共查询到20条相似文献,搜索用时 31 毫秒
1.
Guo R Ding M Zhang SL Xu GJ Zhao FK 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》2008,178(2):209-215
Cellulase genes have been reported not only from fungi, bacteria and plant, but also from some invertebrate animals. Here,
two cellulase (endo-β-1,4-glucanase, EC 3.2.1.4) genes, eg27I and eg27II, were cloned from the freshwater snail Ampullaria crossean cDNA using degenerate primers. The nucleotide sequences of the two genes shared 94.5% identity. The open reading frames of
both genes consisted of 588 bp, encoding 195 amino acids. Both EG27I and EG27II belong to the glycoside hydrolase family 45,
and each lacks a carbohydrate-binding module. The presence of introns demonstrated a eukaryotic origin of the EG27 gene, and,
in addition, successful cloning of EG27 cDNA supported endogenous production of EG27 cellulase by Ampullaria crossean. Investigation of the EG27 cDNA from A. crossean will provide further information on GHF45 cellulases. 相似文献
2.
Purification and Characterization of Two Endo-β-1,4-glucanases from Mollusca, Ampullaria crossean 总被引:2,自引:0,他引:2
Li YH Guo R Yin QY Ding M Zhang SL Xu GJ Zhao FK 《Acta biochimica et biophysica Sinica》2005,37(10):702-708
Two novel endo-β-1,4-glucanases, EG45 and EG27, were isolated from the gastric juice of mollusca, Ampullaria crossean, by anion exchange, hydrophobic interaction, gel filtration and a second round of anion exchange chromatography. The purified proteins EG45 and EG27 appeared as a single band on sodium dodecylsulfate polyacrylamide gel electrophoresis with a molecular mass of 45 kDa and 27 kDa, respectively. The optimum pH for CMC activity was 5.5 for EG45 and 4.4-4.8 for EG27. The optimum temperature range for EG27 was broad, between 50℃ and 60 ℃; for EG45 it was 50 ℃. The analysis on the stability of these two endo-β-1,4-glucanases showed that EG27 was acceptably stable at pH 3.0-11.0 even when the incubation time was prolonged to 24 h at 30 ℃, whereas EG45 remained relatively stable at pH 5.0-8.0. About 85% of the activity of EG27 could be retained upon incubation at 60 ℃ for 24 h. However, less than 10% residual activity of EG45 was detected at 50 ℃. Among different kinds of substrates, both enzymes showed a high preference for carboxymethyl cellulose. EG45, in particular, showed a carboxymethyl cellulose hydrolytic activity of 146.5 IU/mg protein. Both enzymes showed low activities to xylan (from oat spelt) and Sigmacell 101, and they were inactive to p-nitrophenyl-β-D-cellobioside, salicin and starch. 相似文献
3.
G. Sakellaris S. Nikolaropoulos A. E. Evangelopoulos 《Journal of applied microbiology》1989,67(1):77-85
Lactobacillus plantarum produced extracellular polygalacturonase in a medium containing 1.5% low methyl-pectin (w/v) and 0.5% glucose (w/v) as inducers. The enzyme was purified (approximately 70-fold) by ammonium sulphate fractionation, Sephadex G-100 gel filtration and DEAE-cellulose ion exchange chromatography. Two peaks (PG I and PG II) of enzymic activity were obtained from the DEAE-cellulose column. The molecular mass of PG I was similar to that of PG II (32 000 Da). The K m values of PG I and PG II for sodium polypectate were calculated to be 1.63 mg/ml and 1.78 mg/ml respectively. Their isoelectric points were about pH 5.5. The pH optimum was 4.5, while the optimum temperature was 35°C for both PG I and PG II. The two purified enzymes had similar endo modes of action on polygalacturonic acid, as determined by comparison of viscosity reduction and reducing group release. 相似文献
4.
