共查询到20条相似文献,搜索用时 239 毫秒
1.
Matsuo T Kobayashi T Kimura Y Tsuchiyama M Oh T Sakamoto T Adachi S 《Biotechnology letters》2008,30(12):2151-2156
Glyceryl ferulate was synthesized by the condensation of ferulic acid with glycerol using Pectinase PL “Amano” from Aspergillus niger, which contained ferulic acid esterase, to improve the water-solubility of ferulic acid. The optimum reaction medium was
glycerol/0.1 M acetate buffer, pH 4.0, (98:2 v/v). The enzyme immobilized onto Chitopearl BCW3003 exhibited the highest activity
among the those immobilized onto various kinds of Chitopearl BCW resins. The optimum temperature for the immobilized enzyme
was 50°C, and it could be reused at least five times without a significant loss in activity for the synthesis of glyceryl
ferulate in batch reaction. Storage of the reaction mixture at 25°C improved the molar fraction of glyceryl ferulate relative
to the dissolved ferulic residues. 相似文献
2.
A hypothetical protein AN1772.2 of Aspergillus nidulans was found to have a 56% identity with a known type C ferulic acid esterase (FAE) from Talaromyces stipitatus. In addition, it contained a 13-amino acid conserved region flanking the characteristic G-X-S-X-G motif of a serine esterase,
suggesting a FAE function for the protein. The putative FAE was successfully cloned from the genomic DNA and expressed in
Saccharomyces cerevisiae. The recombinant protein exhibited high FAE activities. Therefore, its function as an FAE was unequivocally determined. About
86% of the enzyme activity was found in the growth medium, indicating that the native signal peptide was effective in the
yeast expression system. The recombinant FAE was purified to its homogeneity, and subsequently characterized. The FAE is stable
over an unusually wide range of pH (4.0–9.5), has a pH optimum of 7.0, and a temperature optimum of 45°C. A substrate specificity
profiling reveals that the enzyme is a type B FAE, despite its strong sequence homology with type C FAEs, raising an interesting
question on the role of the conserved region in substrate specificity. 相似文献
3.
B. L. García A. S. Ball J. Rodríguez M. I. Pérez-Leblic M. E. Arias J. L. Copa-Patiño 《Applied microbiology and biotechnology》1998,50(2):213-218
Streptomyces avermitilis CECT 3339 produces extracellular ferulic acid esterase (FAE) activity during growth on a range of lignocellulose substrates.
Maximal levels of FAE activity were detected in culture filtrates from S. avermitilis CECT 3339 grown in media containing wheat bran and yeast extract as carbon and nitrogen sources respectively. Biochemical
characterization of this enzyme activity revealed that it was 100-fold higher when wheat bran was pretreated with Celluclast
(a mix of hydrolytic enzymes). FAE was found to be end-product-inhibited. Characterization of the properties of the enzyme
showed that FAE exhibited an activity optimum pH at 6 with pH stability between pH 6 and 8. The optimum temperature was 50 °C
while the temperature stability was between 30 °C and 40 °C, with rapid inactivation at 60 °C and above. The characteristics
and stability of FAE from S. avermitilis CECT 3339 suggest a potential role for this enzyme in combination with endoxylanases for the upgrading of plant-residue silage
and for biopulping.
Received: 17 November 1997 / Received revision: 13 March 1998 / Accepted: 13 April 1998 相似文献
4.
5.
Sushil Nagar Vijay Kumar Gupta Davender Kumar Lalit Kumar Ramesh Chander Kuhad 《Journal of industrial microbiology & biotechnology》2010,37(1):71-83
This paper reports the production of a cellulase-free and alkali-stable xylanase in high titre from a newly isolated Bacillus pumilus SV-85S using cheap and easily available agro-residue wheat bran. Optimization of fermentation conditions enhanced the enzyme
production to 2995.20 ± 200.00 IU/ml, which was 9.91-fold higher than the activity under unoptimized basal medium (302.2 IU/ml).
Statistical optimization using response-surface methodology was employed to obtain a cumulative effect of peptone, yeast extract,
and potassium nitrate (KNO3) on enzyme production. A 23 central composite design best optimized the nitrogen source at the 0 level for peptone and yeast extract and at the −α level
for KNO3, along with 5.38-fold increase in xylanase activity. Addition of 0.1% tween 80 to the medium increased production by 1.5-fold.
