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1.
Fu D Li Z Huang H Yuan T Shi P Luo H Meng K Yang P Yao B 《Applied microbiology and biotechnology》2011,90(4):1295-1302
The maximum activity of Yersinia enterocolitica phytase (YeAPPA) occurs at pH 5.0 and 45 °C, and notably, its specific activity (3.28 ± 0.24 U mg−1) is 800-fold less than that of its Yersinia kristeensenii homolog (YkAPPA; 88% amino acid sequence identity). Sequence alignment and molecular modeling show that the arginine at position
79 (Arg79) in YeAPPA corresponding to Gly in YkAPPA as well as other histidine acid phosphatase (HAP) phytases is the only
non-conserved residue near the catalytic site. To characterize the effects of the corresponding residue on the specific activities
of HAP phytases, Escherichia coli EcAPPA, a well-characterized phytase with a known crystal structure, was selected for mutagenesis—its Gly73 was replaced
with Arg, Asp, Glu, Ser, Thr, Leu, or Tyr. The results show that the specific activities of all of the corresponding EcAPPA
mutants (17–2,400 U mg−1) were less than that of the wild-type phytase (3,524 U mg−1), and the activity levels were approximately proportional to the molecular volumes of the substituted residues’ side chains.
Site-directed replacement of Arg79 in YeAPPA (corresponding to Gly73 of EcAPPA) with Ser, Leu, and Gly largely increased the
specific activity, which further verified the key role of the residue at position 79 for determining phytase activity. Thus,
a new determinant that influences the catalytic efficiency of HAP phytases has been identified. 相似文献
2.
Sanni P. Voutilainen Harry Boer Marika Alapuranen Janne J?nis Jari Vehmaanper? Anu Koivula 《Applied microbiology and biotechnology》2009,83(2):261-272
Two different types of approach were taken to improve the hydrolytic activity towards crystalline cellulose at elevated temperatures
of Melanocarpus albomyces Cel7B (Ma Cel7B), a single-module GH-7 family cellobiohydrolase. Structure-guided protein engineering was used to introduce an additional
tenth disulphide bridge to the Ma Cel7B catalytic module. In addition, a fusion protein was constructed by linking a cellulose-binding module (CBM) and a linker
from the Trichoderma reesei Cel7A to the C terminus of Ma Cel7B. Both approaches proved successful. The disulphide bridge mutation G4C/M70C located near the N terminus, close to the
entrance of the active site tunnel of Ma Cel7B, led to improved thermostability (ΔT
m = 2.5°C). By adding the earlier found thermostability-increasing mutation S290T (ΔT
m = 1.5°C) together with the disulphide bridge mutation, the unfolding temperature was increased by 4°C (mutant G4C/M70C/S290T)
compared to that of the wild-type enzyme, thus showing an additive effect on thermostability. Both disulphide mutants had
increased activity towards microcrystalline cellulose (Avicel) at 75°C, apparently solely because of their improved thermostability.
The addition of a CBM also improved the thermostability (ΔT
m = 2.5°C) and caused a clear (sevenfold) increase in the hydrolysis activity of Ma Cel7B towards Avicel at 70°C. 相似文献
3.
In our previous studies, the yeast Endomyces fibuliger LU677 was found to degrade amygdalin in bitter apricot seeds. The present investigation shows that E. fibuliger LU677 produces extracellular β-glycosidase activity when grown in malt extract broth (MEB). Growth was very good at 25 °C
and 30 °C and slightly less at 35 °C. When grown in MEB of pH 5 and pH 6 with addition of 0, 10 or 100 ppm amygdalin, E. fibuliger produced only slightly more biomass at pH 5, and was only slightly inhibited in the presence of amygdalin. Approximately,
60% of the added amygdalin was degraded (fastest at 35 °C) during an incubation period of 5 days. Supernatants of cultures
grown at 25 °C and pH 6 for 5 days were tested for the effects of pH and temperature on activity (using amygdalin, linamarin
and prunasin as substrates). Prunase activity had two pH optima (pH 4 and pH 6), amygdalase and linamarase only one each at
pH 6 and pH 4–5 respectively. The linamarase activity evolved earlier than amygdalase (2 days and 4 days respectively). The
data thus indicate the presence of at least two different glycosidases having different pH optima and kinetics of excretion.
