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1.
Jia Ouyang Shen Wang Yan Wang Xin Li Mu Chen Qiang Yong Shiyuan Yu 《World journal of microbiology & biotechnology》2011,27(4):751-758
The purpose of this study was to produce a Trichoderma reesei xylanase (XYN2) in Pichia pastoris and to test its potential application for pulp bleaching. The recombinant xylanase was purified by a two-step process of
ultrafiltration and gel filtration chromatography. The molecular mass of the recombinant enzyme was 21 and 25 kDa by SDS–PAGE
analysis, due to different glycosylation of the native protein. The optimum pH and temperature of the recombinant XYN2 was
5.0 and 50 °C. Enzyme activity was stable at 50 °C and at pH 5.0–7.0. The bleaching ability of the recombinant xylanase was
also studied at 50 °C and pH 6.0, using wheat straw pulp. Biobleaching of the xylanase produced chlorine dioxide savings of
up to 60%, while retaining brightness at the control level and led to a lower kappa number and small enhancements in tensile,
burst and tear strength of pulp fibers. 相似文献
2.
P Shi Z Qiu Y Bai T Yuan H Huang X Pan P Yang W Zhang B Yao 《World journal of microbiology & biotechnology》2012,28(2):687-692
A new xylanase gene, xynBM4, was cloned from Streptomyces megasporus DSM 41476 and expressed in Pichia pastoris. The full-length gene consists of 1,443 bp and encodes 480 amino acids including a putative 49-residue signal peptide. The
deduced amino acid sequence of xynBM4 shows the highest identity of 66.3% to the xylanase Xys1L from Streptomyces halstedii JM8. The purified recombinant XYNBM4 had a high specific activity of 350.7 U mg-1 towards soluble wheat arabinoxylan, exhibited optimal activity at pH 6.0 and 57°C, showed broad pH adaptability (>75% of
the maximum activity at pH 2.5–9.0), was resistant to neutral proteases and most chemicals, and produced simple products.
The hydrolysis products of birchwood xylan and corncob xylan were predominantly xylobiose (76.9 and 90.8%, respectively) and
no xylose. These characteristics suggest that XYNBM4 has potential in various applications, especially in the food industry. 相似文献
3.
Seven lipolytic genes were isolated and sequenced from a metagenomic library that was constructed following biomass enrichment
in a fed-batch bioreactor submitted to high temperature (50–70°C) and alkaline pH (7–8.5). Among those sequences, lipIAF1-6 was chosen for further study and cloned in Streptomyces lividans 10–164. The G+C content within the sequence was 64.3%. The encoded protein, LipIAF1-6, was related to various putative lipases
previously identified in different genome sequences. Homology of LipIAF-6 with the different lipases did not exceed 31%. The
optimum pH (8.5) and temperature (60°C) of the purified enzyme were in agreement with the enrichment conditions. Furthermore,
the enzyme was thermostable for as long as 30 min at 70°C. The maximum activity of the purified lipase was 4,287 IU/mg towards
p-nitrophenyl (p-NP) butyrate (60°C; pH 8.5). LipIAF1-6 does not seem to need the presence of metal ions for its activity. The enzyme was
slightly inhibited by 10 mM CoCl2 (14%), HgCl2 (12%), and dithiothreitol (DTT) (15%). The serine protease inhibitor phenylmethylsulphonyl fluoride (PMSF) reduced activity
by 39% and 71% when incubated at concentrations of 1 and 10 mM, respectively. Finally, LipIAF1-6 was stable in different organic
solvents, and against several surfactants and oxidative agents commonly found in detergent formulations. These results are
quite encouraging for further use of this enzyme in different industrial processes. 相似文献
4.
A gene (arf) encoding an α-l-arabinofuranosidase (ARF) that hydrolyzes arabinose substituted on xylan was isolated from Penicillium sp. The gene was predicted to encode 339 amino acid residues showing 71–75% homology to GH family 54. E. coli expressed ARF showed optimal activity at 50°C and pH 5–6 on wheat arabinoxylan. The hydrolysis activities on oat spelt xylan
by ARF and xylanase were 1.67-fold higher than that of xylanase alone. The synergistic effects of ARF and commercial enzymes
(xylanase and cellulase) on popping-pretreated rice straw were 1.15–1.51-fold higher amounts of sugars released in the [ARF + xylanase + cellulase]
mixture than in the mixtures [ARF + xylanase], [ARF + cellulase], and [xylanase + cellulase]. Moreover, the liberation of
arabinose by ARF was enhanced 2.1–2.9-fold in a reaction with xylanase and cellulase as compared with [xylanase + cellulase]
and ARF alone. 相似文献
5.
