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1.
The acylneuraminate lyase gene from Clostridium perfringens A99 was cloned on a 3.3 kb HindIII DNA fragment identified by screening the chromosomal DNA of this species by hybridization with an oligonucleotide probe
that had been deduced from the N-terminal amino acid sequence of the purified protein, and another probe directed against
a region that is conserved in the acylneuraminate lyase gene of Escherichia coli and in the putative gene of Clostridium tertium. After cloning, three of the recombinant clones expressed lyase activity above the background of the endogenous enzyme of
the E. coli host. The sequenced part of the cloned fragment contains the complete acylneuraminate lyase gene (ORF2) of 864 bp that encodes
288 amino acids with a calculated molecular weight of 32.3 kDa. The lyase structural gene follows a non-coding region with
an inverted repeat and a ribosome binding site. Upstream from this regulatory region another open reading frame (ORF1) was
detected. The 3′-terminus of the lyase structural gene is followed by a further ORF (ORF3). A high homology was found between
the amino acid sequences of the sialate lyases from Clostridium perfringens and Haemophilus influenzae (75% identical amino acids) or Trichomonas vaginalis (69% identical amino acids), respectively, whereas the similarity to the gene from E. coli is low (38% identical amino acids). Based on our new sequence data, the ‘large’ sialidase gene and the lyase gene of C. perfringens are not arranged next to each other on the chromosome of this species.
This revised version was published online in November 2006 with corrections to the Cover Date. 相似文献
2.
The first complete primary structure of a mammalian sialate-pyruvate lyase, namely of the enzyme from porcine kidney, was elucidated by a combination of different PCR techniques followed by sequencing of the resulting fragments. The primers used were either deduced from four porcine lyase peptides or from an alignment of human and mouse expressed sequence tags (ESTs), which were found to be homologous to already known microbial lyase sequences, and cDNA alone or after ligation with a plasmid vector served as a template. The lyase primary structure consists of 319 amino acids with a calculated protein molecular mass of approximately 35 kDa, which fits well to the value determined for the native enzyme. The porcine lyase sequence made it possible to assemble several ESTs from mouse and man in order to obtain the complete putative lyase genes. The three mammalian sequences reveal a high degree of homology both on the nucleotide (83% of the nucleotides are identical between all three sequences) and on the amino-acid level (72% of the amino acids are identical between all three sequences), and thus form a tightly related group. In contrast, the identity between the lyase primary structures from pig kidney and the microbial enzyme from Clostridium perfringens is much less pronounced (25%). Thirty-one amino acids were found to be absolutely conserved in all lyase sequences. Among them are two amino acids (lysine 173 and tyrosine 143 in the porcine lyase) that are most important for the catalytic reaction. After expression cloning, recombinant enzyme activity was expressed in Escherichia coli BL21(DE3)pLysS, which confirms the identity of the cloned sequence and verifies one of the putative human and murine sequences. After SDS/PAGE of a cell extract of the expression clone, a band of 35kDa was stained on the gel. 相似文献
3.
Yasuhiro Ohta Makoto Shimosaka Kousaku Murata Yoji Tsukada Akira Kimura 《Applied microbiology and biotechnology》1986,24(5):386-391
Summary The gene for N-acetylneuraminate lyase [N-acetylneuraminate pyruvate-lyase; NPL] of Escherichia coli C600 was cloned onto pBR322 as a 9.8 kilobase HindIII fragment of chromosomal DNA and the hybrid plasmid was designated pMK2. The gene in the hybrid plasmid was subcloned in pBR322 as a 1.2 kilobase HindIII-EcoRI fragment and the resultant hybrid plasmid was designated pMK6. NPL activity level was increased more than 5-fold in the pMK6-bearing strain compared with that of the wild type, when the cells were grown on a medium containing inducer (N-acetylneuraminate: NANA). The transformants harbouring pMK6 also showed higher activity even in the absence of inducer. The NPL produced by pMK6-bearing cells was structurally and immunologically the same as that purified from E. coli C600. 相似文献
4.
