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1.
Validimír Ken Sylva Paoutová Petr Sedmera Viktor Rylko Zdenk eháek 《FEMS microbiology letters》1986,37(1):31-34
Abstract A new cloning vehicle, pEAP37 was constructed to develop the excretion system of Escherichia coli . This plasmid, derived from pEAP1, carried the penicillinase gene from an alkalophilic Bacillus sp. and the chloramphenicol acetyltransferase gene from pBR329 as selective markers. The Bacillus N-4 cellulase gene, Bacillus 1139 cellulase gene and Aeromonas sp. 212 xylanase L gene was inserted into pEAP37, and the distribution of the plasmid-encoded enzymes was analyzed. Most of these enzyme activities were found in the periplasmic space of E. coli when these extracellular enzyme genes were inserted into pBR322. On the other hand, most of these activities were observed in the culture medium when inserted into pEAP37. 相似文献
2.
Purification and Partial Characterization of Alkaline Xylanase from Escherichia coli Carrying pCX311
Hiroshi Honda Toshiaki Kudo Koki Horikoshi 《Bioscience, biotechnology, and biochemistry》2013,77(11):3165-3169
Xylanase produced by E. coli HB 101 carrying plasmid pCX311, which contains the xylanase A gene of alkalophilic Bacillus sp. strain C-125, was purified by ammonium sulfate precipitation, DEAE-cellulose column chromatography and Sephadex G-75 gel filtration. The purified enzyme had a molecular weight of 43,000. The pH and temperature optima for its activity were 6~10 and 70°C, respectively. The enzyme retained full activity after incubation at 50°C for 10 min. These enzymatic properties of the xylanase were almost the same as those of xylanase A. But this enzyme was less stable than xylanase A at low pHs. Furthermore, we could purify a larger amount of alkaline xylanase from E. coli than from alkalophilic Bacillus sp. strain C-125. 相似文献
3.
将大肠杆菌HB101嗜碱转化子中质粒pGCA所携带的嗜碱基因亚克隆至双元载体pBI121质粒中,构建了植物表达载体pLGC重组质粒。用其转化大肠杆菌HB101获得了能在碱性和卡那霉素抗性平板上生长的转化子,再通过三亲交配法将亚克隆质粒pLGC转化进农杆菌LBA4404,又获得能在碱性平板和卡那霉素及利福平双抗平板上生长的转化子,Southern杂交结果表明HB101转化子亚克隆质粒pLGC是由来自于嗜碱芽孢杆菌NTT36染色体DNA和双元载体pBI121组成,且农杆菌LBA4404转化子含有来自大肠杆菌亚克隆转化子的pLGC质粒。 相似文献
4.
Molecular cloning of a xylanase gene from Bacteroides succinogenes and its expression in Escherichia coli. 总被引:10,自引:5,他引:5
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A gene coding for xylanase synthesis in Bacteroides succinogenes was isolated by cloning, with Escherichia coli HB101 as the host. After partial digestion of B. succinogenes DNA with Sau3A, fragments were ligated into the BamHI site of pBR322 and transformed into E. coli HB101. Of 14,000 colonies screened, 4 produced clear halos on Remazol brilliant blue-xylan agar. Plasmids from two stable clones recovered exhibited identical restriction enzyme patterns, with the same 9.4-kilobase-pair (kbp) insert. The plasmid was designated pBX1. After subcloning of restriction enzyme fragments, a 3-kbp fragment was found to code for xylanase activity in either orientation when inserted into pUC18 and pUC19. The original clone possessed approximately 10-fold higher xylanase activity than did clones harboring the 3-kbp insert in pUC18, pUC19, or pBR322. The enzyme was partially secreted into the periplasmic space of E. coli. The periplasmic enzyme of the BX1 clone had 2% of the activity on carboxymethyl cellulose and less than 0.2% of the activity on p-nitrophenyl xyloside and a range of other substrates that it exhibited on xylan. The xylanase gene was not subject to catabolite repression by glucose or induction by either xylan or xylose. The xylanase activity migrated as a single broad band on nondenaturing polyacrylamide gels. The Km of the pBX1-encoded enzyme was 0.22% (wt/vol) of xylan, which was similar to that for the xylanase activity in an extracellular enzyme preparation from B. succinogenes. Based on these data it appears that the xylanase gene expressed in E. coli is fully functional and codes for an enzyme with properties similar to the B. succinogenes enzyme(s). 相似文献
5.
Molecular cloning and expression of the xylanase gene of alkalophilic Bacillus sp. strain C-125 in Escherichia coli. 总被引:6,自引:1,他引:5
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The gene for xylanase A of alkalophilic Bacillus sp. strain C-125 was cloned in Escherichia coli with pBR322. The plasmid pCX311 contained 2.6- and 2.0-kilobase-pair HindIII fragments. The characteristics of the purified pCX311-encoded xylanase were the same as those of purified xylanase A from alkalophilic Bacillus sp. strain C-125. 相似文献
6.
