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1.
Ooi T Shibata T Sato R Ohno H Kinoshita S Thuoc TL Taguchi S 《Applied microbiology and biotechnology》2007,76(2):377-386
Three endoxylanase genes were cloned from the thermophilic fungus Chaetomium thermophilum CBS 730.95. All genes contained the typical consensus sequence of family 11 glycoside hydrolases. Genomic copies of Ct xyn11A, Ct xyn11B, and Ct xyn11C were expressed in the filamentous fungus T. reesei under the control of the strong T. reesei cel7A (cellobiohydrolase 1, cbh1) promoter. The molecular masses of the Ct Xyn11A, Ct Xyn11B, and Ct Xyn11C proteins on sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS-PAGE) were 27, 23, and 22 kDa, respectively. Ct Xyn11A was produced almost as efficiently as the
homologous xylanase II from a corresponding single-copy transformant strain. Ct Xyn11B production level was approximately
half of that of Ct Xyn11A. The amount of Ct Xyn11C was remarkably lower. Ct Xyn11A had the highest temperature optimum and
stability of the recombinant xylanases and the highest activity at acid-neutral pH (pH 5–7). It was the most suitable for
industrial bleaching of kraft pulp at high temperature. 相似文献
2.
Kazuyoshi Ohta Hidenori Tanaka Daisuke Yamakawa Hironori Hamasuna Hirohisa Fujimoto 《Journal of industrial microbiology & biotechnology》2011,38(8):967-973
An extracellular xylanase XynI of glycoside hydrolase family 11 from the dimorphic fungus Aureobasidium pullulans ATCC 20524 possesses an N-terminal extension of 34 amino acids (Ohta et al., J. Biosci. Bioeng. 92:262–270, 2001). The N-terminal
extension includes three sites (Ala-X-Ala-X-Ala-X-Ala) that are potentially cleavable by signal peptidase I of Escherichia
coli. The A. pullulans
xynI signal sequence was fused in frame to the mature protein region of the equivalent xylanase gene xynA from the filamentous fungus Penicillium citrinum. The gene fusion xynI::A was inserted into the plasmid pET-26b(+) to yield pEXP401. An E. coli BL21(DE3) transformant harboring the pEXP401 exhibited xylanase activity (per ml of the culture) of 16.8 U in the fraction
of culture supernatant as well as 4.29 U in the fraction of cell-free extract after 12 h of growth with isopropyl-β-d-thiogalactopyranoside at 30°C. N-terminal amino acid sequence analysis of the secreted recombinant proteins revealed cleavage
at four distinct sites within the N-terminal extension of XynI, two of which conformed to the Ala-X-Ala motif prior to the
cleavage site. The XynA proteins secreted into the culture medium showed high specific activities from 506 to 651 U/mg, which
were twofold higher than that of the native enzyme. 相似文献
3.
Xylanase A of Thermotoga neapolitana contains binding domains both at the N- and C-terminal ends of the catalytic domain. In the N-terminal position it contains
two carbohydrate-binding modules (CBM) which belong to family 22. These CBMs bind xylan but not to cellulose. The gene encoding
the mature peptide of these CBMs was fused with an alkaline active GH10 xylanase from Bacillus halodurans S7 and expressed in Escherichia coli. The (His)6 tagged hybrid protein was purified by immobilized metal affinity chromatography and characterized. Xylan binding by the chimeric
protein was influenced by NaCl concentration and pH of the binding medium. Binding increased with increasing salt concentration
up to 200 mM. Higher extent of binding was observed under acidic conditions. The fusion of the CBM structures enhanced the
hydrolytic efficiency of the xylanase against insoluble xylan, but decreased the stability of the enzyme. The optimum temperature
and pH for the activity of the xylanase did not change. 相似文献
4.