Enzymes implicated in cysteine and methionine metabolism such as cystathionine β‐lyase (CBL; EC 4.4.1.8), a pyridoxal‐5′‐phosphate (PLP)‐dependent carbon–sulfur lyase, have been shown to play a central role in the generation of sulfur compounds. This work describes the unprecedented cloning and characterization of the metC‐cystathionine β‐lyase from the axillary‐isolated strain Staphylococcus haemolyticus AX3, in order to determine its activity and its involvement in amino acid biosynthesis, and in the generation of sulfur compounds in human sweat. The gene contains a cysteine/methionine metabolism enzyme pattern, and also a sequence capable to effect β‐elimination. The recombinant enzyme was shown to cleave cystathionine into homocysteine and to convert methionine into methanethiol at low levels. No odor was generated after incubation of the recombinant enzyme with sterile human axillary secretions; sweat components were found to have an inhibitory effect. These results suggest that the generation of sulfur compounds by Staphylococci and the β‐lyase activity in human sweat are mediated by enzymes other than the metC gene or by the concerted activities of more than one enzyme. 相似文献
5.
The ICL1 gene encoding isocitrate lyase was cloned from the dimorphic fungus Yarrowia lipolytica by complementation of a mutation (acuA3) in the structural gene of isocitrate lyase of Escherichia coli. The open reading frame of ICL1 is 1668 by long and contains no introns in contrast to currently sequenced genes from other filamentous fungi. The ICL1 gene encodes a deduced protein of 555 amino acids with a molecular weight of 62 kDa, which fits the observed size of the purified monomer of isocitrate lyase from Y. lipolytica. Comparison of the protein sequence with those of known pro- and eukaryotic isocitrate lyases revealed a high degree of homology among these enzymes. The isocitrate lyase of Y. lipolytica is more similar to those from Candida tropicalis and filamentous fungi than to Sacharomyces cerevisiae. This enzyme of Y. lipolytica has the putative glyoxysomal targeting signal S-K-L at the carboxy-terminus. It contains a partial repeat which is typical for eukaryotic isocitrate lyases but which is absent from the E. coli enzyme. Surprisingly, deletion of the ICL1 gene from the genome not only inhibits the utilization of acetate, ethanol, and fatty acids, but also reduces the growth rate on glucose. 相似文献
6.
《Bioscience, biotechnology, and biochemistry》2013,77(2):214-220
PmHNL, a hydroxynitrile lyase from Japanese apricot ume (Prunus mume) seed was purified to homogeneity by ammonium sulfate fractionation and chromatographic steps. The purified enzyme was a monomer with molecular mass of 58 kDa. It was a flavoprotein similar to other hydroxynitrile lyases of the Rosaceae family. It was active over a broad temperature, and pH range. The N-terminal amino acid sequence (20 amino acids) was identical with that of the enzyme from almond (Prunus dulcis). Based on the N-terminal sequence of the purified enzyme and the conserved amino acid sequences of the enzymes from Pr. dulcis, inverse PCR method was used for cloning of a putative PmHNL (PmHNL2) gene from a Pr. mume seedling. Then the cDNA for the enzyme was cloned. The deduced amino acid sequence was found to be highly similar (95%) to that of an enzyme from Pr. serotina, isozyme 2. The recombinant Pichia pastoris transformed with the PmHNL2 gene secreted an active enzyme in glycosylated form. 相似文献
7.
Snehasish Basu Arunava Roy Abhrajyoti Ghosh Amit Bera Dhrubajyoti Chattopadhyay Krishanu Chakrabarti 《Biodegradation》2011,22(1):153-161
After 24 h of incubation with only purified pectate lyase isolated from Bacillus pumilus DKS1 (EF467045), the weight loss of the ramie fibre was found to be 25%. To know the catalytic residue of pectate lyase the
pel gene encoding a pectate lyase from the strain Bacillus pumilus DKS1 was cloned in E. coli XL1Blue and expressed in E. coli BL21 (DE3) pLysS. The pel gene was sequenced and showed 1032 bp length. After purification using CM-Sepharose the enzyme showed molecular weight of
35 kDa and maximal enzymatic activity was observed at 60°C and a pH range of 8.5–9.0. Both Ca2+ and Mn2+ ions were required for activity on Na-pectate salt substrates, while the enzyme was strongly inhibited by Zn2+ and EDTA. The deduced nucleotide sequence of the DKS1 pectate lyase (EU652988) showed 90% homology to pectate lyases from
Bacillus pumilus SAFR-032 (CP000813). The 3D structure as well as the catalytic residues was predicted using EasyPred software and Catalytic
Site Atlas (CSA), respectively. Site directed mutagenesis confirmed that arginine is an essential catalytic residue of DKS1
pectate lyase. 相似文献
8.