Junpei Zhou Pengjun Shi Rui Zhang Huoqing Huang Kun Meng Peilong Yang Bin Yao 《Journal of industrial microbiology & biotechnology》2011,38(4):523-530
A pH-stable and protease-resistant xylanase (XynB119) was identified from Streptomyces sp. TN119, a strain isolated from the gut luminal contents of longhorned beetle (Batocera horsfieldi) larvae. Using the GC TAIL-PCR method, the 1,026-bp coding gene (xynB119) with 67.3% GC content was successfully cloned and expressed in Escherichia coli. It encodes a 341-residue polypeptide with a calculated molecular mass of 35.9 kDa, including a putative 41-residue signal
peptide, a catalytic domain of glycosyl hydrolase (GH) family 11, a short Gly/Pro-rich linker, and a family 2 cellulose-binding
domain (CBM 2). The deduced amino acid sequence is most similar to (61.9% identity) an endo-1,4-β-xylanase from Streptomyces thermoviolaceus OPC-520. Purified recombinant XynB119 exhibited peak activity at 50°C and pH 7.0, remained stable over a broad pH range (retaining >70%
activity after incubation at pH 1.0–11.0 for 1 h at 37°C without substrate), had strong protease resistance (retaining >90%
activity after proteolytic treatment at 37°C for 1 h) and SDS resistance (at 100 mM). These properties make XynB119 promising
for application in the feed industry and valuable for basic research. Compared to r-XynB119, the r-XynB119 derivative without
CBM 2 and linker region (r-XynB119d) exhibited a decreased pH stability of >25% at extreme pHs (pH 1.0–3.0 and pH 11.0–12.0). 相似文献
6.
Andrzej K. Siwicki Zdzisław Zakęś Elżbieta Terech-Majewska Krzysztof Kazuń Agnieszka Lepa Edward Głąbski 《Reviews in Fish Biology and Fisheries》2010,20(3):435-439
Influence of beta-1.3/1.6-glucan (Macrogard) on the innate immunity and protection against Aeromonas hydrophila in tench (Tinca tinca (L.)) was assessed. Macrogard was fed at doses of 0, 0.5, 1 and 2 g kg−1 of pellets for 1 month. The blood, spleen and head kidney from 10 fish of each group were separated and analysed for immunity
parameters. Twenty tench from each group were infected with A. hydrophila. Macrogard at doses 1 and 2 g kg−1 of feed significantly (P < 0.05) increased the phagocytic activity of macrophages and proliferative response of mitogens stimulated lymphocytes. The
same doses significantly (P < 0.05) increased lysozyme activity and Ig level in serum, compared to the control and dose 0.5 g kg−1 of feed. The challenge test showed that Macrogard reduced mortality of tench after experimental infection (5–35%). 相似文献
7.
Berrin JG Ajandouz el H Georis J Arnaut F Juge N 《Applied microbiology and biotechnology》2007,74(5):1001-1010
Two genes encoding family 11 endo-(1,4)-β-xylanases from Penicillium griseofulvum (PgXynA) and Penicillium funiculosum (PfXynC) were heterologously expressed in Escherichia coli as glutathione S-transferase fusion proteins, and the recombinant enzymes were purified after affinity chromatography and
proteolysis. PgXynA and PfXynC were identical to their native counterparts in terms of molecular mass, pI, N-terminal sequence,
optimum pH, and enzymatic activity towards arabinoxylan. Further investigation of the rate and pattern of hydrolysis of PgXynA
and PfXynC on wheat soluble arabinoxylan showed the predominant production of xylotriose and xylobiose as end products. The
initial rate data from the hydrolysis of short xylo-oligosaccharides indicated that the catalytic efficiency increased with
increasing chain length (n) of oligomer up to n = 6, suggesting that the specificity region of both Penicillium xylanases spans about six xylose units. In contrast to PfXynC, PgXynA was found insensitive to the wheat xylanase inhibitor
protein XIP-I. 相似文献
8.
