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1.
A bacterial strain with high cellulase activity (0.26 U/ml culture medium) was isolated from hot spring, and classified and
named as B. subtilis DR by morphological and 16SrDNA gene sequence analysis. A thermostable endocellulase, CelDR, was purified from the isolated
strain. The optimum temperature of the enzyme reaction was 50°C, and CelDR retained 70% of its maximum activity at 75°C after
incubation for 30 min. The putative gene celDR, consisting an open reading frame (ORF) of 1,524 nucleotides and encoding a protein of 508 amino acids with a molecular
weight of 55 kDa, was purified from B. subtilis DR and cloned into pET-28a for expression. The cellulase production in E. coli BL21 (DE3) was enhanced to approximately three times that of the wild-type strain. 相似文献
2.
Potent HaeIII-like DNA restriction activity was detected in cell-free extracts of Caldicellulosiruptor bescii DSM 6725 using plasmid DNA isolated from Escherichia coli as substrate. Incubation of the plasmid DNA in vitro with HaeIII methyltransferase protected it from cleavage by HaeIII nuclease
as well as cell-free extracts of C. bescii. The gene encoding the putative restriction enzyme was cloned and expressed in E. coli with a His-tag at the C-terminus. The purified protein was 38 kDa as predicted by the 981-bp nucleic acid sequence, was optimally
active at temperatures between 75°C and 85°C, and was stable for more than 1 week when stored at 35°C. The cleavage sequence
was determined to be 5′-GG/CC-3′, indicating that CbeI is an isoschizomer of HaeIII. A search of the C. bescii genome sequence revealed the presence of both a HaeIII-like restriction endonuclease (Athe 2438) and DNA methyltransferase
(Athe 2437). Preliminary analysis of other Caldicellulosiruptor species suggested that this restriction/modification activity is widespread in this genus. A phylogenetic analysis based
on sequence alignment and conserved motif searches identified features of CbeI distinct from other members of this group and
classified CbeI as a member of a novel subfamily of HaeIII-like enzymes. 相似文献
3.
The gene encoding NADP+-dependent isocitrate dehydrogenase (IDH; EC 1.1.1.42) of a psychrophilic bacterium, Psychromonas marina, was cloned and sequenced. The open reading frame of the gene encoding IDH of P. marina (PmIDH) was 2229 bp in length and corresponded to a polypeptide composed of 742 amino acids. The molecular mass of IDH was calculated as 80,426 Da. The deduced amino acid sequence of PmIDH exhibited high degrees of homology with the monomeric IDH from other bacteria such as Colwellia maris (62% identity) and Azotobacter vinelandii (AvIDH) (64%). His-tagged PmIDH overexpressed in Escherichia coli cells was purified and characterized. The optimum temperature of PmIDH activity was about 35 °C; however, the enzyme lost 74% of the activity after incubation for 10 min at 30 °C, indicating that this enzyme is thermolabile. Chimeric enzymes produced through domain swapping between PmIDH and mesophilic AvIDH were constructed and their optimum temperatures and thermostability were determined. The results suggest that regions 2 and 3, especially region 3, of the two IDHs are involved in their catalytic activities and optimum temperature and thermostability for activity. 相似文献
4.
Whole-genome sequence analysis of Bacillus halodurans ATCC BAA-125 revealed an isomerase gene ( rhaA) encoding an l-rhamnose isomerase ( l-RhI). The identified l
-RhI gene was cloned from B. halodurans and over-expressed in Escherichia coli. DNA sequence analysis revealed an open reading frame of 1,257 bp capable of encoding a polypeptide of 418 amino acid residues
with a molecular mass of 48,178 Da. The molecular mass of the purified enzyme was estimated to be ∼48 kDa by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and 121 kDa by gel filtration chromatography, suggesting that the enzyme is a homodimer.
The enzyme had an optimal pH and temperature of 7 and 70°C, respectively, with a k
cat of 8,971 min −1 and a k
cat/ K
m of 17 min −1 mM −1 for l-rhamnose. Although l-RhIs have been characterized from several other sources, B. halodurans
l-RhI is distinguished from other l-RhIs by its high temperature optimum (70°C) with high thermal stability of showing 100% activity for 10 h at 60°C. The half-life
of the enzyme was more than 900 min and ∼25 min at 60°C and 70°C, respectively, making B. halodurans
l-RhI a good choice for industrial applications. This work describes one of the most thermostable l-RhI characterized thus far. 相似文献
5.