Chuan He Hisashi Muramatsu Shin-ichiro Kato 《Bioscience, biotechnology, and biochemistry》2017,81(11):2145-2151
Ulvan is a sulfated polysaccharide found in the cell wall of the green algae Ulva. We first isolated several ulvan-utilizing Alteromonas sp. from the feces of small marine animals. The strain with the highest ulvan-degrading activity, KUL17, was analyzed further. We identified a 55-kDa ulvan-degrading protein secreted by this strain and cloned the gene encoding for it. The deduced amino acid sequence indicated that the enzyme belongs to polysaccharide lyase family 24 and thus the protein was named ulvan lyase. The predicted molecular mass of this enzyme is 110 kDa, which is different from that of the identified protein. By deletion analysis, the catalytic domain was proven to be located on the N-terminal half of the protein. KUL17 contains two ulvan lyases, one long and one short, but the secreted and cleaved long ulvan lyase was demonstrated to be the major enzyme for ulvan degradation. 相似文献
9.
Cells of Clostridium sporosphaeroides which were grown on citrate contained citrate lyase and citrate lyase acetylating enzyme, but no detectable citrate synthase and citrate lyase deacetylase activities. Citrate lyase from C. sporosphaeroides was purified to homogeneity as judged by polyacrylamide gel electrophoresis and high performance liquid chromatography. In contrast to the enzyme from Clostridium sphenoides, the addition of l-glutamate was not necessary for activity and stabilization of the enzyme. The purified enzyme had a specific activity of 34 U/mg protein and was comparable to other citrate lyases with respect to its molecular weight and subunit composition. Electron microscopic investigations showed that similar to the lyase from C. sphenoides and in contrast to all other citrate lyases examined so far, the majority of the enzyme molecules was present in star form. 相似文献
10.
Summary Hydroperoxide lyase converts fatty acid hydroperoxides to oxo-fatty acids which, after further oxidation, are suitable for the synthesis of higher polyamides. An improved method utilizing an acetone powder step for isolation of this enzyme from the unicellular alga Chlorella was developed. Using this procedure a five fold increase in hydroperoxide lyase activity from Chlorella pyrenoidosa was obtained compared to previously used extraction methods. Other Chlorella species were assayed, and it was found that C. fusca also contained significant hydroperoxide lyase activity. 相似文献
11.
Yusuf Abou-Jawdah Hagop Atamian Georges Nemer Linda Kfoury Nancy Choukrallah Lucia Hanna 《Biocontrol Science and Technology》2008,18(6):573-581
A Lebanese isolate of a Beauveria species originally isolated from a hymenopterous insect pest was found to be very effective against the pine processionary moth Thaumetopoea wilkinsoni. In laboratory bioassays, high mortality rates of the five larval stages were attained with spore concentrations of about 5000–50,000 spores/larva. The efficacy of kill on the first three larval stages was similar to the growth regulator diflubenzuron, but it was significantly higher on the fourth and fifth larval stages. Sequences of the DNA lyase gene and the EF-1α gene were used for molecular characterisation of this Beauveria isolate. The DNA lyase gene showed more polymorphism than the previously reported ITS region and EF-1α gene. This constitutes the first report on the possibility of using the DNA lyase gene as a molecular tool in fungal taxonomy. 相似文献
12.
Plants are sensitive to D-serine, but functional expression of the dsdA gene, encoding D-serine ammonia lyase, from Escherichia coli can alleviate this toxicity. Plants, in contrast to many other organisms, lack the common pathway for oxidative deamination of D-amino acids. This difference in metabolism has major consequences for plant responses to D-amino acids, since several D-amino acids are toxic to plants even at relatively low concentrations. Therefore, introducing an enzyme specific for a phytotoxic D-amino acid should generate a selectable characteristic that can be screened. Here we present the use of the dsdA gene as a selectable marker for transformation of Arabidopsis. D-serine ammonia lyase catalyses the deamination of D-serine into pyruvate, water and ammonium. dsdA transgenic seedlings can be clearly distinguished from wild type, having an unambiguous phenotype immediately following germination when selected on D-serine containing medium. The dsdA marker allows flexibility in application of the selective agent: it can be applied in sterile plates, in foliar sprays or in liquid culture. Selection with D-serine resistance was compared with selection based on kanamycin resistance, and was found to generate similar transformation frequencies but also to be more unambiguous, more rapid and more versatile with respect to the way the selective agent can be supplied. 相似文献
13.