Anaerobic fungi belonging to the family Neocallimastigaceae are native inhabitants in the rumen of the most herbivores, such
as cattle, sheep and goats. A member of this unique group, Neocallimastix sp. GMLF2 was isolated from cattle feces and screened for its xylanase encoding gene using polymerase chain reaction. The
gene coding for a xylanase (xyn2A) was cloned in Escherichia coli and expression was monitored. To determine the enzyme activity, assays were conducted for both fungal xylanase and cloned
xylanase (Xyl2A) for supernatant and cell-associated activities. Optimum pH and temperature of the enzyme were found to be
6.5 and 50°C, respectively. The enzyme was stable at 40°C and 50°C for 20 min but lost most of its activity when temperature
reached 60°C for 5-min incubation time. Rumen fungal xylanase was mainly released to the supernatant of culture, while cloned
xylanase activity was found as cell-associated. Multiple alignment of the amino acid sequences of Xyl2A with published xylanases
from various organisms suggested that Xyl2A belongs to glycoside hydrolase family 11. 相似文献
5.
Liu JR Duan CH Zhao X Tzen JT Cheng KJ Pai CK 《Applied microbiology and biotechnology》2008,81(2):225-233
A gene encoding a xylanase, named xynS20, was cloned from the ruminal fungus Neocallimastix patriciarum. The DNA sequence of xynS20 revealed that the gene was 1,008 bp in size and encoded amino acid sequences with a predicted molecular weight of 36 kDa.
The amino acid sequence alignment showed that the highest sequence identity (28.4%) is with insect gut xylanase XYL6805. According
to the sequence-based classification, a putative conserved domain of glycosyl hydrolase family 11 was detected at the N-terminus
of XynS20 and a putative conserved domain of family 1 carbohydrate-binding module (CBM) was observed at the C-terminus of
XynS20. An Asn-rich linker sequence was found between the N-terminal catalytic domain and the C-terminal CBM of XynS20. To
examine the activity of the gene product, xynS20 gene was cloned as an oleosin-fused protein, expressed in Escherichia coli, affinity-purified by formation of artificial oil bodies, released from oleosin by intein-mediated peptide cleavage, and
finally harvested by concentration of the supernatant. The specific activity of purified XynS20 toward oat spelt xylan was
1,982.8 U mg−1. The recombinant XynS20 was stable in the mild acid pH range from 5.0 to 6.0, and the optimum pH was 6.0. The optimal reaction
temperature of XynS20 was 45°C; at temperatures below 30 and above 55°C, enzyme activity was less than 50% of that at the
optimal temperature. 相似文献
6.
Ning-Ning Song Yan Zheng Shi-Jin E Duo-Chuan Li 《Journal of microbiology (Seoul, Korea)》2009,47(1):123-130
A superoxide dismutase (SOD) gene of Thermoascus aurantiacus var. levisporus, a thermophilic fungus, was cloned, sequenced, and expressed in Pichia pastoris and its gene product was characterized. The coding sequence predicted a 231 residues protein with a unique 35 amino acids
extension at the N-terminus indicating a mitochondrial-targeting sequence. The content of Mn was 2.46 μg/mg of protein and
Fe was not detected in the purified enzyme. The enzyme was found to be inhibited by NaN3, but not by KCN or H2O2. These results suggested that the SOD in Thermoascus aurantiacus var. levisporus was the manganese superoxide dismutase type. In comparison with other MnSODs, all manganese-binding sites were also conserved
in the sequence (H88, H136, D222, H226). The molecular mass of a single band of the enzyme was estimated to be 21.7 kDa. The
protein was expressed in tetramer form with molecular weight of 68.0 kDa. The activity of purified protein was 2,324 U/mg.
The optimum temperature of the enzyme was 55°C and it exhibited maximal activity at pH 7.5. The enzyme was thermostable at
50 and 60°C and the half-life at 80°C was approximately 40 min. 相似文献
7.
The foodborne pathogen Bacillus cereus can form biofilms on various food contact surfaces, leading to contamination of food products. To study the mechanisms of
biofilm formation by B. cereus, a Tn5401 library was generated from strain UW101C. Eight thousand mutants were screened in EPS, a low nutrient medium. One mutant
(M124), with a disruption in codY, developed fourfold less biofilm than the wild-type, and its defective biofilm phenotype was rescued by complementation.