The gene encoding an endo-β-1,4-xylanase from an Indonesian indigenous Bacillus licheniformis strain I5 was amplified using PCR, cloned, and expressed in Escherichia coli. The nucleotide sequence of a 642 bp DNA fragment was determined, revealing one open reading frame that encoded a xylanase.
Based on the nucleotide sequence, calculated molecular mass of the enzyme was 23 kDa. This xylanase has a predicted typical
putative signal peptide; however, in E. coli, the active protein was located mainly in intracellular form. Neither culture supernatant of recombinant E. coli nor periplasmic fraction has significantly detectable xylanase activity. The deduced amino acid of the gene has 91% identity
with that of Bacillus subtilis endoxylanase. Optimal activity of the recombinant enzyme was at pH 7 and 50°C 相似文献
6.
A gene encoding extracellular lipase was cloned and characterized from metagenomic DNA extracted from hot spring soil. The
recombinant gene was expressed in E. coli and expressed protein was purified to homogeneity using hydrophobic interactions chromatography. The mature polypeptide consists
of 388 amino acids with apparent molecular weight of 43 kDa. The enzyme displayed maximum activity at 50°C and pH 9.0. It
showed thermal stability up to 40°C without any loss of enzyme activity. Nearly 80% enzyme activity was retained at 50°C even
after incubation for 75 min. However above 50°C the enzyme displayed thermal instability. The half life of the enzyme was
determined to be 5 min at 60°C. Interestingly the CD spectroscopic study carried out in the temperature range of 25–95°C revealed
distortion in solution structure above 35°C. However the intrinsic tryptophan fluorescence spectroscopic study revealed that
even with the loss of secondary structure at 35°C and above the tertiary structure was retained. With p-nitrophenyl laurate as a substrate, the enzyme exhibited a K
m
, V
max
and K
cat
of 0.73 ± 0.18 μM, 239 ± 16 μmol/ml/min and 569 s −1 respectively. Enzyme activity was strongly inhibited by CuCl 2, HgCl 2 and DEPC but not by PMSF, eserine and SDS. The protein retained significant activity (~70%) with Triton X-100. The enzyme
displayed 100% activity in presence of 30% n-Hexane and acetone. 相似文献
7.
A gene from Withania somnifera (winter cherry), encoding a highly stable chloroplastic Cu/Zn superoxide dismutase (SOD), was cloned and expressed in Escherichia coli. The recombinant enzyme (specific activity of ~4,200 U mg −1) was purified and characterized. It retained ~90 and ~70% residual activities after 1 h at 80 and 95°C, respectively. At
95°C, thermal inactivation rate constant ( K
d) of the enzyme was 2.46 × 10 −3 min −1 and half-life of heat inactivation was 4.68 h. The enzyme was stable against a broad pH range (2.5–11.0). It also showed
a high degree of resistance to detergent, ethanol and protease digestion. This recombinant Cu/Zn SOD could therefore have
useful applications . 相似文献
8.
In this study, the gene encoding Bacillus sp. HJ171 uracil-DNA glycosylase ( Bsp HJ171 UDG) was cloned and sequenced. The Bsp HJ171 UDG gene consists of a 738-bp DNA sequence, which encodes for a protein that is 245-amino-acid residues in length.
The deduced amino acid sequence of the Bsp HJ171 UDG had a high sequence similarity with other bacterial UDGs. The molecular mass of the protein derived from this amino
acid sequence was 27.218 kDa. The Bsp HJ171 UDG gene was expressed under the control of a T7 lac promoter in the pTYB1 plasmid in Escherichia coli BL21 (DE3). The expressed enzyme was purified in one step using the Intein Mediated Purification with an Affinity Chitin-binding
Tag purification system. The optimal temperature range, pH, NaCl concentration, and KCl concentration of the purified enzyme
was 20–25°C, 8.0, 25 and 25 mM, respectively. The half-life of the enzyme at 40°C and 50°C were approximately 131 and 45 s,
respectively. These heat-labile characteristics enabled Bsp HJ171 UDG to control carry-over contamination in the polymerase chain reaction product (PCR) without losing the PCR product.
G.A. Kim and M.S. Lee contributed equally to this work. 相似文献
9.
A thermostable homodimeric isocitrate dehydrogenase from the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus was purified and characterized. The mol. mass of the isocitrate dehydrogenase subunit was 42 kDa as determined by SDS-PAGE.