G A Nechaeva 《Biokhimii?a (Moscow, Russia)》1986,51(10):1688-1695
Preparations of soluble (I) and membrane-bound (II) acid RNAse with Mr 68,000 and 72,000 Da, respectively, and purified about 2000-fold were isolated from lysosome-rich fractions of rat brain large hemispheres. RNAase II differed from RNAase I by a lower temperature stability. The pH optimum (pH 5.8-6.1), temperature optimum and substrate specificity of RNAase I and II appeared to be identical. The Km values of RNAases I and II for poly(U) are 166 and 160 micrograms/ml; those for RNA--1200 and 1250 mu k/ml, respectively. RNAases I and II extensively hydrolyze soluble, polymeric RNA, rRNA from brain and yeast and poly(U) but do not influence poly(C), poly(A), poly(G), tRNA and DNA. Monovalent cations (K+, Na+, NH4+) activate both RNAase forms. 相似文献
5.
Hiroshi Tsujibo Katsushiro Miyamoto Toru Hasegawa Yoshihiko Inamori 《Journal of applied microbiology》1990,69(4):520-529
T sujibo , H., M iyamoto , K., H asegawa , T. & I namori , Y. 1990. Purification and characterization of two types of alkaline serine proteases produced by an alkalophilic actinomycete. Journal of Applied Bacteriology 69 , 520–529.
Two types of alkaline serine proteases were isolated from the culture filtrate of an alkalophilic actinomycete, Nocardiopsis dassonvillei OPC-210. The enzymes (protease I and protease II) were purified by acetone precipitation, DEAE-Sephadex A-50, CM-Sepharose CL-6B, Sephadex G-75 and phenyl-Toyopearl 650 M column chromatography. The purified enzymes showed a single band on sodium dodecyl sulphate polyacrylamide gel electrophoresis. The molecular weights of proteases I and II were 21000 and 36000, respectively. The pIs were 6.4 (protease I) and 3.8 (protease II). The optimum pH levels for the activity of two proteases were pH 10–12 (protease I) and pH 10.5 (protease II). The optimum temperature for the activity of protease I was 70°C and that for protease II was 60°C. Protease I was stable in the range of pH 4.0–8.0 up to 60°C and protease II was stable in the range of pH 6.0–12.0 up to 50°C. 相似文献
Two types of alkaline serine proteases were isolated from the culture filtrate of an alkalophilic actinomycete, Nocardiopsis dassonvillei OPC-210. The enzymes (protease I and protease II) were purified by acetone precipitation, DEAE-Sephadex A-50, CM-Sepharose CL-6B, Sephadex G-75 and phenyl-Toyopearl 650 M column chromatography. The purified enzymes showed a single band on sodium dodecyl sulphate polyacrylamide gel electrophoresis. The molecular weights of proteases I and II were 21000 and 36000, respectively. The pIs were 6.4 (protease I) and 3.8 (protease II). The optimum pH levels for the activity of two proteases were pH 10–12 (protease I) and pH 10.5 (protease II). The optimum temperature for the activity of protease I was 70°C and that for protease II was 60°C. Protease I was stable in the range of pH 4.0–8.0 up to 60°C and protease II was stable in the range of pH 6.0–12.0 up to 50°C. 相似文献
6.
