanoic acid (OH‐C22) as the lipid moiety. A systematic study was conducted to further understand the fermentative production of SLs containing OH‐C22 (C22‐SL) by R. bogoriensis. Shake‐flask studies showed that R. bogoriensis consumed glucose at a slow pace. HPLC analysis of the C22‐SL products from shake‐flask fermentations at different glucose concentrations showed a correlation between glucose depletion and the extent of C22‐SL deacetylation. A large‐scale bioreactor fermentation resulted in the isolation of C22‐SL at a volumetric product yield of 51 g/L. HPLC analysis of C22‐SL product from the bioreactor fermentation corroborated the finding that glucose depletion correlated with extensive deacetylation of C22‐SL. The antimicrobial activity of C22‐SL was established for the first time to be stronger than the C18‐SL from Candida bombicola against Propionibacterium acnes in a plate assay. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:867–874, 2015 相似文献