Following separation by SDS-PAGE, A. fulgidus isocitrate dehydrogenase could be renatured and detected in situ by activity staining. The enzyme showed dual coenzyme specificity
with a high preference for NADP +. Optimal temperature for activity was 90° C or above, and a half-life of 22 min was found for the enzyme when incubated at
90° C in a 50 mM Tricine-KOH buffer (pH 8.0). Based on the N-terminal amino acid sequence, the gene encoding the isocitrate
dehydrogenase was cloned. DNA sequencing identified the icd gene as an open reading frame encoding a protein of 412 amino acids with a molecular mass corresponding to that determined
for the purified enzyme. The deduced amino acid sequence closely resembled that of the isocitrate dehydrogenase from the archaeon
Caldococcus noboribetus (59% identity) and bacterial isocitrate dehydrogenases, with 57% identity with isocitrate dehydrogenase from Escherichia coli. All the amino acid residues directly contacting substrate and coenzyme (except Ile-320) in E. coli isocitrate dehydrogenase are conserved in the enzyme from A. fulgidus. The primary structure of A. fulgidus isocitrate dehydrogenase confirmes the presence of Bacteria-type isocitrate dehydrogenases among Archaea. Multiple alignment
of all the available amino acid sequences of di- and multimeric isocitrate dehydrogenases from the three domains of life shows
that they can be divided into three distinct phylogenetic groups.
Received: 6 February 1997 / Accepted: 12 June 1997 相似文献
10.
A gene, aga-MJ11, encoding an α-galactosidase (EC 3.2.1.22) was cloned from Pedobacter nyackensis MJ11 CGMCC 2503, expressed in Escherichia coli, and biochemically characterized. The gene consisted of 2,163 nucleotides encoding a 720 amino acid–protein with a theoretical
molecular weight of 82.6 kDa. The deduced amino acid sequence of Aga-MJ11 shared the highest identity of 51% to an α-galactosidase
from Parabacteroides distasonis (YP_001301506), which belongs to glycoside hydrolase (GH) family 36. Purified recombinant Aga-MJ11-H showed optimal activity
at pH 5.5 and 40°C, was stable at pH 4.0–10.0, retained ~80% of the maximum activity at 30°C (the optimum temperature for
freshwater fish), exhibited tolerance to some proteases, and had a wide substrate specificity (pNPG, melidiose, stachyose
and raffinose). All these features make Aga-MJ11 potentially useful for applications in aquaculture. The enzyme studied in
the present work may represent a novel GH-36 α-galactosidase from the genus Pedobacter.
X. Liu and K. Meng contributed equally to this work. 相似文献
11.
A gene encoding endochitinase from Trichoderma virens UKM-1 was cloned and expressed in E. coli BL21 (DE3). Both the endochitinase gene and its cDNA sequences were obtained. The endochitinase gene encodes 430 amino acids
from an open reading frame comprising of 1,690 bp nucleotide sequence with three introns. The endochitinase was expressed
as soluble and active enzyme at 20°C when induced with 1 mM IPTG. Maximum activity was observed at 4 h of post-induction time.
SDS-PAGE showed that the purified endochitinase exhibited a single band with molecular weight of 42 kDa. Biochemical characterization
of the enzyme displayed a near neutral pH characteristic with an optimum pH at 6.0 and optimum temperature at 50°C. The enzyme
is stable between pH 3.0–7.0 and is able to retain its activity from 30 to 60°C. The presence of Mg 2+ and Ca 2+ ions increased the enzyme activity up to 20%. The purified enzyme has a strong affinity towards colloidal chitin and low
effect on ethyl cellulose and D-cellubiose which are non-chitin related substrates. HPLC analysis from the chitin hydrolysis
showed the release of (GlcNAc) 3, (GlcNAc) 2 and GlcNAc, in which (GlcNAc) 2 was the main product. 相似文献
12.
The gene encoding a cold-active and xylose-stimulated β-glucosidase of Marinomonas MWYL1 was synthesized and expressed in Escherichia coli. The recombinant enzyme (reBglM1) was purified and characterized. The molecular mass of the purified reBglM1 determined by
SDS-PAGE agree with the calculated values (50.6 Da). Optima of temperature and pH for enzyme activity were 40°C and 7.0, respectively.