In the presence of amygdalin, lower glycosidase activities were generally produced. However, the amygdalin was degraded from
the start of the growth, strongly indicating an uptake of amygdalin by the cells. The temperature optimum for all activities
was at 40 °C. Activities of amygdalase (assayed at pH 4) and linamarase (at pH 6) evolving during the growth of E. fibuliger were generally higher in cultures grown at 25 °C and 30 °C. TLC analysis of amygdalin degradation products show a two-stage
sequential mechanism as follows: (1) amygdalin to prunasin and (2) prunasin to cyanohydrin.
Received: 16 September 1997 / Received revision: 6 October 1997 / Accepted: 14 October 1997 相似文献
4.
A number of nutritional factors influencing growth and glucose oxidase (EC 1.1.3.4) production by a newly isolated strain
of Penicillium pinophilum were investigated. The most important factors for glucose oxidase production were the use of sucrose as the carbon source,
and growth of the fungus at non-optimal pH 6.5. The enzyme was purified to apparent homogeneity with a yield of 74%, including
an efficient extraction step of the mycelium mass at pH 3.0, cation-exchange chromatography and gel filtration. The relative
molecular mass (M
r) of native glucose oxidase was determined to be 154 700 ± 4970, and 77 700 for the denatured subunit. Electron-microscopic
examinations revealed a sandwich-shaped dimeric molecule with subunit dimensions of 5.0 × 8.0 nm. Glucose oxidase is a glycoprotein
that contains tightly bound FAD with an estimated stoichiometry of 1.76 mol/mol enzyme. The enzyme is specific for d-glucose, for which a K
m value of 6.2 mM was determined. The pH optimum was determined in the range pH 4.0–6.0. Glucose oxidase showed high stability
on storage in sodium citrate (pH 5.0) and in potassium phosphate (pH 6.0), each 100 mM. The half-life of the activity was
considerably more than 305 days at 4 °C and 30 °C, and 213 days at 40 °C. The enzyme was unstable at temperatures above 40 °C
in the range pH 2.0–4.0 and at a pH above 7.0.
Received: 18 November 1996 / Received revision: 3 March 1997 / Accepted: 7 March 1997 相似文献
5.
Phytases are used to improve phosphorus nutrition of food animals and reduce their phosphorus excretion to the environment.
Due to favorable properties, Escherichia coli AppA2 phytase is of particular interest for biotechnological applications. Directed evolution was applied in the present
study to improve AppA2 phytase thermostability for lowering its heat inactivation during feed pelleting (60–80°C). After a
mutant library of AppA2 was generated by error-prone polymerase chain reaction, variants were expressed initially in Saccharomyces cerevisiae for screening and then in Pichia pastoris for characterizing thermostability. Compared with the wild-type enzyme, two variants (K46E and K65E/K97M/S209G) showed over
20% improvement in thermostability (80°C for 10 min), and 6–7°C increases in melting temperatures (T
m). Structural predictions suggest that substitutions of K46E and K65E might introduce additional hydrogen bonds with adjacent
residues, improving the enzyme thermostability by stabilizing local interactions. Overall catalytic efficiency (k
cat / K
m) of K46E and K65E/K97M/S209G was improved by 56% and 152% than that of wild type at pH 3.5, respectively. Thus, the catalytic
efficiency of these enzymes was not inversely related to their thermostability. 相似文献
6.