Addition of 0.1% casamino acids to EPS prolonged the duration of biofilms in the wild-type but not codY mutant. When decoyinine, a GTP synthesis inhibitor, was added to EPS, biofilm formation was decreased in the wild-type but
not the mutant. The codY mutant produced three times higher protease activity than the wild-type. Zymogram and SDS-PAGE data showed that production
of the protease (∼130 kDa) was repressed by CodY. Addition of proteinase K to EPS decreased biofilm formation by the wild-type.
Using a dpp-lacZ fusion reporter system, it was shown that that the B. cereus CodY can sense amino acids and GTP levels. These data suggest that by responding to amino acids and intracellular GTP levels
CodY represses production of an unknown protease and is involved in biofilm formation. 相似文献
8.
The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae. 相似文献
9.
The KgmB methylase (the kanamycin–gentamicin resistance methylase from Streptomyces tenebrarius) acts at G-1405 of 16S rRNA within the sequence CGUCA that is also found 6 bp in front of ribosomal binding site of the kgmB gene. The kgmBlacZ gene and operon fusions were used in order to test for translational autoregulation of kgmB gene. Overexpression of kgmB either in cis or in trans drastically decreased the level of expression of the fusion protein. However, mutagenesis eliminated any role for the CGUCA sequence in translational autoregulation. Hence, the role of second putative regulatory sequence (CGCCC) that was shown to be involved in regulation of another methylase, Sgm (sisomicin–gentamicin methylase gene from Micromonospora zionensis) was examined. It was shown that the Sgm methylase can also decrease the level of expression of the kgmBlacZ fusion protein. 相似文献
10.
11.
Genome sequence analysis of Xanthomonas
oryzae pv. oryzae has revealed a cluster of 12 ORFs that are closely related to the gum gene cluster of Xanthomonas campestris pv. campestris. The gum gene cluster of X. oryzae encodes proteins involved in xanthan production; however, there is little experimental evidence supporting this. In this
study, biochemical analyses of xanthan produced by a defined set of X. oryzae
gum mutant strains allowed us to preliminarily assign functions to most of the gum gene products: biosynthesis of the pentasaccharide repeating unit for GumD, GumM, GumH, GumK, and GumI, xanthan polymerization
and transport for GumB, GumC, GumE, and GumJ, and modification of the pentasaccharide repeating unit for GumF, GumG, and GumL.
In addition, we found that the exopolysaccharides are essential but not specific for the virulence of X. oryzae.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.
Sang-Yoon Kim and Jeong-Gu Kim contributed equally to this work. 相似文献
12.
13.
Yin Li Zhiqiang Liu Fengjie Cui Yingying Xu Hui Zhao 《World journal of microbiology & biotechnology》2007,23(6):837-843
A new strain of Penicillium sp. ZH-30 that produces xylanase was isolated from soil. According to the morphology and comparison of internal transcribed
spacer (ITS) rDNA gene sequence, the strain Penicillium sp. ZH-30 was identified as a strain of Penicillium oxalicum. When xylan or wheat bran was used as substrate at 30°C for 3 days under submerged cultivation, xylanase production was 5.3
and 13.3 U ml−1, respectively. The temperature and pH for optimum activity were 50°C and 5.0–6.0, respectively. 相似文献
14.
Fiki AE Metabteb GE Bellebna C Wartmann T Bode R Gellissen G Kunze G 《Applied microbiology and biotechnology》2007,74(6):1292-1299
The yeast Arxula
adeninivorans provides an attractive expression platform and can be exploited as gene source for biotechnologically interesting proteins.
In the following study, a striking example for the combination of both aspects is presented. The transaldolase-encoding A. adeninivorans ATAL gene, including its promoter and terminator elements, was isolated and characterized. The gene includes a coding sequence
of 963 bp encoding a putative 321 amino acid protein of 35.0 kDa. The enzyme characteristics analyzed from isolates of native
strains and recombinant strains overexpressing the ATAL gene revealed a molecular mass of ca. 140 kDa corresponding to a tetrameric structure, a pH optimum of ca. 5.5, and a temperature
optimum of 20°C. The preferred substrates for the enzyme include d-erythrose-4-phosphate and d-fructose-6-phosphate, whereas d-glyceraldehyde is not converted. The ATAL expression level under salt-free conditions was observed to increase in media supplemented with 5% NaCl rendering the ATAL promoter attractive for moderate heterologous gene expression under high-salt conditions. Its suitability was assessed for
the expression of a human serum albumin (HSA) reporter gene. 相似文献
15.