The trifunctional enzyme (XAR–XYN) associating the Thermoanaerobacter ethanolicus xylosidase-arabinosidase (XAR) with the Thermomyces lanuginosus xylanase (XYN) was produced in E. coli to study the effect of the physical association of the fusion partners on the enzymatic efficiency. Recombinant XAR, XYN and XAR–XYN were purified to homogeneity and characterized. The optimal pH and temperature of the XAR–XYN were found to be similar to those of the XAR and XYN, except for less temperature optimum of α-arabinosidase activity. Its pH and xylanase activity exhibited more stable than those of the XAR and XYN. Finally, the XAR–XYN was tested for degradation of oat spelt xylan and wheat bran, the XAR–XYN was found to be more facile than the corresponding free enzyme degradation of wheat bran but provided little or no advantage on purified xylan. Furthermore cooperation within a trifunctional enzyme containing linker SAGSSAAGSGSG between each partner was achieved, leading to a trifunctional enzyme with enhanced enzymatic efficiency on arabinoxylan. 相似文献
9.
The recombinant Bordetella pertussis CyaA pore-forming (CyaA-PF) fragment was previously shown to be expressed separately in Escherichia coli as a soluble precursor that can be in vivo palmitoylated to exert haemolytic activity. In this study, PCR-based mutagenesis
was employed to investigate the contributions to haemolysis of five predicted helices within the N-terminal hydrophobic region
of the CyaA-PF fragment. Single proline substitutions were made for alanine near the centre of each predicted helix as a means
of disrupting local secondary structure. All mutant proteins were over-expressed in E. coli as a 126-kDa soluble protein at levels comparable to the wild-type. Marked reductions in haemolytic activity against sheep
erythrocytes of mutants, A510P, A538P, A583P and A687P pertaining to the putative helices 1500–522, 2529–550, 3571–593 and 5678–698, respectively, were observed. However, a slight decrease in haemolytic activity was found for the proline replacement in
the predicted helix 4602–627 (A616P). MALDI–TOF–MS and LC–MS–MS analyses verified the palmitoylation at Lys983 of all five mutants as identical to that of the CyaA-PF wild-type protein, indicating that toxin modification via this acylation
was not affected by the mutations. Altogether, these results suggest that structural integrity of the predicted helices 1,
2, 3 and 5, but not helix 4, is important for haemolytic activity, particularly for the putative transmembrane helices 2 and
3 that might conceivably be involved in pore formation of the CyaA-PF fragment. 相似文献
10.
Duwoon Kim Keun Sik Baik Seong Chan Park Seon-Jun Kim Tai-Sun Shin Sung-Joo Jung Myung-Joo Oh Chi Nam Seong 《Journal of industrial microbiology & biotechnology》2009,36(11):1375-1382
Pseudoalteromonas sp. NO3 was isolated from the hemolymph of diseased sea squirts (Halocynthia rorentzi) with symptoms of soft tunic syndrome. The strain was found to produce an extracellular cellulase (CelY) that consisted of
a 1,476 bp open reading frame encoding 491 amino acid residues with an approximate molecular mass of 52 kDa. Homologies of
the deduced amino acid sequence of celY with the products of the celA, celX, celG and cel5Z genes were 92.6, 93.3, 92.6, and 59.1%, respectively. Additionally, CelY had 50–80% remnant catalytic activity at temperatures
of 10–20°C. Highest carboxymethyl cellulose (CMC) hydrolysis was observed at pH 8.0 and 40°C. CMC activity was determined
by zymogram active staining and different degraded product profiles for CelY were obtained when cellotetraose, cellopentaose,
and CMC were used as substrates. This study identified a transglycosylation activity in CelY that allows the enzyme to digest
G4 to G2 and G3 without the production of G1. 相似文献
11.