Yang HM Yao B Meng K Wang YR Bai YG Wu NF 《Journal of industrial microbiology & biotechnology》2007,34(3):213-218
Substitution of the N-terminus of Streptomyces olivaceoviridis xylanase XYNB to generate mutant TB has been previously shown to increase the thermostability of the enzyme. To further improve
the stability of this mutant, we introduced a disulfide bridge (C109–C153) into the TB mutant, generating TS. To assess the
effect of the disulfide bridge in the wild-type enzyme, the S109C-N153C mutation was also introduced into XYNB, resulting
in XS. The mutants were expressed in Pichia pastoris, the recombinant enzymes were purified, and the effect of temperature and pH on enzymatic activity was characterized. Introduction
of the disulfide bridge (C109–C153) into XYNB (XS variant) and TB (TS variant) increased the thermostability up to 2.8-fold
and 12.4-fold, respectively, relative to XYNB, after incubation at 70°C, pH 6.0, for 20 min. In addition, a synergistic effect
of the disulfide bridge and the N-terminus replacement was observed, which extended the half-life of XYNB from 3 to 150 min.
Moreover, XS and TS displayed better resistance to acidic conditions compared with the respective enzymes that did not contain
a disulfide bridge. 相似文献
7.
M. Coletta Alessandra Gambacurta Maria Elisabetta Clementi Fulvio Erba Francesca Polizio Mattia Falconi Franca Ascoli 《Journal of biological inorganic chemistry》1999,4(6):678-683
The pH and temperature dependence of both the kinetic and thermodynamic properties of the Thr72→Ile mutant of Scapharca
inaequivalvis homodimeric hemoglobin were investigated between pH 2 and 10 and between 8 °C and 36 °C, in comparison with the wild-type
recombinant protein. Results demonstrate pH-independent O2-binding properties, at least between pH 5 and 10, with the higher affinity of the mutant being related to a less negative
entropy change. This observation may relate to a variation in the number of water molecules involved in the intersubunit communication.
Furthermore, the kinetic properties of ligand association and dissociation seem to be in keeping with possible structural
alterations of water molecules at the subunit interface occurring in the Thr72→Ile mutant as well as with amino acid residues
involved in the modulation of reactivity and cooperativity at the level of (1) the proximal side of the heme pocket and of
(2) the heme propionates bridging the two subunits.
Received: 25 February 1999 / Accepted: 9 August 1999 相似文献
8.
A highly thermostable endo-(1,4)-β-mannanase from the marine bacterium Rhodothermus marinus 总被引:2,自引:0,他引:2
Rhodothermus marinus ATCC 43812, a thermophilic bacterium isolated from marine hot springs, possesses hydrolytic activities for depolymerising
substrates such as carob-galactomannan. Screening of expression libraries identified mannanase-positive clones. Subsequently,
the corresponding DNA sequences were determined, eventually identifying a coding sequence specifying a 997 amino acid residue
protein of 113 kDa. Analyses revealed an N-terminal domain of unknown function and a C-terminal mannanase domain of 550 amino
acid residues with homology to known mannanases of glycosidase family 26. Action pattern analysis categorised the R. marinus mannanase as an endo-acting enzyme with a requirement for at least five sugar moieties for effective catalytic activity.
When expressed in Escherichia coli, purified gene product with catalytic activity was mainly found as two protein fragments of 45 kDa and 50 kDa. The full-length
protein of 113 kDa was only detected in crude extracts of R. marinus, while truncated protein-containing fractions of the original source resulted in a major active protein of 60 kDa. Biochemical
analysis of the mannanase revealed a temperature and pH optimum of 85 °C and pH 5.4, respectively. Purified, E. coli-produced protein fragments showed high heat stability, retaining more than 70% and 25% of the initial activity after 1 h
incubation at 70 °C and 90 °C, respectively. In contrast, R. marinus-derived protein retained 87% activity after 1 h at 90 °C. The enzyme hydrolysed carob-galactomannan (locust bean gum) effectively
and to a smaller extent guar gum, but not yeast mannan.
Received: 5 November 1999 / Received revision: 19 January 2000 / Accepted: 23 January 2000 相似文献
9.