Szucs P Skinner JS Karsai I Cuesta-Marcos A Haggard KG Corey AE Chen TH Hayes PM 《Molecular genetics and genomics : MGG》2007,277(3):249-261
The epistatic interaction of alleles at the VRN-H1 and VRN-H2 loci determines vernalization sensitivity in barley. To validate the current molecular model for the two-locus epistasis,
we crossed homozygous vernalization-insensitive plants harboring a predicted “winter type” allele at either VRN-H1 (Dicktoo) or VRN-H2 (Oregon Wolfe Barley Dominant), or at both VRN-H (Calicuchima-sib) loci and measured the flowering time of unvernalized F2 progeny under long-day photoperiod. We assessed whether the spring growth habit of Calicuchima-sib is an exception to the
two-locus epistatic model or contains novel “spring” alleles at VRN-H1 (HvBM5A) and/or VRN-H2 (ZCCT-H) by determining allele sequence variants at these loci and their effects relative to growth habit. We found that (a) progeny
with predicted “winter type” alleles at both VRN-H1 and VRN-H2 alleles exhibited an extremely delayed flowering (i.e. vernalization-sensitive) phenotype in two out of the three F2 populations, (b) sequence flanking the vernalization critical region of HvBM5A intron 1 likely influences degree of vernalization sensitivity, (c) a winter habit is retained when ZCCT-Ha has been deleted, and (d) the ZCCT-H genes have higher levels of allelic polymorphism than other winterhardiness regulatory genes. Our results validate the model
explaining the epistatic interaction of VRN-H2 and VRN-H1 under long-day conditions, demonstrate recovery of vernalization-sensitive progeny from crosses of vernalization-insensitive
genotypes, show that intron length variation in VRN-H1 may account for a continuum of vernalization sensitivity, and provide molecular markers that are accurate predictors of “winter
vs spring type” alleles at the VRN-H loci. 相似文献
16.
Chenyan Zhou Dongfeng Li Minchen Wu Wu Wang 《World journal of microbiology & biotechnology》2008,24(8):1393-1401
The xylanase gene xyn II from Aspergillus usamii E001 was placed under the control of an alcohol oxidase promoter (AOX1) in the plasmid pPIC9K and integrated into the genome of a methylotrophic yeast, P. pastoris GS115, by electroporation. His+ transformants were screened for on the basis of their resistance to G418 and activity assay. A transformant, P. pastoris GSC12, which showed resistance to over 6 mg G418/ml and highest xylanase activity was selected. Recombinant xylanase was
secreted by P. pastoris GSC12 24 h after methanol induction of shake-flask cultures, and reached a final yield of 3139. About 68 U/mg 120 h after
the induction. The molecular mass of this xylanase was estimated to be 21 kDa by SDS-PAGE. The optimum pH and temperature
were 4.2 and 50 °C, respectively. Xylanase was stable below 50 °C and within pH 3.0–7.0. Its activity was increased by EDTA
and Co2+ ion and strongly inhibited by Mn2+, Li+ and Ag+ ions. The K
m and V
max values with birchwood xylan as the substrate were found to be 5.56 mg/ml and 216 μmol/mg/min, respectively. This is the first
report on expression and characterization of xylanase from A. usamii in P. pastoris. The hydrolysis products consisted of xylooligosaccharides together with a small amount of xylose. This property made the
enzyme attractive for industrial purposes, as relatively pure xylooligosaccharides could be obtained. 相似文献
17.