Molecular cloning of a xylanase gene from Bacteroides succinogenes and its expression in Escherichia coli 总被引:4,自引:0,他引:4
A Sipat K A Taylor R Y Lo C W Forsberg P J Krell 《Applied and environmental microbiology》1987,53(3):477-481
A gene coding for xylanase synthesis in Bacteroides succinogenes was isolated by cloning, with Escherichia coli HB101 as the host. After partial digestion of B. succinogenes DNA with Sau3A, fragments were ligated into the BamHI site of pBR322 and transformed into E. coli HB101. Of 14,000 colonies screened, 4 produced clear halos on Remazol brilliant blue-xylan agar. Plasmids from two stable clones recovered exhibited identical restriction enzyme patterns, with the same 9.4-kilobase-pair (kbp) insert. The plasmid was designated pBX1. After subcloning of restriction enzyme fragments, a 3-kbp fragment was found to code for xylanase activity in either orientation when inserted into pUC18 and pUC19. The original clone possessed approximately 10-fold higher xylanase activity than did clones harboring the 3-kbp insert in pUC18, pUC19, or pBR322. The enzyme was partially secreted into the periplasmic space of E. coli. The periplasmic enzyme of the BX1 clone had 2% of the activity on carboxymethyl cellulose and less than 0.2% of the activity on p-nitrophenyl xyloside and a range of other substrates that it exhibited on xylan. The xylanase gene was not subject to catabolite repression by glucose or induction by either xylan or xylose. The xylanase activity migrated as a single broad band on nondenaturing polyacrylamide gels. The Km of the pBX1-encoded enzyme was 0.22% (wt/vol) of xylan, which was similar to that for the xylanase activity in an extracellular enzyme preparation from B. succinogenes. Based on these data it appears that the xylanase gene expressed in E. coli is fully functional and codes for an enzyme with properties similar to the B. succinogenes enzyme(s). 相似文献
7.
Keisuke Nagahisa Satoshi Ezaki Shinsuke Fujiwara Tadayuki Imanaka Masahiro Takagi 《FEMS microbiology letters》1999,178(1):183-190
The Escherichia coli 3-ketoacyl-ACP reductase gene (fabGEc) was cloned using a PCR technique to investigate the metabolic link between fatty acid metabolism and polyhydroxyalkanoate (PHA) production. Three plasmids respectively harboring fabGEc and the poly-3-hydroxyalkanoate synthesis genes phaCAc and phaC1Ps from Aeromonas caviae and Pseudomonas sp. 61-3 respectively were constructed and introduced into E. coli HB101 strain. On a two-stage cultivation using dodecanoate as the sole carbon source, recombinant E. coli HB101 strains harboring fabGEc and phaC genes accumulated PHA copolymers (about 8 wt% of dry cell weight) consisting of several (R)-3-hydroxyalkanoate units of C4, C6, C8, and C10. It has been suggested that overexpression of the fabGEc gene leads to the supply of (R)-3-hydroxyacyl-CoA for PHA synthesis via fatty acid degradation. 相似文献
8.
Cloning, sequencing, and expression of a xylanase gene from the anaerobic ruminal bacterium Butyrivibrio fibrisolvens. 总被引:13,自引:3,他引:10
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A gene coding for xylanase activity, xynA, from the anaerobic ruminal bacterium Butyrivibrio fibrisolvens 49 was cloned into Escherichia coli JM83 by using plasmid pUC19. The gene was located on a 2.3-kilobase (kb) DNA insert composed of two adjacent EcoRI fragments of 1.65 and 0.65 kb. Expression of xylanase activity required parts of both EcoRI segments. In E. coli, the cloned xylanase enzyme was not secreted and remained cell associated. The enzyme exhibited no arabinosidase, cellulase, alpha-glucosidase, or xylosidase activity. The isoelectric point of the cloned protein was approximately 9.8, and optimal xylanase activity was obtained at pH 5.4. The nucleotide sequence of the 1,535-base-pair EcoRV-EcoRI segment from the B. fibrisolvens chromosome that included the xynA gene was determined. An open reading frame was found that encoded a 411-amino-acid-residue polypeptide of 46,664 daltons. A putative ribosome-binding site, promoter, and leader sequence were identified. Comparison of the XynA protein sequence with that of the XynA protein from alkalophilic Bacillus sp. strain C-125 revealed considerable homology, with 37% identical residues or conservative changes. The presence of the cloned xylanase gene in other strains of Butyrivibrio was examined by Southern hybridization. The cloned xylanase gene hybridized strongly to chromosomal sequences in only two of five closely related strains. 相似文献
9.