N. Kitamoto M. Go T. Shibayama T. Kimura Y. Kito K. Ohmiya N. Tsukagoshi 《Applied microbiology and biotechnology》1996,46(5-6):538-544
Two endo-1,4-β-glucanase genes, designated celA and celB, from a shoyu koji mold Aspergillus oryzae KBN616, were cloned and characterized. The celA gene comprised 877 bp with two introns. The CelA protein consisted of 239 amino acids and was assigned to the cellulase family
H. The celB gene comprised 1248 bp with no introns. The CelB protein consisted of 416 amino acids and was assigned to the cellulase family
C. Both genes were overexpressed under the promoter of the A. oryzae taka-amylase A gene for purification and enzymatic characterization of CelA and CelB. CelA had a molecular mass of 31 kDa,
a pH optimum of 5.0 and temperature optimum of 55 °C, whereas CelB had a molecular mass of 53 kDa, a pH optimum of 4.0 and
temperature optimum of 45 °C.
Received: 3 July 1996 / Accepted: 15 July 1996 相似文献
10.
M. J. Artolozaga E. Kubátová J. Volc H. M. Kalisz 《Applied microbiology and biotechnology》1997,47(5):508-514
Pyranose 2-oxidase (P2O) was purified 43-fold to apparent homogeneity from the basidiomycete Phanerochaete chrysosporium using liquid chromatography on phenyl Sepharose, Mono Q (twice) and phenyl Superose. The native enzyme has a molecular mass
of about 250 kDa (based on native PAGE) and is composed of four identical subunits of 65 kDa. It contains three isoforms of
isoelectric point (pI) 5.0, 5.05 and 5.15 and does not appear to be a glycoprotein. P2O is optimally stable at pH 8.0 and
up to 60 °C. It is active over a broad pH range (5.0–9.0) with maximum activity at pH 8.0–8.5 and at 55 °C, and a broad substrate
specificity. d-Glucose is the preferred substrate, but 1-β-aurothioglucose, 6-deoxy-d-glucose, l-sorbose, d-xylose, 5-thioglucose, d-glucono-1,5-lactone, maltose and 2-deoxy-d-glucose are also oxidised at relatively high rates. A Ping Pong Bi Bi mechanism was demonstrated for the P2O reaction at
pH 8.0, with a catalytic constant (k
cat) of 111.0 s−1 and an affinity constant (K
m) of 1.43 mM for d-glucose and 83.2 μM for oxygen. Whereas the steady-state kinetics for glucose oxidation were unaffected by the medium at
pH ≥ 7.0, at low pH both pH and buffer composition affected the P2O kinetics with the k
cat/K
m value decreasing with decreasing pH. The greatest effect was observed in acetate buffer (0.1 M, pH 4.5), where the k
cat decreased to 60.9 s−1 and the K
m increased to 240 mM. The activity of P2O was completely inhibited by 10 mM HgCl2, AgNO3 and ZnCl2, and 50% by lead acetate, CuCl2 and MnCl2.
Received: 28 August 1996 / Received revision: 25 November 1996 / Accepted: 29 November 1996 相似文献
11.
Ethanol production by recombinant Escherichia coli strain FBR5 from dilute acid pretreated wheat straw (WS) by separate hydrolysis and fermentation (SHF) and simultaneous saccharification
and fermentation (SSF) was studied. The yield of total sugars from dilute acid (0.5% H2SO4) pretreated (160 °C, 10 min) and enzymatically saccharified (pH 5.0, 45 °C, 72 h) WS (86 g/l) was 50.0 ± 1.4 g/l. The hydrolyzate
contained 1,184 ± 19 mg furfural and 161 ± 1 mg hydroxymethyl furfural per liter. The recombinant E. coli FBR5 could not grow at all at pH controlled at 4.5 to 6.5 in the non-abated wheat straw hydrolyzate (WSH) at 35 °C. However,
it produced 21.9 ± 0.3 g ethanol from non-abated WSH (total sugars, 44.1 ± 0.4 g/l) in 90 h including the lag time of 24 h
at controlled pH 7.0 and 35 °C. The bioabatement of WS was performed by growing Coniochaeta ligniaria NRRL 30616 in the liquid portion of the pretreated WS aerobically at pH 6.5 and 30 °C for 15 h. The bacterium produced 21.6 ± 0.5 g
ethanol per liter in 40 h from the bioabated enzymatically saccharified WSH (total sugars, 44.1 ± 0.4 g) at pH 6.0. It produced
24.9 ± 0.3 g ethanol in 96 h and 26.7 ± 0.0 g ethanol in 72 h per liter from bioabated WSH by batch SSF and fed-batch SSF,
respectively. SSF offered a distinct advantage over SHF with respect to reducing total time required to produce ethanol from
the bioabated WS. Also, fed-batch SSF performed better than the batch SSF with respect to shortening the time requirement
and increase in ethanol yield. 相似文献
12.