Optimum pH for xylanase was 6.0. The enzyme was 100% stable over the pH range from 5 to 11 for 1 h at 37°C and it lost no
activity, even after 3 h of incubation at pH 7, 8, and 9. Optimum temperature for the enzyme was 50°C, but the enzyme displayed
78% residual activity even at 65°C. The enzyme retained 50% activity after an incubation of 1 h at 60°C. Characteristics of
B. pumilus SV-85S xylanase, including its cellulase-free nature, stability in alkali over a long duration, along with high-level production,
are particularly suited to the paper and pulp industry. 相似文献
6.
A fragment coding for a putative extracellular α-amylase, from the genomic library of the yeast Saccharomycopsis fibuligera KZ, has been subcloned into yeast expression vector pVT100L and sequenced. The nucleotide sequence revealed an ORF of 1,485 bp
coding for a 494 amino acid residues long protein with 99% identity to the α-amylase Sfamy from S. fibuligera HUT 7212. The S. fibuligera KZ α-amylase (Sfamy KZ) belongs to typical extracellular fungal α-amylases classified in the glycoside hydrolase family 13,
subfamily 1, as supported also by clustering observed in the evolutionary tree. Sfamy KZ, in addition to the essential GH13
α-amylase three-domain arrangement (catalytic TIM barrel plus domains B and C), does not contain any distinct starch-binding
domain. Sfamy KZ was expressed as a recombinant protein in Saccharomyces cerevisiae and purified to electrophoretic homogeneity. The enzyme had a molecular mass 53 kDa and contained about 2.5% of carbohydrate.
The enzyme exhibited pH and temperature optima in the range of 5–6 and 40–50 °C, respectively. Stable adsorption of the enzyme
to starch granules was not detected but a low degradation of raw starch in a concentration-dependent manner was observed. 相似文献
7.
Jaya Ram Simkhada Hyo Jeong Lee So Young Jang Seung Sik Cho Eun Jung Park Jae Kyung Sohng Jin Cheol Yoo 《Biotechnology letters》2009,31(3):429-435
A 60 kDa phospholipase D (PLD) was obtained from Streptomyces olivochromogenes by one-step chromatography on Sepharose CL-6B. Maximal activity was at pH 8 and 75°C and the enzyme was stable from pH 7
to 13 and from 55 to 75°C. Thermal and pH stability with temperature optimum of the enzyme were highest among Streptomyces PLDs reported so far. The activity was Ca2+-dependent and enhanced by detergents. The Km and Vmax values for phosphatidylcholine were 0.6 mM and 650 μmol min−1 mg−1, respectively. In addition, the enzyme also revealed transphosphatidylation activity, which was optimum at pH 8 and 50°C.
The first 15 amino acid residues of the N terminal sequence were ADYTPGAPGIGDPYY, which are significantly different from the
other known PLDs. The enzyme may therefore be a novel PLD with potential application in the lipid industry. 相似文献
8.
Jianshe Wang Yingguo Bai Peilong Yang Pengjun Shi Huiying Luo Kun Meng Huoqing Huang Jun Yin Bin Yao 《World journal of microbiology & biotechnology》2010,26(5):917-924
A xylanase gene, xynE2, was cloned from thermoalkaline Anoxybacillus sp. E2 and was expressed in Escherichia coli BL21 (DE3). The gene consisted of 987 bp and encoded a 328-residue xylanase with a calculated molecular weight of 38.8 kDa.
On the basis of amino acid sequence similarities, this enzyme was assigned as a member of glycoside hydrolase family 10. Purified
recombinant XynE2 showed maximal activity at pH 7.8 and 65°C, and was thermostable at 60°C. The enzyme was highly active and
stable over a broad pH range, showing more than 90% of maximal activity at pH 6.6–pH 8.6 and retaining more than 80% of activity
at pH 4.6–pH 12.0, 37°C for 1 h, respectively. These favorable properties make XynE2 a good candidate in the pulp and paper
industries. This is the first report on gene cloning, expression and characterization of a xylanase from the genus Anoxybacillus. 相似文献
9.
Huang HK Tokashiki M Maeno S Onaga S Taira T Ito S 《Journal of industrial microbiology & biotechnology》2012,39(1):55-62
A heat-labile phenolic acid decarboxylase from Candida guilliermondii (an anamorph of Pichia guilliermondii) was purified to homogeneity by simple successive column chromatography within 3 days. The molecular mass was 20 kDa by sodium
dodecyl sulfate–polyacrylamide gel electrophoresis and 36 kDa by gel-filtration chromatography, suggesting that the purified
enzyme is a homodimer. The optimal pH and temperature were approximately 6.0 and 25°C. Characteristically, more than 50% of
the optimal activity was observed at 0°C, suggesting that this enzyme is cold-adapted. The enzyme converted p-coumaric acid, ferulic acid, and caffeic acid to corresponding products with high specific activities of approximately 600,
530, and 46 U/mg, respectively. The activity was stimulated by Mg2+ ions, whereas it was completely inhibited by Fe2+, Ni2+, Cu2+, Hg2+, 4-chloromericuribenzoate, N-bromosuccinimide, and diethyl pyrocarbonate. The enzyme was inducible and expressed inside the cells moderately by ferulic
acid and p-coumaric acid and significantly by non-metabolizable 6-hydroxy-2-naphthoic acid. 相似文献
10.