10.
Cloning of a xylanase gene from Fibrobacter succinogenes 135 and its expression in Escherichia coli 总被引:2,自引:0,他引:2
A genomic library consisting of 4- to 7-kb EcoRI DNA fragments from Fibrobacter succinogenes 135 was constructed using a phage vector, lambda gtWES lambda B, and Escherichia coli ED8654 as the host bacterium. Two positive plaques, designated lambda FSX101 and lambda FSX102, were identified. The inserts were 10.5 and 9.8 kb, respectively. A 2.3-kb EcoRI fragment that was subcloned from lambda FSX101 into pBR322 also showed xylanase activity. Southern blot analysis showed that the cloned EcoRI fragment containing the xylanase gene had originated from F. succinogenes 135. The cloned endo-(1,4)-beta-D-xylanase gene (pFSX02) was expressed constitutively in E. coli HB101 when grown on LB and on M9 medium containing either glucose or glycerol as the carbon source. Most of the beta-D-xylanase activity was located in the periplasmic space. Zymogram activity stains of nondenaturing polyacrylamide gels and isoelectric focusing gels showed that several xylanase isoenzymes were present in the periplasmic fraction of the E. coli clone FSX02 and they probably were due to posttranslational modification of a single gene product. Comparison of the FSX02 xylanase and the xylanase from the extracellular culture fluids of F. succinogenes 135 and S85 for their ability to degrade oat spelt xylan showed that, for equal units of beta-D-xylanase activity, hydrolysis by the cloned gene product was more complete. However, unlike the unfractionated mixture of xylanases from F. succinogenes 135 and S85, the enzyme from E. coli FSX02 was unable to release arabinose from oat spelt xylan. 相似文献
11.
The gene for an alkaline endoglucanase from the alkalophilic Bacillus sp. KSM-64 was cloned into the HindIII site of pBR322 and expressed in Escherichia coli HB101. The nucleotide sequence of a 4.1-kb region of the HindIII insert had two open reading frames, ORF-1 and ORF-2. The protein deduced from ORF-1 was composed of 244 amino acids with an M(r) of 27,865. Subcloning analysis proved that the alkaline endoglucanase was encoded by ORF-2 (822 amino acids with an M(r) of 91,040). Upstream from ORF-2, there were three consensus like sequences of the sigma A-type promoter of Bacillus subtilis, a putative Shine-Dalgarno sequence (AGGAGGT), and a catabolite repression operator-like sequence (TGTAAGCGGTTAACC). The HindIII insert was subcloned into a shuttle vector, pHY300PLK, and the encoded alkaline endoglucanase gene was highly expressed both in E. coli and B. subtilis. One of the three promoter-like sequences in ORF-2 could be suitable for high levels of enzyme expression in both host organisms. 相似文献
12.
Construction of a novel suicide vector: selection for Escherichia coli HB101 recombinants carrying the DNA insert. 总被引:2,自引:0,他引:2
We constructed a new type of cloning vector, pERISH2, that transforms Escherichia coli HB101 only when a foreign DNA fragment is ligated into the cloning site of the plasmid vector. Plasmid pERISH2 carries the rcsB gene which is derived from the chromosome of E. coli HB101 and is involved in the regulation of colanic acid production. When E. coli HB101 is transformed by this vector carrying the intact rcsB gene, the gene product RcsB blocks bacterial growth. However, if the rcsB gene is inactivated by the insertion of a foreign DNA fragment, this recombinant plasmid no longer inhibits the growth of E. coli HB101. Although E. coli HB101 is not stably transformed by pERISH2, E. coli K-12 strains such as JM109 and C600 can harbor this vector. Therefore, pERISH2 can be amplified in JM109 and be prepared from this strain in a large quantity using conventional methods. A chromosomal gene library of Klebsiella pneumoniae is constructed easily and efficiently by the utilization of this new cloning vector. 相似文献
13.
Cloning and DNA sequencing of xyaA, a gene encoding an endo-beta-1,4-xylanase from an alkalophilic Bacillus strain (N137).
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The gene xyaA encoding an alkaline endo-beta 1,4-xylanase from an alkalophilic Bacillus sp. strain (N137) isolated in our laboratory was cloned and expressed in Escherichia coli. The nucleotide sequence of a 1,656-bp DNA fragment containing xyaA was determined, revealing one open reading frame of 993 bp that encodes a xylanase (XyaA) of 39 kDa. This xylanase lacks a typical putative signal peptide, yet the protein is found in the Bacillus culture supernatant. In Escherichia coli, the active protein is located mainly in the periplasmic space. The xylanase activity of the cloned XyaA is an endo-acting enzyme that shows optimal activity at pH 8 and 40 degrees C. This activity is stable at a pH between 6 and 11. Incubations of XyaA at 40 degrees C for 1 h destroyed 45% of the activity. 相似文献
14.