A Clostridium thermocellum gene, xynX, coding for a xylanase was cloned and the complete nucleotide sequence was determined. The xylanase gene of Clostridium thermocellum consists of an ORF of 3261 nucleotide encoding a xylanase (XynX) of 1087 amino acid residues (116 kDa). Sequence analysis
of XynX showed a multidomain structure that consisted of four different domains: an N-terminal thermostabilizing domain homologous
to sequences found in several thermophilic enzymes, a catalytic domain homologous to family 10 glycosyl hydrolases, a duplicated
cellulose-binding domain (CBD) homologous to family IX CBDs, and a triplicated S-layer homologous domain. A deletion mutant
of xynX having only the catalytic region produced a mutant enzyme XynX-C which retained catalytic activity but lost thermostability.
In terms of half-life at 70 °C, the thermostability of XynX-C was about six times lower than that of the other mutant enzyme,
XynX-TC, produced by a mutant containing both the thermostabilizing domain and the catalytic domain. The optimum temperature
of XynX-C was about 5–10 °C lower than that of XynX-TC.
Received: 12 January 2000 / Received revision: 24 April 2000 / Accepted: 1 May 2000 相似文献
13.
The thermotolerant fungus, Aspergillus niger NCIM 563, was used for production of extracellular phytase on agricultural residues: wheat bran, mustard cake, cowpea meal,
groundnut cake, coconut cake, cotton cake and black bean flour in solid state fermentation (SSF). Maximum enzyme activity
(108 U g−1 dry mouldy bran, DMB) was obtained with cowpea meal. During the fermentation phytic acid was hydrolysed completely with a
corresponding increase in biomass and phytase activity within 7 days. Phosphate in the form of KH2PO4 (10 mg per 100 g of agriculture residue) increased phytase activity. Among various surfactants added to SSF, Trition X-100
(0.5%) exhibited a 30% increase in phytase activity. The optimum pH and temperature of the crude enzyme were 5.0 and 50°C
respectively. Phytase activity (86%) was retained in buffer of pH 3.5 for 24 h. The enzyme retained 75% of its activity on
incubation at 55°C for 1 h. In the presence of 1 mM K+ and Zn2+, 95% and 55% of the activity were retained. Scanning electron microscopy showed a high density growth of fungal mycelia on
wheat bran particles during SSF. Journal of Industrial Microbiology & Biotechnology (2000) 24, 237–243.
Received 07 June 1999/ Accepted in revised form 18 December 1999 相似文献
14.
The lipA gene, a structural gene encoding for protein of molecular mass 48 kDa, and lipB gene, encoding for a lipase-specific chaperone with molecular mass of 35 kDa, of Pseudomonas aeruginosa B2264 were co-expressed in heterologous host Escherichia coli BL21 (DE3) to obtain in vivo expression of functional lipase. The recombinant lipase was expressed with histidine tag at
its N terminus and was purified to homogeneity using nickel affinity chromatography. The amino acid sequence of LipA and LipB
of P. aeruginosa B2264 was 99–100% identical with the corresponding sequence of LipA and LipB of P. aeruginosa LST-03 and P. aeruginosa PA01, but it has less identity with Pseudomonas cepacia (Burkholderia cepacia) as it showed only 37.6% and 23.3% identity with the B. cepacia LipA and LipB sequence, respectively. The molecular mass of the recombinant lipase was found to be 48 kDa. The recombinant
lipase exhibited optimal activity at pH 8.0 and 37°C, though it was active between pH 5.0 and pH 9.0 and up to 45°C. K
m and V
max values for recombinant P. aeruginosa lipase were found to be 151.5 ± 29 μM and 217 ± 22.5 μmol min−1 mg−1 protein, respectively. 相似文献
15.