Klebsiella pneumoniae genes for citrate lyase and citrate lyase ligase: localization, sequencing, and expression 总被引:2,自引:0,他引:2
In the course of studies on anaerobic citrate metabolism in Klebsiella pneumoniae, the DNA region upstream of the gene for the sodium-dependent citrate carrier (dtS) was investigated. Nucleotide sequence analysis revealed a cluster of five new genes that were oriented inversely to citS and probaby form an operon. The genes were named citCDEFG. Based on known protein sequence data, the gene products derived from citD, citE and citF could be identified as the λ-, β-, and α-subunits of citrate lyase, respectively. This enzyme catalyses the cleavage of citrate to oxaloacetate and acetate. The gene product derived from citC (calculated Mr 36476) exhibited no obvious similarity to other proteins. In the presence of acetate and ATP, cell extracts from a citC-expressing Escherichia coli strain were able to reactivate purified citrate lyase from K. pneumoniae that had been inactivated by chemical deacetylation of the prosthetic group. This represents 5-phosphoribosyi-dephospho-acetyl-coenzyme A which is covalently bound to serine-14 of the acyl carrier protein (λ-subunit). CitC was thus identified as acetate:SH-citrate lyase ligase. The function of the gene product derived from citG (Mr 32 645) has not yet been identified. Expression of the CitCDEFG gene cluster in E. coli led to the formation of citrate lyase which was active only in the presence of acetyl-coenzyme A, a compound known to substitute for the prosthetic group. These and other data strongly indicated that the enzyme synthesized in E. coli lacked its prosthetic group. Thus, additional genes besides citCDEFG appear to be required for the formation of holo-citrate lyase. 相似文献
14.
15.
The alyPEEC gene encoding alginate lyase from marine bacterium Pseudoalteromonas elyakovii IAM 14594 was subcloned into pBAD24 with arabinose promoter and sequenced, and overexpressed in TOP10 strain of E. coli after arabinose induction. Expression levels of alyPEEC gene in E. coli cells were over 39.6-fold higher than those in P. elyakovii IAM 14594 cells. The molecular mass of purified alginate lyase from the engineered E. coli cells was estimated to be 32.0 kDa. Optimum pH and temperature of the alginate lyase activity were 7.0 and 30 °C, respectively.
The enzyme was unstable on heating and in acidic and alkaline solution. The enzyme activity was stimulated by the MgCl2, NaCl, KCl, CaCl2, BaCl2 and MnCl2, but was inhibited by the addition of 1.0 mM of EGTA, EDTA, SDS, ZnSO4, AgNO3, and CoCl2. All the alginate, polyM and polyG could be converted into oligosaccharides with more than tetrasaccharides by the purified
recombinant alginate lyase, suggesting that the recombinant alginate lyase produced by the engineered E. coli has highly potential application in seaweed genetics, food and pharmaceutical industries. 相似文献
16.
G. A. Eroshenko G. N. Odinokov L. M. Kookleva N. Yu. Shavina V. V. Kutyrev 《Russian Journal of Genetics》2011,47(10):1181-1187
Comparative analysis of nucleotide sequences of genes participating in melibiose fermentation and isocitrate lyase production
was conducted in 90 natural Yersinia pestis strains of main and nonmain subspecies. It was ascertained that the lack of the ability to utilize disaccharide melibiose
in strains of the main subspecies is caused by integration of the insertion sequence IS285 at 73 bp from the beginning of
the structural gene melB that encodes the transport protein galactoside permease. In contrast, strains of nonmain subspecies (caucasica, altaica, hissarica and ulegeica) contain the intact gene melB and are capable of fermenting melibiose. Differences in the manifestation of the other differential trait, production of
isocitrate lyase, are connected with the presence in strains of the main species of mutation (insertion of two nucleotides
+CC) in the regulatory gene iclR encoding repressor protein of the acetate operon, which is the reason for constitutive synthesis of this enzyme. Strains
of nonmain subspecies do not contain mutations in gene iclR, and this correlates in these strains with their capacity for inducible synthesis of isocitrate lyase. 相似文献
17.