Tony Marcio da Silva Alexandre Maller André Ricardo de Lima Damásio Michele Michelin Richard John Ward Izaura Yoshico Hirata João Atilio Jorge Héctor Francisco Terenzi Maria Lourdes T. M. de Polizeli 《Journal of industrial microbiology & biotechnology》2009,36(12):1439-1446
A glucoamylase from Aspergillus niveus was produced by submerged fermentation in Khanna medium, initial pH 6.5 for 72 h, at 40°C. The enzyme was purified by DEAE-Fractogel
and Concanavalin A-Sepharose chromatography. The enzyme showed 11% carbohydrate content, an isoelectric point of 3.8 and a
molecular mass of 77 and 76 kDa estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or Bio-Sil-Sec-400
gel filtration, respectively. The pH optimum was 5.0–5.5, and the enzyme remained stable for at least 2 h in the pH range
of 4.0–9.5. The temperature optimum was 65°C and retained 100% activity after 240 min at 60°C. The glucoamylase remained completely
active in the presence of 10% methanol and acetone. After 120 min hydrolysis of starch, glucose was the unique product formed,
confirming that the enzyme was a glucoamylase (1,4-alpha-d-glucan glucohydrolase). The K
m was calculated as 0.32 mg ml−1. Circular dichroism spectroscopy estimated a secondary structure content of 33% α-helix, 17% β-sheet and 50% random structure,
which is similar to that observed in the crystal structures of glucoamylases from other Aspergillus species. The tryptic peptide sequence analysis showed similarity with glucoamylases from A. niger, A. kawachi,
A. ficcum, A. terreus, A. awamori and A. shirousami. We conclude that the reported properties, such as solvent, pH and temperature stabilities, make A. niveus glucoamylase a potentially attractive enzyme for biotechnological applications. 相似文献
11.
Rogério S. Pereira Solange I. Mussatto Inês C. Roberto 《Journal of industrial microbiology & biotechnology》2011,38(1):71-78
The inhibitory action of acetic acid, ferulic acid, and syringaldehyde on metabolism of Candida guilliermondii yeast during xylose to xylitol bioconversion was evaluated. Assays were performed in buffered and nonbuffered semidefined
medium containing xylose as main sugar (80.0 g/l), supplemented or not with acetic acid (0.8–2.6 g/l), ferulic acid (0.2–0.6 g/l),
and/or syringaldehyde (0.3–0.8 g/l), according to a 23 full factorial design. Since only individual effects of the variables were observed, assays were performed in a next step
in semidefined medium containing different concentrations of each toxic compound individually, for better understanding of
their maximum concentration that can be present in the fermentation medium without affecting yeast metabolism. It was concluded
that acetic acid, ferulic acid, and syringaldehyde are compounds that may affect Candida guilliermondii metabolism (mainly cell growth) during bioconversion of xylose to xylitol. Such results are of interest and reveal that complete
removal of toxic compounds from the fermentation medium is not necessary to obtain efficient conversion of xylose to xylitol
by Candida guilliermondii. Fermentation in buffered medium was also considered as an alternative to overcome the inhibition caused by these toxic compounds,
mainly by acetic acid. 相似文献
12.
A new superoxide dismutase (SOD) gene from the thermophilic fungus Chaetomium thermophilum (Ctsod) was cloned and expressed in Pichia pastoris and its gene product was characterized. The specific activity of the purified CtSOD was 2,170 U/mg protein. The enzyme was
inactivated by KCN and H2O2 but not by NaN3, confirming that it belonged to the type of Cu, ZnSOD. The amino acid residues involved in coordinating copper and zinc were
conserved. The recombinant CtSOD exhibited optimum activity at pH 6.5 and 60°C. The enzyme retained 65% of the maximum activity
at 70°C for 60 min and the half-life was 22 and 7 min at 80 and 90°C, respectively. The recombinant yeast exhibited higher
stress resistance than the control yeast cells to salt and superoxide-generating agents, such as paraquat and menadione. 相似文献
13.