A total of sixteen spontaneously generated, independent suppressor mutants was isolated from a mutant (divE42) of Escherichia coli K12 that is defective in cell division. One of the suppressor mutants, designated TR4, had a novel phenotype: it was able
to grow at 42° C but not at 32° C. The Kohara genomic library was screened for complementing clones. Clone 148 was able to
complement the mutation responsible for the cold-sensitive phenotype, and the gene for trigger factor (tig), which encodes a ribosome-associated peptidyl-prolyl cis/trans isomerase, was identified as the mutated gene by deletion analysis with the insert DNA from clone 148. DNA sequencing revealed
that the mutation in the tig gene of the TR4 suppressor mutant was a single nucleotide insertion (+A) at a distance of 834 nucleotides from the initiation
codon for this enzyme. When the wild-type tig gene was introduced into the TR4 suppressor mutant, the bacteria were able to grow at 32° C but not at 42° C, an indication
that the intergenic suppressor mutation was recessive to the wild-type allele. A model is proposed that accounts for the phenotypes
of the divE42 mutant and the TR4 suppressor mutant.
Received: 3 March 1998 / Accepted: 14 July 1998 相似文献
16.
M. Graf A. Brunella M. Kittelmann K. Laumen O. Ghisalba 《Applied microbiology and biotechnology》1997,47(6):650-657
A new amidohydrolase deacetylating several N-acetyl-1-phenylethylamine derivatives (R)-specifically was found in Arthrobacter aurescens AcR5b. The strain was isolated from a wet haystack by enrichment culture with (R)-N-acetyl-1-phenylethylamine as the sole carbon source. (R) and (S )-N-acetyl-1-phenylethylamine do not serve as inducers for acylase formation. By improving the growth conditions the enzyme production
was increased 47-fold. The amidohydrolase was purified to homogeneity leading to a 5.2-fold increase of the specific activity
with a recovery of 67%. A molecular mass of 220 kDa was estimated by gel filtration. Sodium dodecyl sulfate/polyacrylamide
gel electrophorosis shows two subunits with molecular masses of 16 kDa and 89 kDa. The optimum pH and temperature were pH 8
and 50 °C, respectively. The enzyme was stable in the range of pH 7–9 and at temperatures up to 30 °C. The enzyme activity
was inhibited by Cu2+, Co2+, Ni2+, and Zn2+, and this inhibition was reversed by EDTA.M
Received: 20 September 1996 / Received version: 23 December 1996 / Accepted: 30 December 1996 相似文献
17.
The biodegradation of tributyl phosphate (Bu3-P, TBP), releasing phosphate at a high enough concentration locally to precipitate uranium from solution, was demonstrated
by a mixed culture consisting primarily of pseudomonads. The effect of various parameters on Bu3-P biodegradation by growing cells is described. Growth at the expense of Bu3-P as the carbon and phosphorus source occurred over a pH range from 6.5 to 8, and optimally at pH 7. Bu3-P biodegradation was optimal at 30 °C, reduced at 20 °C and negligible at 4 °C and 37 °C. Incorporation of Cu or Cd inhibited,
and Ni, Co and Mn reduced its degradation. Inorganic phosphate (above 10 mM) and kerosene (up to 1 g/l) reduced Bu3-P biodegradation significantly, but nitrate had no effect. Sulphate (10–100 mM) was inhibitory. When pregrown biomass was used
the fastest rates of tributyl and dibutyl phosphate biodegradation were 25 μmol h−1 mg protein−1 and 37 μmol h−1 mg protein−1 respectively. Microcarrier-immobilised biomass decontaminated uranium-bearing acid mine waste water by uranium phosphate
precipitation at the expense of Bu3-P hydrolysis in the presence of 35 mM SO4
2−. At pH 4.5, 79% of the UO2
2+ was removed at a flow rate of 1.4 ml/h on a 7-ml test column.
Received: 2 June 1997 / Received revision: 15 September 1997 / Accepted: 19 September 1997 相似文献
18.
