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1.
An agar-degrading Thalassomonas bacterium, strain JAMB-A33, was isolated from the sediment off Noma Point, Japan, at a depth of 230 m. A novel -agarase from the isolate was purified to homogeneity from cultures containing agar as a carbon source. The molecular mass of the purified enzyme, designated as agaraseA33, was 85 kDa on both SDS-PAGE and gel-filtration chromatography, suggesting that it is a monomer. The optimal pH and temperature for activity were about 8.5 and 45°C, respectively. The enzyme had a specific activity of 40.7 U/mg protein. The pattern of agarose hydrolysis showed that the enzyme is an endo-type -agarase, and the final main product was agarotetraose. The enzyme degraded not only agarose but also agarohexaose, neoagarohexaose, and porphyran. 相似文献
2.
《Journal of Fermentation and Bioengineering》1990,69(2):129-131
Cellulomonas sp. isolated from soil produces a high level of α-mannosidase (α-mannanase) inductively in culture fluid. The enzyme had two different molecular weight forms, and the properties of the high-molecular-weight form were reported previously (Takegawa, K. et al.: Biochim. Biophys. Acta, 991, 431–437, 1989). The low-molecular-weight α-mannosidase was purified to homogeneity by polyacrylamide gel electrophoresis. The molecular weight of the enzyme was over 150,000 by gel filtration. Unlike the high-molecular-weight form, the low-molecular-weight enzyme readily hydrolyzed α-1,2- and α-1,3-linked mannose chains. 相似文献
3.
Secreted β-galactosidase from a Flavobacterium sp. isolated from a low-temperature environment 总被引:1,自引:0,他引:1
Sørensen HP Porsgaard TK Kahn RA Stougaard P Mortensen KK Johnsen MG 《Applied microbiology and biotechnology》2006,70(5):548-557
The bacterial strain Flavobacterium sp. 4214 isolated from Greenland was found to express β-galactosidase (EC 3.2.1.23) at temperatures below 25°C. A chromosomal
library of Flavobacterium sp. 4214 was constructed in Escherichia coli, and the gene gal4214-1 encoding a β-galactosidase of 1,046 amino acids (114.3 kDa) belonging to glycosyl hydrolase family 2 was isolated. This was
the only gene encoding β-galactosidase activity that was identified in the chromosomal library. Expression levels in both
Flavobacterium sp. 4214 and in initial recombinant E. coli strains were insufficient for biochemical characterization. However, a combination of T7 promoter expression and introduction
of an E. coli host that complemented rare transfer RNA genes yielded 15 mg of β-galactosidase per liter of culture. Gal4214-1-His protein
was found to be active in monomeric conformation. The protein was secreted from the cytoplasm, probably through an N-terminal
signaling sequence. The Gal4214-1-His protein was found to have optimum activity at a temperature of 42°C, but with short-term
stability at temperatures above 25°C. 相似文献
4.
Qiaojuan Yan Luo Tang Shaoqing Yang Peng Zhou Shuping Zhang Zhengqiang Jiang 《Process Biochemistry》2012,47(3):472-478
The purification and characterization of an extracellular α-l-arabinofuranosidase (α-l-AFase) from Chaetomium sp. was investigated in this report. The α-l-AFase was purified to homogeneity with a purification fold of 1030. The purified α-l-AFase had a specific activity of 20.6 U mg?1. The molecular mass of the enzyme was estimated to be 52.9 kDa and 51.6 kDa by SDS–PAGE and gel filtration, respectively. The optimal pH and temperature of the enzyme were pH 5.0 and 70 °C, respectively. The enzyme was stable over a broad pH range of 4.0–10.0 and also exhibited excellent thermostability, i.e., the residual activities reached 75% after treatment at 60 °C for 1 h. The enzyme showed strict substrate specificity for the α-l-arabinofuranosyl linkage. The Km and Vmax values for p-nitrophenyl (pNP)-α-l-arabinofuranoside were calculated to be 1.43 mM and 68.3 μmol min?1 mg?1 protein, respectively. Furthermore, the gene encoding α-l-AFase was cloned and sequenced and found to contain a catalytic domain belonging to the glycoside hydrolase (GH) family 43 α-l-AFase. The deduced amino acid sequence of the gene showed the highest identity (67%) to the putative α-l-AFase from Neurospora crassa. This is the first report on the purification, characterization and gene sequence of an α-l-AFase from Chaetomium sp. 相似文献
5.
Molecular cloning and characterization of a novel γ-CGTase from alkalophilic Bacillus sp. 总被引:1,自引:0,他引:1
Hirano K Ishihara T Ogasawara S Maeda H Abe K Nakajima T Yamagata Y 《Applied microbiology and biotechnology》2006,70(2):193-201
We found a novel cyclodextrin glucanotransferase (CGTase) from alkalophilic Bacillus sp. G-825-6. The enzyme was expressed in the culture broth by recombinant Bacillus subtilis KN2 and was purified and characterized. The enzyme named CGTase825-6 showed 95% amino acid sequence identity with a known
enzyme β-/γ-CGTase from Bacillus firmus/lentus 290-3. However, the product specificity of CGTase825-6 differed from that of β-/γ-CGTase. CGTase825-6 produced γ-cyclodextrin
(CD) as the main product, but degradation of γ-CD was observed with prolonged reaction. The product specificity of the enzyme
was positioned between γ-CGTase produced by Bacillus clarkii 7364 and B. firmus/lentus 290-3 β-/γ-CGTase. It showed that the difference of product specificity was dependent on only 28 amino acid residues in 671
residues in CGTase825-6. We compared the amino acid sequence of CGTase825-6 and those of other CGTases and constructed a protein
structure model of CGTase825-6. The comparison suggested that the diminished loop (Val138-Asp142) should provide subsite -8
for γ-CD production and that Asp142 might have an important role in product specificity. CGTase825-6 should be a useful tool
to produce γ-CD and to study the differences of producing mechanisms between γ-CD and β-CD. 相似文献
6.