The enzyme exhibited about 20% activity at 5°C and was stable over the range of pH 5.5–10.0. The presence of xylose significantly
enhanced enzyme activity even at higher concentrations up to 600 mM, with maximal stimulatory effect (about 1.45-fold) around
300 mM. The enzyme is active with both glucosides and galactosides and showed high catalytic efficiency ( k
cat = 500.5 s −1) for oNPGlc. These characterizations enable the enzyme to be a promising candidate for industrial applications. 相似文献
13.
The gene encoding an FMN-dependent NADH azoreductase, AzrG, from thermophilic Geobacillus stearothermophilus was cloned and functionally expressed in recombinant Escherichia coli. Purified recombinant AzrG is a homodimer of 23 kDa and bore FMN as a flavin cofactor. The optimal temperature of AzrG was
85 °C for the degradation of Methyl Red (MR). AzrG remained active for 1 h at 65 °C and for 1 month at 30 °C, demonstrating
both superior thermostability and long-term stability of the enzyme. AzrG efficiently decolorized MR, Ethyl Red at 30 °C.
Furthermore, the enzyme exhibited a wide-range of degrading activity towards several tenacious azo dyes, such as Acid Red
88, Orange I, and Congo Red. These results suggested the sustainable utilization of G. stearothermophilus as an azo-degrading strain for AzrG carrying whole-cell wastewater treatments for azo pollutants under high temperature conditions. 相似文献
14.
An OmpA family protein (FopA) previously reported as one of the major outer membrane proteins of an acidophilic iron-oxidizing
bacterium Acidithiobacillus ferrooxidans was characterized with emphasis on the modification by heat and the interaction with peptidoglycan. A 30-kDa band corresponding
to the FopA protein was detected in outer membrane proteins extracted at 75°C or heated to 100°C for 10 min prior to sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). However, the band was not detected in outer membrane proteins
extracted at ≤40°C and without boiling prior to electrophoresis. By Western blot analysis using the polyclonal antibody against
the recombinant FopA, FopA was detected as bands with apparent molecular masses of 30 and 90 kDa, suggesting that FopA existed
as an oligomeric form in the outer membrane of A. ferrooxidans. Although the fopA gene with a sequence encoding the signal peptide was successfully expressed in the outer membrane of Escherichia coli, the recombinant FopA existed as a monomer in the outer membrane of E. coli. FopA was detected in peptidoglycan-associated proteins from A. ferrooxidans. The recombinant FopA also showed the peptidoglycan-binding activity. 相似文献
15.
A thermophilic Geobacillus bacterium secreting high activity of endo-glucanase (EC 3.2.1.4) was isolated from rice straw compost supplemented with pig
manure. A full-length gene of 1,104 bp, celA, encoding this glycosyl hydrolase family 5 endo-glucanase of 368 amino acids was isolated. No related gene from Geobacillus has been reported previously. The recombinant CelA expressed in Escherichia coli had an optimal activity at 65°C and pH 5.0, and it exhibited tenfold greater specific activity than the commercially available
Trichoderma reesei endo-glucanase. CelA displayed activity over a broad temperature range from 45 to 75°C and was a thermostable enzyme with
90% activity retained after heating at 65°C for 6 h. Interestingly, CelA activity could be enhanced by 100% in the presence
of 2 mM MnSO 4. CelA had high specific activity over β- d-glucan from barley and Lichenan, making it a potentially useful enzyme in biofuel and food industries. 相似文献
16.
A novel bacterial strain with high cellulase activity (2.82 U/ml) was isolated, and then identified by its morphological character
and 16S rRNA sequence, and named Bacillus subtilis strain I15. The extracellular thermostable cellulase exhibited the maximum activity at 60°C and pH 6.0. It was very stable
since more than 90% of original CMCase activity was maintained at 65°C after incubation for 2 h. The cellulase gene, celI15, was cloned and extracellularly expressed by Escherichia coli BL21 (DE3), which encoded the extracellular protein of about 52 kDa. The extracellular activity of CelI15 from E. coli BL21 was up to about 6.78 U/ml, and all the other properties were almost the same as that from the wild-type strain. 相似文献
17.