Luo H Wang K Huang H Shi P Yang P Yao B 《Journal of industrial microbiology & biotechnology》2012,39(4):547-555
In this article, we firstly report a highly alkali-tolerant fungal β-mannanase from Humicola
insolens Y1. The full-length cDNA of the β-mannanase, designated as man5A, has an open reading frame of 1,233 bp that encodes a 411-amino acid polypeptide (Man5A) with a calculated molecular mass
of 42.3 kDa. The deduced sequence of Man5A comprises a putative 20-residue signal peptide and a catalytic domain belonging
to glycoside hydrolase family 5, and displays 61–85% identities with hypothetical proteins and 32–39% with experimentally
verified fungal β-mannanases. Purified recombinant Man5A produced by Pichia pastoris has a specific activity of 1,122 U mg−1 and exhibits optimal activity at pH 5.5 and 70°C. Distinct from other reported fungal β-mannanases, Man5A is highly alkali
tolerant, exhibiting 45 and 36% of the maximal activity at pH 8.0 and 9.0, respectively, and more than 10% activity even at
pH 10.0. Moreover, Man5A has excellent pH stability at pH 5.0–12.0 and is highly thermostable at 50°C. The higher frequency
of alkaline amino acids (Arg and Lys), greater pKa values of the catalytic residues, and more positively charged residues on the surface of Man5A might be the causes. Man5A
has strong resistance to various neutral and alkaline proteases, retaining more than 97% of the activity after proteolytic
treatment for 1 h. The superior characteristics of Man5A make it more advantageous for the application in the kraft pulp industry. 相似文献
12.
Sørensen HR Jørgensen CT Hansen CH Jørgensen CI Pedersen S Meyer AS 《Applied microbiology and biotechnology》2006,73(4):850-861
A novel α-l-arabinofuranosidase (α-AraF) belonging to glycoside hydrolase (GH) family 43 was cloned from Humicola insolens and expressed in Aspergillus oryzae. 1H-NMR analysis revealed that the novel GH43 enzyme selectively hydrolysed (1→3)-α-l-arabinofuranosyl residues of doubly substituted xylopyranosyl residues in arabinoxylan and in arabinoxylan-derived oligosaccharides. The optimal activity of the cloned enzyme was at pH 6.7 and 53 °C. Two other novel α-l-arabinofuranosidases (α-AraFs), both belonging to GH family 51, were cloned from H. insolens and from the white-rot basidiomycete Meripilus giganteus. Both GH51 enzymes catalysed removal of (1→2) and (1→3)-α-l-arabinofuranosyl residues from singly substituted xylopyranosyls in arabinoxylan; the highest arabinose yields were obtained with the M. giganteus enzyme. Combinations (50:50) of the GH43 α-AraF from H. insolens and the GH51 α-AraFs from either M. giganteus or H. insolens resulted in a synergistic increase in arabinose release from water-soluble wheat arabinoxylan in extended reactions at pH 6 and 40 °C. This synergistic interaction between GH43 and GH51 α-AraFs was also evident when a GH43 α-AraF from a Bifidobacterium sp. was supplemented in combination with either of the GH51 enzymes. The synergistic effect is presumed to be a result of the GH51 α-AraFs being able to catalyse the removal of single-sitting (1→2)–α-l-arabinofuranosyls that resulted after the GH43 enzyme had catalysed the removal of (1→3)–α-l-arabinofuranosyl residues on doubly substituted xylopyranosyls in the wheat arabinoxylan. 相似文献
13.
Jianshe Wang Yingguo Bai Pengjun Shi Huiying Luo Huoqing Huang Jun Yin Bin Yao 《World journal of microbiology & biotechnology》2011,27(2):207-213
A xylanase gene, xynA4-2, was obtained from the genome sequence of thermoacidophilic Alicyclobacillus sp. A4 and expressed in Escherichia coli BL21 (DE3). xynA4-2 encodes a mature protein of 411 residues with a calculated molecular weight of 46.8 kDa. Based on the amino acid sequence
similarities (highest identity of 61%), the enzyme was confined into glycoside hydrolase family 10. The purified recombinant
XynA4-2 exhibited maximum activity at pH 6.2 and 55°C. The enzyme was stable over a broad pH range, retaining more than 90%
of the original activity at pH 5.8–12.0, 37°C for 1 h. The substrate specificity of XynA4-2 was relatively narrow, exhibiting
100, 93, and 35% of the relative activity towards birchwood xylan, oat spelt xylan, and wheat arabinoxylan, respectively.
