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1.
Purification and properties of a novel raw starch degrading cyclomaltodextrin glucanotransferase from Bacillus firmus 总被引:3,自引:0,他引:3
B. N. Gawande A. Goel A. Y. Patkar S. N. Nene 《Applied microbiology and biotechnology》1999,51(4):504-509
A novel raw starch degrading cyclomaltodextrin glucanotransferase (CGTase; E.C. 2.4.1.19), produced by Bacillus firmus, was purified to homogeneity by ultrafiltration, affinity and gel filtration chromatography. The molecular weight of the
pure protein was estimated to be 78 000 and 82 000 Da, by SDS-PAGE and gel filtration, respectively. The pure enzyme had a
pH optimum in the range 5.5–8.5. It was stable over the pH range 7–11 at 10 °C, and at pH 7.0 at 60 °C. The optimum temperature
for enzyme activity was 65 °C. In the absence of substrate, the enzyme rapidly lost its activity above 30 °C. K
m and k
cat for the pure enzyme were 1.21 mg/ml and 145.17 μM/mg per minute respectively, with soluble starch as the substrate. For cyclodextrin
production, tapioca starch was the best substrate used when gelatinized, while wheat starch was the best substrate used when
raw. This CGTase could degrade raw wheat starch very efficiently; up to 50% conversion to cyclodextrins was obtained from
150 g/l starch without using any additives. The enzyme produced α-, β- and γ-cyclodextrins in the ratio of 0.2:9.2:0.6 and
0.2:8.6:1.2 from gelatinized tapioca starch and raw wheat starch with 150 g/l concentration respectively, after 18 h incubation.
Received: 25 September 1998 / Received revision: 15 December 1998 / Accepted: 21 December 1998 相似文献
2.
High-level expression and secretion of methyl parathion hydrolase in Bacillus subtilis WB800 总被引:4,自引:0,他引:4
The methyl parathion hydrolase (MPH)-encoding gene mpd was placed under the control of the P43 promoter and Bacillus subtilis nprB signal peptide-encoding sequence. High-level expression and secretion of mature, authentic, and stable MPH were achieved using the protease-deficient strain B. subtilis WB800 as the host. 相似文献
3.
Two genes encoding EngB endoglucanase and mini-CbpA1 scaffolding protein of Clostridium cellulovorans were constructed and coexpressed in Bacillus subtilis WB800. The resulting minicellulosomes were isolated by gel filtration chromatography and characterized. Biochemical and immunological evidence indicated that fraction II contained minicellulosomes consisting of mini-CbpA1 and EngB. The in vivo synthesis of minicellulosomes suggests that it will be possible in the future to insert into B. subtilis cellulosomal genes that will allow growth on cellulosic materials and the production of various designer cellulosomes with specific functions. 相似文献
4.
Hee-Yeon Cho Hideaki Yukawa Masayuki Inui Roy H. Doi Sui-Lam Wong 《Applied microbiology》2004,70(9):5704-5707
Two genes encoding EngB endoglucanase and mini-CbpA1 scaffolding protein of Clostridium cellulovorans were constructed and coexpressed in Bacillus subtilis WB800. The resulting minicellulosomes were isolated by gel filtration chromatography and characterized. Biochemical and immunological evidence indicated that fraction II contained minicellulosomes consisting of mini-CbpA1 and EngB. The in vivo synthesis of minicellulosomes suggests that it will be possible in the future to insert into B. subtilis cellulosomal genes that will allow growth on cellulosic materials and the production of various designer cellulosomes with specific functions. 相似文献
5.
Molecular cloning, DNA nucleotide sequencing, and expression in Bacillus subtilis cells of the Bacillus macerans cyclodextrin glucanotransferase gene. 总被引:5,自引:2,他引:5
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T Takano M Fukuda M Monma S Kobayashi K Kainuma K Yamane 《Journal of bacteriology》1986,166(3):1118-1122
The gene for cyclodextrin glucanotransferase from Bacillus macerans was cloned in an Escherichia coli bacteriophage, lambda D69, and was recloned in a Bacillus subtilis plasmid, pUB110. Starting from an ATG initiation codon, a unique reading frame was shown to extend for 2,142 base pairs (714 amino acids). The nucleotide sequence revealed that the enzyme is composed of two identical subunits. 相似文献
6.