Cardoso PG Ribeiro JB Teixeira JA de Queiroz MV de Araújo EF 《Journal of industrial microbiology & biotechnology》2008,35(3):159-166
The pectin lyase (PL) is an industrially important enzyme since it is used for maceration and clarification in the process
of fruit juice production in food industries. In order to increase the yields of pectin lyase we cloned the plg1 (pectin lyase 1) from Penicillium griseoroseum gene under the control of the strong constitutive promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (gpdA) and the terminator region of the tryptophan synthetase (trpC) gene from Aspergillus nidulans (plasmid pAN52-Plg1) and transformed this construct into the P. griseoroseum strain PG63. One of the pAN52-Plg1 multi-copy transformants (strain 105) grown in culture medium containing glucose or sugar
cane juice showed PL activities of 4,804 or 5,202 U ml−1 respectively, which represented 57- and 132-fold increases. In addition, the apparent specific activity of PL produced by
this strain was much higher than the one observed for a commercial pectinase preparation. Evaluation of the extracellular
proteins in the culture supernatant of strain 105 by SDS-PAGE showed the presence of a clear and strong band of approximately
40 kDa that probably corresponds to PL. The enzyme yields reported here demonstrate that the system we developed is able to
express pectin lyase at levels comparable to, or exceeding, previously reported data. 相似文献
18.
Summary An extracellular endopolygalacturonate lyase of Cytophaga johnsonii was purified from the culture filtrate. It appeared to be homogeneous as judged by polyacrylamide gel electrophoresis at pH 8.6 as well as pH 4.3. The purified enzyme had a pH optimum around 9.0 and required Ca++ ions for its maximum activity. The apparent K
mfor polygalacturonic acid was found to be 0.22%. Both paper and column chromatography indicated formation and accumulation of an unsaturated monomer along with unsaturated di-, tri-, tetra- and pentamers from polygalacturonic acid by the enzyme action, indicating that the enzyme cleaved the substrate randomly in a non-hydrolytic manner. The glycosidic linkage next to the non-reducing end of polygalacturonic acid was not resistant to attack by this enzyme unlike in other known polygalacturonate lyases.Abbreviations PG lyase
Polygalacturonate lyase
- Tris
Tris (hydroxymethyl) aminomethane 相似文献
19.
R. F. Matagne 《Molecular & general genetics : MGG》1978,160(1):95-99
Summary In Chlamydomonas reinhardi, the arg-7 cistron is the structural gene for the enzyme argininosuccinate lyase which catalyzes the last reaction in the biosynthesis of arginine.Fourteen mutants (nine previously analyzed and five new mutants) defective in the lyase have been investigated so far: they all map within a cistron (length: 1.0–1.6 recombination units) of the linkage group I and fall within six groups of complementation. The enzyme activity found in the diploids formed by intragenic complementation was always lower than in wild-type haploid or diploid strains. The study of the denaturation curves obtained by heat treatment of the lyase indicates that in some diploids, several enzyme varieties can be present.These results and those previously obtained with diploids formed by intragenic and intergenic complementation (Matagne and Loppes, 1972; Matagne, 1976) are discussed in relation to the recent data showing that the argininosuccinate lyase is a multimeric enzyme probably composed of five identical polypeptide chains (Matagne and Schlösser, 1977) 相似文献
20.
Colletotrichum gloeosporioides produced exo-pectin lyase and protease in a) liquid cultures with incorporated washed cell wall material from unripe or ripe avocado and b) autoclaved immature fruit. The activity of exo-pectin lyase and protease produced in liquid cultures incorporating washed cell walls from immature fruits was almost the same as when washed cell walls from ripe fruits were incorporated. Ripe fruit tissue rotted by the fungus contained exo-pectin lyase, endo-polygalacturonase (endo-PG) and protease. The endo-PG was found to be endogenous to avocado fruit, and had a pH optimum of 5.5. The pH optima of exo-pectin lyase and protease were 8.5 and 7.5 respectively in all three enzyme preparations. All these enzyme preparations completely macerated avocado fruit tissue discs in vitro in less than 3 h of incubation but not potato tuber discs. Neither immature nor ripe fruit contained substances, proteinaceous or otherwise, which could inhibit the exo-pectin lyase or protease activity of these preparations. The results indicated that C. gloeosporioides possesses sufficient enzyme potential to invade cell walls of unripe fruit and that the fruit tissue does not have a mechanism to inactivate such enzymes. 相似文献