Supplementation of XynA4-2 to mash caused the reduction of mash filtration rate (5.6%) and viscosity (4.0%). When combined
with the commercial glucanase from Sunson, higher reduction was detected in the filtration rate (12.0%) and viscosity (17.2%).
These favorable properties make XynA4-2 a good candidate in the brewing industry. 相似文献
14.
J. H. A. Betini M. Michelin S. C. Peixoto-Nogueira J. A. Jorge H. F. Terenzi M. L. T. M. Polizeli 《Bioprocess and biosystems engineering》2009,32(6):819-824
This study describes the production of xylanases from Aspergillus niveus, A. niger, and A. ochraceus under solid-state fermentation using agro-industrial residues as substrates. Enzyme production was improved using a mixture
of wheat bran and yeast extract or peptone. When a mixture of corncob and wheat bran was used, xylanase production from A. niger and A. ochraceus increased by 18%. All cultures were incubated at 30 °C at 70–80% relative humidity for 96 h. For biobleaching assays, 10
or 35 U of xylanase/g dry cellulose pulp were incubated at pH 5.5 for 1 or 2 h, at 55 °C. The delignification efficiency was
20%, the brightness (percentage of ISO) increased two to three points and the viscosity was maintained confirming the absence
of cellulolytic activity. These results indicated that the use of xylanases could help to reduce the amount of chlorine compounds
used in cellulose pulp treatment. 相似文献
15.
A novel protein from Gluconobacter oxydans DSM2003 which shows 60–70% similarity with members of aldo–keto reductase (AKR) superfamily was overexpressed in Escherichia coli BL21 (DE3) and purified by one step affinity chromatography with a Ni-NTA agarose column. The recombinant protein (named
GOX0644) consists of 279 amino acids with an apparent molecular mass of 32 kDa in the soluble fraction, and the gene sequence
encoding the protein GOX0644 is 100% identical to the ORF of gox0644 in G. oxydans 621H (DSM2343). For a detailed analysis of its enzymatic activity, the substrate specificity of the recombinant protein GOX0644
was determined. With NADPH as a cofactor, GOX0644 exhibited better activity to aromatic aldehydes, especially o-chlorobenzaldehyde, compared to aliphatic aldehydes. It showed almost no activity toward glyceraldehyde, xylose, glucose,
and ketones. The protein was unable to oxidize primary- or secondary alcohols. Based on these results, GOX0644 was defined
as a novel NADPH-dependent aldehyde reductase. Kinetic parameters of the protein and the dependence of its activity on temperature
and pH were also determined. 相似文献
16.
The tortoise tick Hyalomma aegyptium has a typical three-host life-cycle. Whereas its larvae and nymphs are less host-specific feeding on a variety of tetrapods,
tortoises of the genus Testudo are principal hosts of adults. Ticks retained this trait also in our study under laboratory conditions, while adults were
reluctant to feed on mammalian hosts. Combination of feeding larvae and nymphs on guinea pigs and feeding of adults on Testudo marginata tortoises provided the best results. Feeding period of females was on average 25 days (range 17–44), whereas males remain
after female engorgement on tortoise host. Female pre-oviposition period was 14 days (3–31), followed by 24 days of oviposition
(18–29). Pre-eclosion and eclosion, both together, takes 31 days (21–43). Larvae fed 5 days (3–9), then molted to nymphs after
17 days (12–23). Feeding period of nymphs lasted 7 days (5–10), engorged nymphs molted to adults after 24 days (19–26). Sex
ratio of laboratory hatched H. aegyptium was nearly equal (1:1.09). The average weight of engorged female was 0.95 (0.72–1.12) g. The average number of laid eggs
was 6,900 (6,524–7,532) per female, it was significantly correlated with weight of engorged female. Only 2.8% of engorged
larvae and 1.8% of engorged nymphs remained un-molted and died. Despite the use of natural host species, feeding success of
females reached only 45%. The whole life-cycle was completed within 147 days (98–215). 相似文献
17.