Recent genome sequencing of Cellvibrio japonicas revealed the presence of two highly homologous ferulic acid esterases (FAEs), encoded by fee1A and fee1B. In this work, the
putative FAE, Fee1B, was successfully cloned and expressed in an E. coli system and the purified enzyme was characterized as a type-D FAE with a pH and temperature optima of 6.5 and 35−40°C, respectively.
Additionally, the two tandem N-terminal carbohydrate binding modules of the multi-domain enzyme were shown to be crucial for optimum enzyme activity. The
potential of the enzyme in biomass processing was demonstrated with its high synergy with a xylanase in the release of reducing
sugar from arabinoxylan and its ability to liberate ferulic acid from various complex xylan substrates. 相似文献
14.
SM Cedrola AC de Melo AM Mazotto U Lins RB Zingali AS Rosado RS Peixoto AB Vermelho 《World journal of microbiology & biotechnology》2012,28(3):1259-1269
The aim of this study is to investigate the culture conditions of chicken feather degradation and keratinolytic enzyme production
by the recently isolated Bacillus subtilis SLC and to evaluate the potential of the SLC strain to recycle feather waste discarded by the poultry industry. The SLC strain
was isolated from the agroindustrial waste of a poultry farm in Brazil and was confirmed to belong to Bacillus subtilis by rDNA gene analysis. There was high keratinase production when the medium was at pH 8 (280 U ml−1). Activity was higher using the inoculum propagated for 72 h on 1% whole feathers supplemented with 0.1% yeast extract. In
the enzymatic extract, the keratinases were active in the pH range from 2.0 to 12.0 with a maximum activity at pH 10.0 and
temperature 60°C. For gelatinase the best pH was 5.0 and the best temperature was 37°C. All keratinases are serine peptidases.
The crude enzymatic extract degraded keratin, gelatin, casein, and hemoglobin. Scanning electron microscopy showed Bacillus cells adhered onto feather surfaces after 98 h of culture and degraded feather filaments were observed. MALDI-TOF mass spectrometric
analysis showed multiple peaks from 522 to 892 m/z indicating feather degradation. The presence of sulfide was detected on
extracellular medium probably participating in the breakdown of sulfide bridges of the feather keratin. External addition
of sulfide increased feather degradation. 相似文献
15.
In this study, the cellulase gene celD from Clostridium thermocellum was cloned into expression vectors pET-20b(+) and pHsh. While high expression can be achieved by means of both these expression
systems, only the pHsh expression system gives soluble proteins. By weakening the mRNA secondary structure and replacing the
rare codons for the N-terminal amino acids of the target protein, the expression level of CelD was increased from 4.1 ± 0.3 to 6.4 ± 0.4 U ml−1 in LB medium. Recombinant CelD was purified by heat treatment followed by Ni–NTA affinity. The purified CelD exhibited the
highest activity at pH 5.4 and 60°C, and retained more than 50% activity after incubation at 70°C for 1 h. The cellulase activity
of CelD was significantly enhanced by Ca2+ but inhibited by EDTA. The favorable properties of CelD offer the potential for genetic modification of strains for biomass
degradation. Presently, one of the major bottlenecks for industrial cellulase users is the high cost of enzyme production.
The high level expression of soluble enzymes from the pHsh expression system offers a novel approach for the production of
cellulases to be used in various agro-industrial processes such as chemical, food and textile. 相似文献
16.
Purification, immobilization and characterization of linoleic acid isomerase on modified palygorskite 总被引:2,自引:0,他引:2
Linoleic acid isomerase from Lactobacillus delbrueckii subsp. bulgaricus 1.1480 was purified by DEAE ion-exchange chromatography and gel filtration chromatography. An overall 5.1% yield and purification
of 93-fold were obtained. The molecular weight of the purified protein was ~41 kDa which was analyzed by SDS-PAGE. The purified
enzyme was immobilized on palygorskite modified with 3-aminopropyltriethoxysilane. The immobilized enzyme showed an activity
of 82 U/g. The optimal temperature and pH for the activity of the free enzyme were 30 °C and pH 6.5, respectively; whereas
those for the immobilized enzyme were 35 °C and pH 7.0, respectively. The immobilized enzyme was more stable than the free
enzyme at 30–60 °C, and the operational stability result showed that more than 85% of its initial activity was retained after
incubation for 3 h. The K
m and V
max values of the immobilized enzyme were found to be 0.0619 mmol l−1 and 0.147 mmol h−1 mg−1, respectively. The immobilized enzyme had high operational stability and retained high enzymatic activity after seven cycles
of reuse at 37 °C. 相似文献
17.
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
18.