Two genes (agal1 and agal2) encoding α-galactosidases were identified by sequence-based screening approaches. The gene agal1 was identified from a data set of a sequenced hot spring metagenome, and the deduced amino-acid sequence exhibited 99% identity to an α-galactosidase from the thermophilic bacterium Dictyoglomus thermophilum. The gene agal2 was identified from the whole genome sequence of the thermophile Meiothermus ruber. The amino-acid sequences exhibited structural motifs typical for glycoside hydrolase (GH) family 36 members and were also differentiated into different subgroups of this family. Recombinant production of the heat-active GH36b enzyme Agal1 (87 kDa) and GH36bt enzyme Agal2 (57 kDa) was carried out in E. coli. Agal1 exhibited a specific activity of 1502.3 U/mg at 80 °C, pH 6.5, and Agal2 225.4 U/mg at 60–70 °C, pH 6.5. Half-lives of 14 h (Agal1) and 39 h (Agal2) were obtained at 50 °C, and Agal1 showed half-lives of 4 and 2 h at 70 and 80 °C, respectively. In addition to the natural substrates melibiose, raffinose, and stachyose, 4NP α-d-galactopyranoside was hydrolyzed. Galactose was also liberated from locust bean gum. Both heat-active enzymes are attractive candidates for application in food and feed industry for high-temperature processes for the degradation of raffinose family oligosaccharides. 相似文献
18.
A novel lipolytic enzyme was isolated from a metagenomic library after demonstration of lipolytic activity on an LB agar plate
containing 1% (w/v) tributyrin. A novel esterase gene ( estIM1), encoding a lipolytic enzyme (EstIM1), was cloned using a shotgun method from a pFosEstIM1 clone of the metagenomic library,
and the enzyme was characterized. The estIM1 gene had an open reading frame (ORF) of 936 base pairs and encoded a protein of 311 amino acids with a molecular mass 34 kDa
and a p I value of 4.32. The deduced amino acid sequence was 62% identical to that of an esterase from an uncultured bacterium (ABQ11271).
The amino acid sequence indicated that EstIM1 was a member of the family IV of lipolytic enzymes, all of which contain a GDSAG
motif shared with similar enzymes of lactic acid microorganisms. EstIM1 was active over a temperature range of 1–50°C, at
alkaline pH. The activation energy for hydrolysis of p-nitrophenyl propionate was 1.04 kcal/mol, within a temperature range of 1–40°C. The activity of EstIM1 was about 60% of maximal
even at 1°C, suggesting that EstIM1 is efficiently cold-adapted. Further characterization of this cold-adapted enzyme indicated
that the esterase may be very valuable in industrial applications. 相似文献
19.
A 2,3-dihydroxybiphenyl (2,3-DHBP) dioxygenase gene from a Rhodococcus sp. strain, named RrbphCI and involved in the degradation of polychlorinated biphenyls (PCBs), was synthesized. RrbphCI was expressed in Escherichia coli and its encoded enzyme was purified. SDS–PAGE analysis indicated that the size of the protein encoded by RrbphCI was about 32 kDa. The activity of the 2,3-DHBP dioxygenase was 82.8 U/mg when the substrate was 2,3-DHBP, with optimum pH
8.0 at 30°C, and optimum temperature was 40°C at pH 8.0. The RrbphCI gene was transformed into Pseudomonas
putida strain EG11, to determine the ability of the enzyme to degrade 2,3-DHBP. The wild type EG11 degraded 61.86% of supplied 2,3-DHBP
and the transformed EG11 (hosting the RrbphCI gene) utilized 52.68% after 2 min of treatment at 30°C. The overexpressed and purified enzyme was able to degrade 2,3-DHBP.
The 2,3-DHBP dioxygenase is a key enzyme in the PCB degradation pathway. RrbphCI and its encoded 2,3-DHBP dioxygenase may have transgenic applications in bioremediation of PCBs. 相似文献
20.
A thermostable glycerol kinase (FGK) was purified 34-fold to homogeneity from Flavobacterium meningosepticum. The molecular masses of the enzyme were 200 kDa by gel filtration and 50 kDa by SDS-PAGE. The Km for glycerol and ATP were 0.088 and 0.030 m M, respectively. The enzyme was stable at 65°C for 10 min and at 37°C for two weeks. The enzyme gene was cloned into Escherichia coli and its complete DNA was sequenced. The FGK gene consists of an open reading frame of 1494-bp encoding a protein of 498 amino acids. The deduced amino acid sequence of the gene had 40-60% similarity to those of glycerol kinases from other origins and the amino acid sequence of the putative active site residue reported for E. coli GK is identical to the corresponding sequence of FGK except for one amino acid residue. 相似文献
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