This paper discusses a number of experiments performed, involving the fusion by an electric field of mesophyll protoplasts
from Solanum tuberosum cv. Bintje, S. tuberosum dihaploid clones 243, 299 and the wild tuberous disease-resistant species S. bulbocastanum and S. pinnatisectum. Three fusion experiments (S. bulbocastanum + S. tuberosum dihaploid 243, S. pinnatisectum + S. tuberosum cv. Bintje and S. pinnatisectum + S. tuberosum dihaploid 299) yielded 542 calli, the 52 ones of which produced shoots. Obtained regenerants were estimated by the flow-cytometry (FC)
and RAPD analysis to determine hybrid plants.The utilisation of the FC as a useful method for detecting somatic hybrids is
also discussed in this paper. The combination S. bulbocastanum + S. tuberosum dihaploid 243 led to the creation of eight somatic hybrids, the combination S. pinnatisectum + S. tuberosum cv. Bintje yielded four somatic hybrids and the combination S. pinnatisectum + S. tuberosum dihaploid 299 resulted in no hybrid regenerants. Morphology in vitro, growth vigour and production of tuber-like structures
were evaluated in hybrid plants. Plants were transferred in vivo for further estimation (acclimatization, habitus evaluation
and tuberization ability). 相似文献
18.
Erkang Yin Yilin Le Jianjun Pei Weilan Shao Qiyin Yang 《World journal of microbiology & biotechnology》2008,24(2):275-280
According to the amino acid sequence, a codon-optimized xylanase gene (xynA1) from Thermomyces lanuginosus DSM 5826 was synthesized to construct the expression vector pHsh-xynA1. After optimization of the mRNA secondary structure in the translational initiation region of pHsh-xynA1, free energy of the 70 nt was changed from −6.56 to −4.96 cal/mol, and the spacing between AUG and the Shine-Dalgarno sequence
was decreased from 15 to 8 nt. The expression level was increased from 1.3 to 13% of total cell protein. A maximum xylanase
activity of 47.1 U/mL was obtained from cellular extract. The recombinant enzyme was purified 21.5-fold from the cellular
extract of Escherichia coli by heat treatment, DEAE-Sepharose FF column and t-Butyl-HIC column. The optimal temperature and pH were 65 °C and pH 6.0,
respectively. The purified enzyme was stable for 30 min over the pH range of 5.0–8.0 at 60 °C, and had a half-life of 3 h
at 65 °C. 相似文献
19.
Bacillus
thuringiensis subsp. kurstaki BUPM255 secretes a chitobiosidase Chi255 having an expected molecular weight of 70.665 kDa. When the corresponding gene,
chi255, was expressed in E. coli, the active form, extracted from the periplasmic fraction of E. coli/pBADchi255, was of about 54 kDa, which suggested that Chi255 was excessively degraded by the action of E. coli proteases. Therefore, in vitro progressive C-terminal Chi255 deleted derivatives were constructed in order to study their
stability and their activity in E. coli. Interestingly, when the chitin binding domain (CBD) was deleted from Chi255, an active form (Chi2555Δ5) of expected size
of about 60 kDa was extracted from the E. coli periplasmic fraction, without the observation of any proteolytic degradation. Compared to Chi255, Chi255Δ5 exhibited a higher
chitinase activity on colloidal chitin. Both of the enzymes exhibit activities at broad pH and temperature ranges with maximal
enzyme activities at pH 5 and pH 6 and at temperatures 50°C and 40°C, respectively for Chi255 and Chi255Δ5. Thus, it was concluded
that the C-terminal deletion of Chi255 CBD might be a nice tool for avoiding the excessive chitinase degradation, observed
in the native chitinase, and for improving its activity. 相似文献
20.
Lee JH Moon YH Kim N Kim YM Kang HK Jung JY Abada E Kang SS Kim D 《Biotechnology letters》2008,30(4):749-754
The gene encoding sucrose phosphorylase (742sp) in Leuconostoc mesenteroides NRRL B-742 was cloned and expressed in Escherichia coli. The nucleotide sequence of the transformed 742sp comprised an ORF of 1,458 bp giving a protein with calculated molecular mass of 55.3 kDa. 742SPase contains a C-terminal amino acid sequence that is significantly different from those of other Leu. mesenteroides SPases. The purified 742SPase had a specific activity of 1.8 U/mg with a K
m of 3 mM with sucrose as a substrate; optimum activity was at 37°C and pH 6.7. The purified 742SPase transferred the glucosyl
moiety of sucrose to cytosine monophosphate (CMP).
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献