Bukhtojarov FE Ustinov BB Salanovich TN Antonov AI Gusakov AV Okunev ON Sinitsyn AP 《Biochemistry. Biokhimii?a》2004,69(5):542-551
Using different chromatographic techniques, eight cellulolytic enzymes were isolated from the culture broth of a mutant strain of Chrysosporium lucknowense: six endoglucanases (EG: 25 kD, pI 4.0; 28 kD, pI 5.7; 44 kD, pI 6.0; 47 kD, pI 5.7; 51 kD, pI 4.8; 60 kD, pI 3.7) and two cellobiohydrolases (CBH I, 65 kD, pI 4.5; CBH II, 42 kD, pI 4.2). Some of the isolated cellulases were classified into known families of glycoside hydrolases: Cel6A (CBH II), Cel7A (CBH I), Cel12A (EG28), Cel45A (EG25). It was shown that EG44 and EG51 are two different forms of one enzyme. EG44 seems to be a catalytic module of an intact EG51 without a cellulose-binding module. All the enzymes had pH optimum of activity in the acidic range (at pH 4.5-6.0), whereas EG25 and EG47 retained 55-60% of the maximum activity at pH 8.5. Substrate specificity of the purified cellulases against carboxymethylcellulose (CMC), beta-glucan, Avicel, xylan, xyloglucan, laminarin, and p-nitrophenyl-beta-D-cellobioside was studied. EG44 and EG51 were characterized by the highest CMCase activity (59 and 52 U/mg protein). EG28 had the lowest CMCase activity (11 U/mg) amongst the endoglucanases; however, this enzyme displayed the highest activity against beta-glucan (125 U/mg). Only EG51 and CBH I were characterized by high adsorption ability on Avicel cellulose (98-99%). Kinetics of Avicel hydrolysis by the isolated cellulases in the presence of purified beta-glucosidase from Aspergillus japonicus was studied. The hydrolytic efficiency of cellulases (estimated as glucose yield after a 7-day reaction) decreased in the following order: CBH I, EG60, CBH II, EG51, EG47, EG25, EG28, EG44. 相似文献
7.
探索获得优良的新型普鲁兰酶基因,丰富普鲁兰酶理论,对实现普鲁兰酶国产化具有重要意义。分析GenBank数据库中蜡样芽胞杆菌假定Ⅰ型、Ⅱ型普鲁兰酶基因序列,从实验室保藏的蜡样芽胞杆菌Bacilluscereus GXBC-3中克隆得到3个普鲁兰酶基因pulA、pulB、pulC,并分别导入大肠杆菌进行胞内诱导表达。纯化重组酶酶学性质研究表明重组酶PulA能水解α-l,6-和α-l,4-糖苷键,为Ⅱ型普鲁兰酶,以普鲁兰糖为底物时,最适反应温度及pH分别为40℃和6.5,比活力为32.89 U/mg;以可溶性淀粉为底物时,最适反应温度及pH分别为50℃和7.0,比活力为25.71 U/mg。重组酶PulB和PulC二者均只能水解α-l,6-糖苷键,为I型普鲁兰酶,以普鲁兰糖为底物时,其最适反应温度及pH分别为45℃、7.0和45℃、6.5,比活力分别为228.54 U/mg和229.65 U/mg。 相似文献
8.
Haruko Tagami Kenji Tayama Tohru Tohyama Masahiro Fukaya Hajime Okumura Yoshiya Kawamura Sueharu Horinouchi Teruhiko Beppu 《FEMS microbiology letters》1988,56(2):161-165
Abstract A type II restriction endonuclease, named Aaa I, was purified from Acetobacter aceti subsp. aceti No. 1023. The optimum pH and temperature were determined to be 8.5 and 37°C, respectively. The enzyme activity was stimulated by the addition of either NaCl or KCl and their optimum concentrations were 100 mM for both cations. Aaa I recognized the hexanucleotide sequence and cleaved it at the positions indicated by the arrows. Aaa I was an isoschizomer of Xma III from Xanthomonas malvacaerum and Eco 52I from Escherichia coli . 相似文献
9.
A newly isolated strain of the fungus, Mucor circinelloides (NRRL 26519), when grown on lactose, cellobiose, or Sigmacell 50 produces complete cellulase (endoglucanase, cellobiohydrolase, and β-glucosidase) system. The extracellular endoglucanase (EG) was purified to homogeneity from the culture supernatant by ethanol precipitation (75%, v/v), CM Bio-Gel A column chromatography, and Bio-Gel A-0.5 m gel filtration. The purified EG (specific activity 43.33 U/mg protein) was a monomeric protein with a molecular weight of 27 000. The optimum temperature and pH for the action of the enzyme were at 55 °C and 4.0–6.0, respectively. The purified enzyme was fully stable at pH 4.0–7.0 and temperature up to 60 °C. It hydrolysed carboxymethyl cellulose and insoluble cellulose substrates (Avicel, Solka-floc, and Sigmacell 50) to soluble cellodextrins. No glucose, cellobiose, and short chain cellooligosaccarides were formed from these substrates. The purified EG could not degrade oat spelt xylan and larch wood xylan. It bound to Avicell, Solka-floc, and Sigmacell 50 at pH 5.0 and the bound enzyme was released by changing the pH to 8.0. The enzyme activity was enhanced by 27±5 and 44±14% by the addition of 5 mM MgCl2 and 0.5 mM CoCl2, respectively, to the reaction mixture. Comparative properties of this enzyme with other fungal EGs are presented. 相似文献
10.