Molecular cloning and nucleotide sequence of the gene encoding an endo-1,4-beta-glucanase from Bacillus sp. KSM-330. 总被引:1,自引:0,他引:1
The gene encoding an acid endo-1,4-beta-glucanase from Bacillus sp. KSM-330 was cloned into the HindIII site of pBR322 and expressed in Escherichia coli HB101. The recombinant plasmid contained a 3.1 kb HindIII insert, 1.8 kb of which was sufficient for the expression of endoglucanase activity in E. coli HB101. Nucleotide sequencing of this region (1816 bp) revealed an open reading frame of 1389 bp. The protein deduced from this sequence was composed of 463 amino acids with an Mr of 51882. The deduced amino acid sequence from amino acids 56 through 75 coincided with the amino-terminal sequence of the endoglucanase, Endo-K, purified from culture of Bacillus sp. KSM-330. The deduced amino acid sequence of Endo-K had 30% homology with that of the celA enzyme from Clostridium thermocellum NCIB 10682 and 25% homology with that of the enzyme from Cellulomonas uda CB4. However, the Endo-K protein exhibited no homology with respect to either the nucleotide or the amino acid sequences of other endoglucanases from Bacillus that had been previously characterized. These results indicate that the gene for Endo-K in Bacillus sp. KSM-330 has evolved from an ancestral gene distinct from that of other Bacillus endoglucanases. 相似文献
15.
The expression of Thermoactinomyces sp. E79 protease gene cloned into E. coli was highly host-dependent and the levels of protease expression was most stable in E. coli RR1 and E. coli HB101. Heating the intracellular extract at 70°C for 15 min converted the inactive recombinant E79 protease to its active mature form and also resulted in purification of the enzyme in a single step. Addition of 10 mM CaCl2 to the E79 protease decreased its autolysis and increased its thermal stability. © Rapid Science Ltd. 1998 相似文献
16.
将类产碱假单胞菌(Pseudomonas pseudoalcaligene)总DNA经Sau3AI部分酶解后的35~50kbDNA片段与经BamHI线性化及CIAP处理过的粘粒pIJ285连接,以大肠杆菌LE392为受体,构建类产碱假单胞菌的基因文库。通过三丁酸甘油酯平板和橄榄油平板法检测克隆子,获得一株具有耐热碱性脂肪酶活性的菌株LE392(pHZ1401)。随后将pHZ1401上的外源DNA片段进行亚克隆,从而获得了具有脂肪酶活性的菌株HB101(pHZ1402)和HB101(pHZ1403),它们分别携带有2.9kb和3.0kb的外源片段。两外源片段约有2kb的重叠区。HB101(pHZ1403)所分泌的脂肪酶活性比HB101(pHZ1402)高4倍,是出发菌的5倍。 相似文献
17.
18.
19.
Excretion of the penicillinase of an alkalophilic Bacillus sp. through the Escherichia coli outer membrane. 总被引:7,自引:3,他引:4
Two plasmids containing the penicillinase gene of alkalophilic Bacillus sp. strain 170, pEAP1 and pEAP2, were constructed. Most of the penicillinase produced by Escherichia coli, which carried these plasmids, was found in the culture medium. This excretion is caused by the cloned DNA fragment which contains some component that changes the outer membrane of E. coli. 相似文献
20.
Cloning and expression in Escherichia coli of the gene for an Arthrobacter beta-(1----3)-glucanase. 总被引:3,自引:2,他引:1
When inserted in the correct orientation at the BamHI site of plasmid YRp7, an 8.6-kilobase BamHI fragment of Arthrobacter sp. strain YCWD3 DNA gave Escherichia coli HB101 cells harboring the recombinant plasmid pBX20 the ability to lyse bakers' yeast cell walls or bakers' yeast glucan in agar medium. An extract of the transformed E. coli cells contained an endo-beta-(1----3)-glucanase with the same activity pattern as that of glucanase I produced by Arthrobacter sp. strain YCWD3. Although part of the glucanase activity was contributed by apparently defective molecules, two protein species were found which had high lytic activity on yeast cell walls and adsorbed to microcrystalline cellulose, and both had a single constituent polypeptide with a molecular weight of about 55,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In these properties the protein species were indistinguishable from those glucanase I protein species of Arthrobacter sp. strain YCWD3 which we believe are nearly the intact molecule. We conclude that the cloned fragment of Arthrobacter sp. strain YCWD3 DNA contains the structural gene for glucanase I. A recombinant plasmid obtained by subcloning a PstI fragment of pBX20 into pBR322 caused the transformed E. coli cells to produce apparently defective glucanase molecules only. This observation serves as additional supporting evidence for our conclusion. 相似文献