J Tchango Tchango D Watier P Eb R Tailliez T Njine J P Hornez 《Journal of industrial microbiology & biotechnology》1997,18(1):26-29
The combined effects of temperature (2–46°C) and pH (1.55–6.25) on the growth of Candida pelliculosa isolated from guava nectar produced in Cameroon were studied using a turbidity method, ie measurement of optical density
at 630 nm. A quadratic polynomial model was constructed to predict the effects and interactions of these two environmental
conditions on the maximal optical density obtained (r
2 = 0.97). The relation between optical density and population density of C. pelliculosa (CFU ml−1) was also established using an exponential regression (r
2 = 0.99). According to the model, maximal growth conditions were 37°C and pH 6.25 for obtaining the maximal optical density
of 1.25 corresponding to about 60 × 106 CFU ml−1. A good agreement of the model was found between the predicted values and the observed values of maximal optical density.
The model was validated by the experimental values of maximal optical density obtained in the growth of C. pelliculosa in commercial guava nectar (pH 3.15).
Received 01 December 1995/ Accepted in revised form 30 August 1996 相似文献
19.
The cellulolytic myxobacterium Sorangium cellulosum is able to efficiently degrade many kinds of polysaccharides, but none of the enzymes involved have been characterized. In
this paper, a xylanase gene (xynA) was cloned from S. cellulosum So9733-1 using thermal asymmetric interlaced PCR. The gene is composed of 1,209 bp and has only 52.27% G + C content, which
is much lower than that of most myxobacterial DNA reported (67–72%). Gene xynA encodes a 402 amino acid protein that contains a single catalytic domain belonging to the glycoside hydrolase family 10.
The novel xylanase gene, xynA, was expressed in Escherichia coli BL21 (DE3) and the recombinant protein (r-XynA) was purified by Ni-affinity chromatography. The r-XynA had the optimum temperature
of 30–35°C and exhibited 33.3% activity at 5°C and 13.7% activity at 0°C. Approximately 80% activity was lost after 20-min
pre-incubation at 50°C. These results indicate that r-XynA is a cold-active xylanase with low thermostability. At 30°C, the
K
m values of r-XynA on beechwood xylan, birchwood xylan, and oat spelt xylan were 25.77 ± 4.16, 26.52 ± 4.78, and 38.13 ± 5.35 mg/mL,
respectively. The purified r-XynA displayed optimum activity at pH 7.0. The activity of r-XynA was enhanced by the presence
of Ca2+. The r-XynA hydrolyzed beechwood xylan, birchwood xylan, and xylooligosaccharides (xylotriose, xylotetraose, and xylopentose)
to produce primarily xylose and xylobiose. To our knowledge, this is the first report on the characterization of a xylanase
from S. cellulosum. 相似文献
20.
Zhang R Yang P Huang H Yuan T Shi P Meng K Yao B 《Applied microbiology and biotechnology》2011,92(2):317-325
A phytase-encoding gene (phyA115) was cloned from Janthinobacterium sp. TN115, a symbiotic bacterial strain isolated from the gut contents of Batocera horsfieldi larvae (Coleoptera: Cerambycidae), and expressed in Escherichia coli. The 1,884-bp full-length gene encodes a 28-residue putative signal peptide and a 599-residue mature protein with a calculated
mass of 64 kDa. The deduced PhyA115 shares low identity with known sequences (47% at most) and contains an N-terminal incomplete
domain (residues 29–297; domain N) and a typical β-propeller phytase domain at the C terminus (residues 298–627; domain C).
Distinct from other β-propeller phytases that have neutral pH optima (pH 6.0–7.5), purified recombinant PhyA115 exhibits maximal
activity at pH 8.5 and 45°C in the presence of 1 mM Ca2+ and is highly active over a wider pH range (pH 6.0–9.0). These results indicate that PhyA115 is a β-propeller phytase that
has application potential in aquaculture feed. To our knowledge, this is the first report of cloning of a phytase gene from
the symbiotic microbes of an insect digestive tract and from the genus Janthinobacterium. The N-terminal incomplete domain is found to have no phytase activity but can influence the pH property of PhyA115. 相似文献