This study reports the purification and characterization of endoglucanases (EG I and EG II) from a newly isolated thermophilic fungus, Melanocarpus sp. MTCC 3922. The molecular weight of EG I and EG II as with SDS-PAGE and pI were approximately 40 and 50 kDa, and approximately 4.0 and 3.6, respectively. EG I and EG II were optimally active at 50 and 70 degrees C, and pH 6.0 and 5.0, respectively. EG I was active over a broad range of pH (5.0-7.0), whereas, loss of activity was observed as the temperature was increased from 50 to 80 degrees C. However, EG II was active over pH 4.0-6.0 and temperature 40-80 degrees C. The presence of mercaptoethanol and SDS inhibited the EG I activity but showed no negative effect on EG II. Both the endoglucanases showed higher activity against barley-beta-glucan as compared to CMC. Km values of EG I and EG II for barley-beta-glucan were lower than CMC. Turn over number (K(cat)) and catalytic efficiency (K(cat)/Km) values of both the endoglucanases were higher with barley-beta-glucan as substrate than CMC. EG I showed affinity for Avicel indicating the presence of cellulose binding domains (CBD) whereas, EG II was found to lack CBD. 相似文献
11.
Microbial transglutaminase (MTG) gene (mtg) from Streptomyces hygroscopicus H197 strain was cloned by PCR and mutated by deleting a specific 84 bp fragment using overlapping extension PCR. The mutant MTG and the wild MTG genes expressed by recombinant plasmid pET32a+-mutant mtg and pET32a+-mtg, respectively, and were harvested by alternating freeze–thaw steps and purified by Ni column. The purified mutant MTG and the wild MTG exhibited 0.22 U/mg and 0.16 U/mg activity, respectively, and 0.69 U/mg and 0.54 U/mg activity, respectively, after activated by trypsin. The molecular weight of mutant MTG was estimated as 67 kDa by SDS-PAGE. Both MTGs showed optimum activity at pH 6–8 for hydroxamate formation from N-CBZ-Gln-Gly and hydroxylamine, and exhibited higher stability at 40°C and 1–3% salinity. The two types of MTG were not stable in the presence of Zn(II), Cu(II), Hg(II), Pb(II), Fe(III), and Ag(I), suggesting that they could possess a thiol group. In addition, the mutant MTG and the wild MTG were strongly affected by ethanol. Furthermore, the mutant MTG was obviously (P < 0.05 or P < 0.01) more stable than the wild MTG at 50°C and 60°C, at pH 4, 5, and 9, at 7% and 9% salinity, 30% and 35% ethanol concentration, and in the presence of Li(I) and Ag(I). The polyhydroxy compounds as protein stabilizers could elevate MTG stability. 相似文献
12.
《Journal of Fermentation and Bioengineering》1994,77(5):548-550
Two types of extracellular lipases (I and II) from Trichosporon fermentans WU-C12 were purified by acetone precipitation and successive chromatographies on Butyl-Toyopearl 650 M, Toyopearl HW-55F and Q-Sepharose FF. The molecular weight of lipase I was 53 kDa by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and 160 kDa by gel filtration, while that of lipase II was 55 kDa by SDS-PAGE and 60 kDa by gel filtration. For the hydrolysis of olive oil, the optimum pH and temperature of both the lipases were 5.5 and 35°C, respectively. The lipases showed stable activities after incubation at 30°C for 24 h in a pH range from 4.0 to 8.0. The thermostability of lipase I for 30 min at a reaction pH of 5.5 was up to 40°C, while that of lipase II under the same conditions was up to 50°C. Both lipases could hydrolyze the 1-, 2-, and 3-positions of triolein, and cleave all three ester bonds, regardless of the position in the triglyceride. 相似文献
13.