High-Level Expression and Secretion of Methyl Parathion Hydrolase in Bacillus subtilis WB800 总被引:6,自引:0,他引:6
The methyl parathion hydrolase (MPH)-encoding gene mpd was placed under the control of the P43 promoter and Bacillus subtilis nprB signal peptide-encoding sequence. High-level expression and secretion of mature, authentic, and stable MPH were achieved using the protease-deficient strain B. subtilis WB800 as the host. 相似文献
7.
Ohdan K Kuriki T Takata H Kaneko H Okada S 《Applied and environmental microbiology》2000,66(7):3058-3064
We constructed two types of chimeric enzymes, Ch1 Amy and Ch2 Amy. Ch1 Amy consisted of a catalytic domain of Bacillus subtilis X-23 alpha-amylase (Ba-S) and the raw starch-binding domain (domain E) of Bacillus A2-5a cyclomaltodextrin glucanotransferase (A2-5a CGT). Ch2 Amy consisted of Ba-S and D (function unknown) plus E domains of A2-5a CGT. Ch1 Amy acquired raw starch-binding and -digesting abilities which were not present in the catalytic part (Ba-S). Furthermore, the specific activity of Ch1 Amy was almost identical when enzyme activity was evaluated on a molar basis. Although Ch2 Amy exhibited even higher raw starch-binding and -digesting abilities than Ch1 Amy, the specific activity was lower than that of Ba-S. We did not detect any differences in other enzymatic characteristics (amylolytic pattern, transglycosylation ability, effects of pH, and temperature on stability and activity) among Ba-S, Ch1 Amy, and Ch2 Amy. 相似文献
8.
9.
一株产环糊精葡萄糖基转移酶的地衣芽孢杆菌的选育、产酶条件及酶学特性 总被引:9,自引:0,他引:9
从土壤分离物中筛选到一株环糊精葡萄糖基转移酶 (CGTase)产生菌 4 0 3,96h发酵酶活为 0 95U mL。经紫外辐射和硫酸二乙酯复合诱变而获得突变株CLS4 0 3,96h发酵酶活达 1 36U mL ,提高 4 3%。该突变菌株被鉴定为地衣芽孢杆菌 (Bacilluslicheniformis) ,产CGTase的最佳碳源为可溶性淀粉 ,最佳氮源为硝酸铵 ,最适初始pH为 6 5 ,最适培养温度为 35℃ ,发酵期间CGTase的产生高峰 (第 96h)滞后于菌体生物量高峰 (第 4 8h) 2d。菌株所产CGTase的最适反应pH为 6 0 ,最适温度为 5 5℃ ,在pH 6 0~ 7 5间和 5 0℃下保持 1h后的剩余酶活均达 90 %以上 ;酶液中适量添加Ca2 能大幅提高CGTase在 5 5℃下的稳定性。经高效液相色谱分析 ,CGTase作用于淀粉后的产物以α 环糊精为主 ,β 环糊精为次 ,二者比例为 2 4 7∶1,环糊精总产率达 2 9 8% ,但产物中不含γ 环糊精 相似文献
10.
A cloning vehicle, pFTB91, for the Bacillus subtilis host was constructed with DNA fragments heterologous to the host chromosome. It consists of three DNA fragments: (i) chromosomal DNA of Bacillus amyloliquefaciens which complements the leuA and ilvC mutations in B. subtilis; (ii) a B. amyloliquefaciens plasmid DNA that supplies an autonomously replicating function; and (iii) a HindIII fragment of Staphylococcus aureus plasmid pTP5 that carries gene tetr, conferring the TetR phenotype. It has sufficiently low DNA homology to prevent its integration into the host chromosome in recombination-competent cells of B. subtilis. It is 9.3 kb, and approx. 10 copies are present per chromosome. The SalI and KpnI sites in the ilvC+ and tetr genes, respectively, could be used for selection of recombinant plasmids by insertional inactivation. The plasmid has unique sites for EcoRI, PstI, and XbaI. 相似文献
11.
12.