Pilar Pérez Aitor Alonso Gloria Zita Rosa Morcuende Rafael Martínez-Carrasco 《Plant Growth Regulation》2011,65(3):439-447
Increases in growth temperature have been observed to affect photosynthesis differently under long-term exposure to ambient-
and twice ambient-air CO2 concentrations. This study investigates the causes of this interaction in wheat (Triticum aestivum L.) grown in the field over two consecutive years under temperature gradient chambers in ambient (370 μmol mol−1) or elevated (700 μmol mol−1) atmospheric CO2 concentrations and at ambient or ambient +4°C temperatures, with either a low or a high nitrogen supply. The photosynthesis-internal
CO2 response curves and the activity, activation state, kcat and amount of Ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) were measured, as well as the soluble protein concentration
in flag leaves at ear emergence and 8–15 days after anthesis. A high nitrogen supply increased Vcmax, the Rubisco amount and activity and soluble protein contents, but did not significantly change the Rubisco kcat. Both elevated CO2 and above ambient temperatures had negative effects on Vcmax and Rubisco activity, but at elevated CO2, an increase in temperature did not decrease Vcmax or Rubisco activity in relation to ambient temperature. The amounts of Rubisco and soluble protein decreased with elevated
CO2 and temperature. The negative impact of elevated CO2 on Rubisco properties was somewhat counteracted at elevated temperatures by an increase in kcat. This effect can diminish the detrimental effects on photosynthesis of combined increases of CO2 and temperature. 相似文献
18.
We investigated seasonal variation of grazing impact of the pigmented nanoflagellates (PNF) with different sizes upon Synechococcus in the subtropical western Pacific coastal waters using grazing experiments with fluorescently labeled Synechococcus (FLS). For total PNF, conspicuous seasonal variations of ingestion rates on Synechococcus were found, and a functional response was observed. To further investigate the impact of different size groups, we separated
the PNF into four categories (<3, 3–5, 5–10, and >10 μm). Our results indicated that the smallest PNF (<3 μm PNF) did not
ingest FLS and was considered autotrophic. PNF of 3–5 μm in size made up most of the PNF community; however, their ingestion
on Synechococcus was too low (0.1–1.9 Syn PNF−1 h−1) to support their growth, and they had to depend on other prey or photosynthesis to survive. The ingestion rate of the 3–5 μm
group exhibited no significant seasonal variation; by contrast, the ingestion rates of 5–10 and >10 μm PNFs showed significant
seasonal variation. During the warm season, 3–5 μm PNF were responsible for the grazing of 12% of Synechococcus production, 5–10 μm PNF for 48%, and >10 μm PNF for 2%. Taken together, our results demonstrate that the PNF of 3–10 μm consumed
most Synechococcus during the warm season and exhibited a significant functional response to the increase in prey concentration. 相似文献
19.
Zhongbiao Tan Jianfang Li Minchen Wu Cunduo Tang Huimin Zhang Junqing Wang 《World journal of microbiology & biotechnology》2011,27(12):2767-2774
A 777-bp cDNA fragment encoding a mature alkaline lipase (LipI) from Penicillium cyclopium PG37 was amplified by RT–PCR, and inserted into the expression plasmid pPIC9 K. The recombinant plasmid, designated as pPIC9 K-lipI,
was linearized with SalI and transformed into Pichia pastoris GS115 (his4, Mut+) by electroporation. MD plate and YPD plates containing G418 were used for screening of the multi-copy P. pastoris transformants (His+, Mut+). One transformant resistant to 4.0 mg/ml of G418, numbered as P. pastoris GSL4-7, expressing the highest recombinant LipI (rLipI) activity was chosen for optimizing expression conditions. The integration
of the gene LipI into the P. pastoris GS115 genome was confirmed by PCR analysis using 5′- and 3′-AOX1 primers. SDS–PAGE and lipase activity assays demonstrated that the rLipI, a glycosylated protein with an apparent molecular
weight of about 31.5 kDa, was extracellularly expressed in P. pastoris. When the P. pastoris GSL4-7 was cultured under the optimized conditions, the expressed rLipI activity was up to 407 U/ml, much higher than that
(10.5 U/ml) expressed with standard protocol. The rLipI showed the highest activity at pH 10.5 and 25°C, and was stable at
a broad pH range of 7.0–10.5 and at a temperature of 30°C or below. 相似文献