Abstract Optimal culture conditions for obtaining low molecular mass endoglucanase (EG) from culture fluids of Trichoderma reesei were determined. The purification of this unglycosylated EG in a two-step procedure is described. In contrast to most cellulases, this EG did not bind to ConA-affinity columns. The unglycosylated fraction of the culture fluid proteins was further purified by preparative isoelectric focusing. Conditions relevant to an enzyme assay for this EG were determined (pH optimum 5.8, temperature optimum 52°C). 相似文献
14.
15.
Bacillus sp. A-001, which produced large amounts of amylase, was isolated from fermenting tef ( Eragrostis tef ) on tryptone soya agar supplemented with 1% starch. The organism grew between pH 4.5 and 10.5 with an optimum at 7–7.5. Growth occurred between 20 and 55°C but the optimum was about 35–40°C. At optimum medium pH (7.5) and a temperature of 35°C the organism entered the stationary phase after about 72 h and amylase production was at its highest (9.6 units ml-1 ) at this time. Enzyme activity was optimal at pH 5.5 and 40°C and showed good thermal stability; it required 110 min to lose 50% of its activity at 70°C. The enzyme hydrolysed native starch (flour from tef, corn and kocho) to various oligosaccharides, including maltotriose, maltose and glucose. 相似文献
16.
Michniewicz A Ullrich R Ledakowicz S Hofrichter M 《Applied microbiology and biotechnology》2006,69(6):682-688
Cerrena unicolor secreted two laccase isoforms with different characteristics during the growth in liquid media. In a synthetic low-nutrient
nitrogen glucose medium (Kirk medium), high amounts of laccase (4,000 U l−1) were produced in response to Cu2+. Highest laccase levels (19,000 U l−1) were obtained in a complex tomato juice medium. The isoforms (Lacc I, Lacc II) were purified to homogeneity with an overall
yield of 22%. Purification involved ultrafiltration and Mono Q separation. Lacc I and II had M
w of 64 and 57 kDa and pI of 3.6 and 3.7, respectively. Both isoforms had an absorption maximum at 608 nm but different pH optima and thermal stability.
Optimum pH ranged from 2.5 to 5.5 depending on the substrate. The pH optima of Lacc II were always higher than those of Lacc
I. Both laccases were stable at pH 7 and 10 but rapidly lost activity at pH 3. Their temperature optimum was around 60°C,
and at 5°C they still reached 30% of the maximum activity. Lacc II was the more thermostable isoform that did not lose any
activity during 6 months storage at 4°C. Kinetic constants (K
m, k
cat) were determined for 2,2′-azino-bis(3-ethylthiazoline-6-sulfonate) (ABTS), 2,6-dimethoxyphenol and syringaldazine. 相似文献
17.
Based on the original thermostable alpha-amylase gene from Bacillus licheniformis, two amino acids were site-directed mutagenised by polymerase chain reaction to obtain a new gene. This gene, with Leu134→Arg
and Ser320→Ala, was substituted for acid-resistant capability previously. To favor purification of the product, high-level
expression and secretion of mature, authentic and stable recombinant mutagenised alpha-amylase were achieved with protease-deficient
strain Bacillus subtilis WB600 as the host. The recombinant mutagenised alpha-amylase with the activity of 4,700 U/mL was then purified by ammonium
sulphate fractionation, anion exchange and gel filtration, consecutively. By multi-step purification, the specific activity
of the recombinant protein was up to 916.7 U/mg with a 187.1-fold purification. The mutagenised protein was found to be more
acid resistant than the native protein. The optimum pH and stable range of pH with the mutagenised protein was 4.5 and 4.0
to 6.5, respectively, compared with pH 6.5 and 5.5 to 7.0 as the favorite pH and pH stability range of the native protein. 相似文献
18.