Dina Rairakhwada Jeong-Woo Seo Mi-young Seo Ohsuk Kwon Sang-Ki Rhee Chul Ho Kim 《Journal of industrial microbiology & biotechnology》2010,37(2):195-204
Although levan produced by Bacillus amyloliquefaciens is known to have efficient immunostimulant property which gives 100% survival of common carp when infected with Aeromonas hydrophila, no detailed reports are available describing kinetic studies of d-glucose production and levan formation. In this study, we cloned and characterized the enzymatic kinetics using levansucrase
expressed in Escherichia coli. Optimum pH for d-glucose production and levan formation was 6.0 and 8.0, respectively, whereas optimum temperature was 30°C and 4°C, respectively.
The K
m and V
max values for levansucrase were calculated to be 47.81 mM sucrose and 57.47 μmole/min mg protein, respectively. Prominent expression
of levansucrase was obtained through xylose induction in Bacillus megaterium, where most of the His6-tagged protein was secreted into the culture broth, giving levansucrase activity of 12,906 U/l. Response-surface methodology
(RSM) was further employed to optimize the fermentation conditions and improve the level of levansucrase production. Maximum
levansucrase activity of 20,251 U/l was obtained in 12 h of fermentation carried out at 28°C, starting induction with 0.735%
xylose when A
600 was 1.2, which was 1.6- and 62-fold higher than those obtained in the nonoptimized conditions for the recombinant strain
and the native strain, respectively. 相似文献
13.
An expression vector, pUBEX, was constructed for extracellular production of heterologous proteins in Bacillus subtilis using a polyhistidine tag on the C-terminal sequence, providing an efficient and easy purification of the protein. A CII protein, a member of Bowman–Birk protease inhibitors, which was expressed as an inactive protein in Escherichia coli, was successfully expressed in Bacillus subtilis using the pUBEX vector and was purified to 6.4 mg l–1 by the immobilized metal affinity chromatography. 相似文献
14.
Mannanase, an extracellular enzyme catalyzes the hydrolysis of hemicelluloses to produce oligosaccharides, has a potential to be applied in food industries. In this study, a mannanase gene from B. subtilis Z-2 was isolated through PCR screening of a genomic DNA library. The nucleotide sequence of the mannanase gene, man, contained an open reading frame of 1.080 bp, which codes for a deduced 26 amino-acid signal peptide and a mature protein with the deduced molecular mass of 38 KDa. The man gene can both be expressed heterologously into the periplasm in plasmid pET22b(+) containing intact signal peptide (pET-NdeI18) and the pelB signal peptide of the pET22b(+)vector (pET-NcoI3). The Escherichia coli BL21 (DE3) containing pET-NcoI3 secreted about twice as much mannanase as that harboring pET-NdeI18. In E. coli DH5alpha, expression of man was under the control of the lac promoter in the pRK415 vector and was much more effective when the Shine-Dalgamo (SD) sequence was changed from GGGGAG to AAGGAG and the start codon was changed from TTG to ATG, respectively. These results suggest that genetic modification of the SD sequence and start codon is practical for high-level expression of mannanase in different bacterial strains. 相似文献
15.
Gene cloning, nucleotide sequence, and expression of a cephalosporin-C deacetylase from Bacillus subtilis.
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The gene encoding a cephalosporin-C deacetylase (CAH) from Bacillus subtilis SHS 0133 was cloned and sequenced. The nucleotide sequence contained an open reading frame encoding a polypeptide consisting of 318 amino acids, the molecular weight of which was in good agreement with the value obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The deduced amino acid sequence contained the common sequence Gly-X-Ser-X-Gly found in many esterases, lipases, and serine proteases. This indicates that CAH is a serine enzyme. A possible promoter sequence which is very similar to the consensus sequences of -35 and -10 regions recognized by B. subtilis RNA polymerase utilizing sigma factor H was found in the 5'-flanking region of the CAH structural gene. Two repeated A+T-rich blocks consisting of 24 bp were also found in the upstream region of the initiation codon. We constructed a series of expression plasmids by inserting the CAH gene into Escherichia coli ATG vectors. The degree of CAH gene expression depended on promoters and vector plasmids, which have different replication origins. The expressed CAH protein was an active form in the soluble fraction obtained after cell disruption. The highest expression level was accomplished with an expression plasmid, pCAH400, which has the trp promoter and the replication origin derived from pAT153. In the fermentation using a 30-liter jar fermentor, the transformant E. coli JM103(pCAH400) produced 440 U of CAH per ml of culture during a 24-h incubation. This value corresponded to 2.1 g of CAH protein in 1 liter of culture broth. 相似文献
16.