Muhammad Aamer Mehmood Xiang Xiao Fauzia Yusuf Hafeez Yingbao Gai Fengping Wang 《World journal of microbiology & biotechnology》2010,26(12):2171-2178
A chitinase gene from Bacillus thuringiensis serovar konkukian S4 was cloned, sequenced, and heterologously expressed in Escherichia coli M15. Recombinant enzyme (Chi74) was purified by Ni-NTA affinity column chromatography. The chi74 gene contains an open reading frame (ORF), with a capacity to encode an endochitinase with a deduced molecular weight 74 kDa
and predicted isoelectric point of 5.67. Comparison of Chi74 with other chitinases has shown that it contains a modular structure
with an N-terminal family 18 catalytic-domain, a Fibronectin-III like domain and a C-terminal carbohydrate binding module
(CBM-II). Turn over rate (K
cat
) of the enzyme was determined using colloidal chitin (28.3 ± 0.70 S−1) as substrate. The Purified enzyme was active at a broad range of pH (pH 3.5–7.5) and temperature (20–70°C) with a peak activity
at pH 5.5 and 55°C. However, the enzyme was found to be stable up to 30°C for longer incubation periods. Moreover, the purified
enzyme was shown to inhibit fungal spore germination and hyphal growth in the pathogenic fungi Fusarium oxysporum and Aspergillus niger. These studies will lead us to develop broad spectrum resistance in the crop plants via co-expression of the chitinases and
the insecticidal proteins. 相似文献
19.
Thermolabile xylanase of the Antarctic yeast Cryptococcus adeliae: production and properties 总被引:4,自引:0,他引:4
Xylanase production by the Antarctic psychrophilic yeast Cryptococcus adeliae was increased 4.3 fold by optimizing the culture medium composition using statistical designs. The optimized medium containing
24.2 g l−1 xylan and 10.2 g l−1 yeast extract and having an initial pH of 7.5 yielded xylanase activity at 400 nkat (nanokatal) ml−1 after 168-h shake culture at 4°C. In addition, very little endoglucanase, β-mannanase, β-xylosidase, β-glucosidase, α-l-arabinofuranosidase, and no filter paper cellulase activities were detected. Among 12 carbon sources tested, maximum xylanase
activity was induced by xylan, followed by lignocelluloses such as steamed wheat straw and alkali-treated bagasse. The level
of enzyme activity produced on other carbon sources appeared to be constitutive. Among the complex organic nitrogen sources
tested, the xylanase activity was most enhanced by yeast extract, followed by soymeal, Pharmamedia (cotton seed protein),
and Alburex (potato protein). A batch culture at 10°C in a 5-l fermenter (3.5-1 working volume) using the optimized medium
gave 385 nkat at 111 h of cultivation. The crude xylanase showed optimal activity at pH 5.0–5.5 and good stability at pH 4–9
(21 h at 4°C). Although the enzyme was maximally active at 45°–50°C, it appeared very thermolabile, showing a half-life of
78 min at 35°C. At 40°–50°C, it lost 71%–95% activity within 5 min. This is the first report on the production as well as
on the properties of thermolabile xylanase produced by an Antarctic yeast.
Received: December 10, 1999 / Accepted: March 23, 2000 相似文献
20.
Fábio C. Sampaio Janaína T. de Faria Flávia M. Lopes Passos Attilio Converti Luis Antônio Minin 《Journal of industrial microbiology & biotechnology》2009,36(2):293-300
Xylose reductase (XR) is the enzyme that catalyzes the first step of xylose metabolism. Although XRs from various yeasts have
been characterized, little is known about this enzyme in Debaryomyces hansenii. In the present study, response surface analysis was used to determine the optimal conditions for D. hansenii UFV-170 XR activity. The influence of pH and temperature, ranging from 4.0 to 8.0 and from 25 to 55°C, respectively, was
evaluated by a 22 central composite design face-centered. The F-test (ANOVA) and the Student’s t test were performed to evaluate the statistical significance of the model and the regression coefficients, respectively.
The NADPH-dependent XR activity varied from 0.502 to 2.53 U mL−1, corresponding to 0.07–0.352 U mg−1, whereas the NADH-dependent one was almost negligible. The model predicted with satisfactory correlation (R
2 = 0.940) maximum volumetric activity of 2.27 U mL−1 and specific activity of 0.300 U mg−1 at pH 5.3 and 39°C, which were fairly confirmed by additional tests performed under these conditions. The enzyme proved very
stable at low temperature (4°C), keeping its activity almost entirely after 360 min, which corresponded to the half-time at
39°C. On the other hand, at temperatures ≥50°C it was lost almost completely after only 20 min. 相似文献