高温α-淀粉酶基因突变体在大肠杆菌、毕赤酵母中的表达 总被引:1,自引:0,他引:1
对地衣芽孢杆菌(Bacillus licheniformis)高温α-淀粉酶(amyE)基因进行改造获得的基因突变体(amyEM),通过PCR扩增,将此基因分别克隆至大肠杆菌表达载体pBV220和毕赤酵母表达载体pPIC9K上,并分别转化大肠杆菌DH5α和毕赤酵母GS115感受态细胞,获得重组大肠杆菌和重组毕赤酵母。通过表达产物的酶活性检测和SDS-PAGE分析,证明突变α-淀粉酶(AmyEM)在大肠杆菌、毕赤酵母中获得有效表达。对重组大肠杆菌产生的α-淀粉酶的粗酶性质分析表明,此酶分子量约为55kDa。其最适反应温度为80℃~90℃,与野生型基因相比,其最适pH均为6.0,但不同的是突变体在pH 5.0~5.5时表现出较高的酶活力;在毕赤酵母细胞的表达产物可分泌至胞外。由于酵母可对蛋白进行糖基化,酶分子量增加到60kDa,最适pH也改变为5.5。此高温α-淀粉酶突变体所具有的在微酸性环境具有较高酶活力的性质,具有重要的潜在工业应用价值。 相似文献
19.
Purification and characterization of the major β-1,4-endoglucanase from Thermomonospora curvata 总被引:1,自引:1,他引:0
The major β-1,4-endoglucanase (EG) of the thermophilic actinomycete, Thermomonospora curvata , contributed over 80% of the total EG activity recovered from cell-free culture fluid after growth on cellulose. The enzyme was purified to electrophoretic homogeneity by ammonium sulphate precipitation, ion-exchange chromatography and size exclusion HPLC. This monomeric enzyme had a specific activity of 750 IU mg−1 when assayed with 2.5% (w/v) carboxymethyl cellulose (CMC) at 70°C, pH 6.0. Highest activity was observed on CMC with a degree of polymerization of 3200. The EG was stable for 48 h at 60°C, pH 6.0 and had a half-life of 30 min at 80°C; temperature and pH optima were 70–73°C and 6.0–6.5, respectively. The mol. wt was 100000 and the pI was 4.0. The K m and V max values were 7.33 mg ml−1 and 833 μmol min−1 , respectively. EG activity was inhibited by Fe2 + , Hg2 + , Ag+ and Pb2 + , and enhanced by dithiothreitol and Zn2 + . The first 12 amino acid residues at the N -terminus were: Asp-Glu-Val-Asp-Glu-Ile-Arg-Asn-Gly-Asp-Phe-Ser. Glutamic and aspartic acid constituted 24% of the total amino acid composition; no amino sugar was found. 相似文献
20.
The enzymatic lignocellulosic biomass conversion into value-added products requires the use of enzyme-rich cocktails, including β-glucosidases that hydrolyze cellobiose and cellooligosaccharides to glucose. During hydrolysis occurs accumulation of monomers causing inhibition of some enzymes; thus, glucose/xylose tolerant β-glucosidases could overcome this drawback. The search of new tolerant enzymes showing additional properties, such as high activity, wide-pH range, and thermal stability is very relevant to improve the bioprocess. We describe a novel β-glucosidase GH1 from the thermophilic Anoxybacillus thermarum (BgAt), which stood out by the robustness combination of great glucose/xylose tolerance, thermal stability, and high Vmax. The recombinant his-tagged-BgAt was overexpressed in Escherichia coli, was purified in one step, showed a high glucose/xylose tolerance, and activity stimulation (presence of 0.4 M glucose/1.0 M xylose). The optimal activity was at 65 °C - pH 7.0. BgAt presented an extraordinary temperature stability (48 h – 50 °C), and pH stability (5.5–8.0). The novel enzyme showed outstanding Vmax values compared to other β-glucosidases. Using p-nitrophenyl-β-d-glucopyranoside as substrate the values were Vmax (7614 U/mg), and KM (0.360 mM). These values suffer a displacement in Vmax to 14,026 U/mg (glucose), 14,886 U/mg (xylose), and KM 0.877 mM (glucose), and 1.410 mM (xylose). 相似文献