The cgt-gene from the alkaliphilic halotolerant Bacillus pseudalcaliphilus 8SB was isolated and sequenced. An open reading frame (ORF) of 2112 bp encoding a polypeptide of 704 amino acids, composed of a 29-amino acid signal sequence and a 675-amino acid mature enzyme was found. The established low level of homology with nucleotide sequences of other Bacillus CGTases (less than 82%) suggested that the cgt-gene from Bacillus pseudalcaliphilus 8SB encodes a new enzyme. The cgt-gene was cloned as a PCR amplicon and thereby the construction of genome library was avoided. This is the first evidence for the use of pJET vector as an expression vector. The opportunity to apply its T7 promoter for efficient extracellular production of heterologous proteins in Escherichia coli BL21 (DE3) was demonstrated. The expression of extracellular recombinant CGTase improved 23-fold, concerning β-CGTase activity and 4.5-fold concerning γ-CGTase activity after IPTG induction and glycine supplementation was achieved. 相似文献
17.
CsCl density gradient fractionation of cell lysates was employed to follow the fate of Escherichia coli, phage T6, and non-glucosylated phage T6 deoxyribonucleic acid (DNA) after uptake by competent cells of Bacillus subtilis 168 thy minus trp minus. Shortly after uptake, most of the radioactive Escherichia coli or non-glucosylated T6 DNA was found in the denatured form; the remainder of the label was associated with recipient DNA. Incubation of the cells after DNA uptake led to the disappearance of denatured donor DNA and to an increase in the amount of donor label associated with recipient DNA. These findings are analogous to those previously reported with homologous DNA. By contrast, T6 DNA, which is poorly taken up, appeared in the native form shortly after uptake and was degraded on subsequent incubation. The nature of the heterologous DNA fragments associated with recipient DNA was investigated with Escherichia coli 2-H and 3-H-labeled DNA. Association of radioactivity with recipient DNA decreased to one-fourth in the presence of excess thymidine; residual radioactivity could not be separated from recipient DNA by shearing (sonic oscillation) and/or denaturation, but was reduced by one-half in the presence of a DNA replication inhibitor. Residual radioactivity associated with donor DNA under these conditions was about 5% of that originally taken up. Excess thymidine, but not the DNA replication inhibitor, also decreased association of homologous DNA label with recipient DNA; but, even in the presence of both of these, the decrease amounted to only 60%. It is concluded that most, or all, of the Escherichia coli DNA label taken up is associated with recipient DNA in the form of mononucleotides via DNA replication. 相似文献
18.
Efficient cloning in Bacillus megaterium: comparison to Bacillus subtilis and Escherichia coli cloning hosts 总被引:3,自引:0,他引:3
Quantitative cloning efficiencies for B. megaterium, B. subtilis , and E. coli were compared. Transformation of B. megaterium is less efficient than transformation of B. subtilis or E. coli . The frequency of recombinant clones was equal in E. coli and B. megaterium ; both somewhat higher than in B. subtilis . Equivalent average insert sizes were found in B. megaterium and E. coli clones, but significantly smaller inserts were obtained in B. subtilis clones. Clones obtained and propagated in B. megaterium were structurally stable when grown under plasmid selection. 相似文献
19.
A Olsson T Hagstr?m B Nilsson M Uhlén S Gatenbeck 《Applied and environmental microbiology》1985,49(5):1084-1089
The Bacillus sphaericus gene coding for penicillin V amidase, which catalyzes the hydrolysis of penicillin V to yield 6-aminopenicillanic acid and phenoxyacetic acid, has been isolated by molecular cloning in Escherichia coli. The gene is contained within a 2.2-kilobase HindIII-PstI fragment and is expressed when transferred into E. coli and Bacillus subtilis. The expression in B. subtilis carrying the recombinant plasmid is approximately two times higher than in the original B. sphaericus strain. A comparison of the purified enzyme from B. sphaericus and the expressed gene product in E. coli minicells suggests that the native enzyme consists of four identical subunits, each with a molecular weight of 35,000. 相似文献