Shi P Yao G Cao Y Yang P Yuan T Huang H Bai Y Yao B 《Enzyme and microbial technology》2011,49(3):277-283
A new β-mannosidase gene, designated as man2S27, was cloned from Streptomyces sp. S27 using the colony PCR method and expressed in Escherichia coli BL21 (DE3). The full-length gene consists of 2499 bp and encodes 832 amino acids with a calculated molecular mass of 92.6 kDa. The amino acid sequence shares highest identity of 62.6% with the mannosidase Man2A from Cellulomonas fimi which belongs to the glycoside hydrolase family 2. Purified recombinant Man2S27 showed optimal activity at pH 7.0 and 50 °C. The specific activity, Km, and kcat values for p-nitrophenyl-β-d-mannopyranoside (p-NP-β-MP) were 35.3 U mg-1, 0.23 mM, and 305 s-1, respectively. Low transglycosylation activity was observed when Man2S27 was incubated with p-NP-β-MP (glycosyl donor) and methyl-α-d-mannopyranoside (p-NP-α-MP) (acceptor) at 50 °C and pH 7.0, and a small amount of methylmannobioside was synthesized. Using locust bean gum as the substrate, more reducing sugars were liberated by the synergistic action of Man2S27 and β-mannanase (Man5S27), and the synergy degree in sequential reactions with Man5S27 firstly and Man2S27 secondly was higher than that in the simultaneous reactions. 相似文献
7.
Summary A -cyclodextrin glucosyltransferase was purified from alkalophilic Bacillus sp. No. 562 over 64-fold with a yield of 32%. Its molecular size was estimated to be 170 kDa by gel filtration and 82 kDa by SDS-PAGE, with a pI of 7.2. The enzyme showed optimum activity at 65 °C and pH 7.0. It was stable from 0 to70 °C and from pH 7.0 to 11.0. The enzyme was specifically inhibited by Fe2+ and Fe3+. 相似文献
8.
Hiroaki Minegishi Yasuhiro Shimane Akinobu Echigo Yukari Ohta Yuji Hatada Masahiro Kamekura Tadashi Maruyama Ron Usami 《Extremophiles : life under extreme conditions》2013,17(6):931-939
An agar-degrading archaeon Halococcus sp. 197A was isolated from a solar salt sample. The agarase was purified by hydrophobic column chromatography using a column of TOYOPEARL Phenyl-650 M. The molecular mass of the purified enzyme, designated as Aga-HC, was ~55 kDa on both SDS-PAGE and gel-filtration chromatography. Aga-HC released degradation products in the order of neoagarohexose, neoagarotetraose and small quantity of neoagarobiose, indicating that Aga-HC was a β-type agarase. Aga-HC showed a salt requirement for both stability and activity, being active from 0.3 M NaCl, with maximal activity at 3.5 M NaCl. KCl supported similar activities as NaCl up to 3.5 M, and LiCl up to 2.5 M. These monovalent salts could not be substituted by 3.5 M divalent cations, CaCl2 or MgCl2. The optimal pH was 6.0. Aga-HC was thermophilic, with optimum temperature of 70 °C. Aga-HC retained approximately 90 % of the initial activity after incubation for 1 hour at 65–80 °C, and retained 50 % activity after 1 hour at 95 °C. In the presence of additional 10 mM CaCl2, approximately 17 % remaining activity was detected after 30 min at 100 °C. This is the first report on agarase purified from Archaea. 相似文献
9.
Young Min Ha Dong Gun Lee Jung-Hoon Yoon Yong-Ha Park Young Jae Kim 《Biotechnology letters》2001,23(17):1435-1438
Bacillus sp. KYJ 963, a local isolate, produced an extracellular amylase with M
r=59 kDa. The amylase was easily purified by adsorption on soluble starch. The analyses of TLC and N-terminal amino acid sequence from the purified protein revealed that the enzyme was a novel -amylase which could not hydrolyze maltose or -cyclodextrin and its N-terminal amino acid sequence was A-V-N-G-Q-S-F-N-S-N-Y-K-T-Y-K-. 相似文献
10.
Purpose of work The purpose of this study is to report a thermostable λ-carrageenase that can degrade λ-carrageenan yielding neo-λ-carrabiose at 75 °C. A thermophilic strain Lc50-1 producing λ-carrageenase was isolated from a hot spring in Indonesia and identified as a Bacillus sp. The λ-carrageenase, Cga-L50, with an apparent molecular weight of 37 kDa and a specific activity of 105 U/mg was purified from the culture supernatant. The optimum pH and temperature of Cga-L50 were 8.0 and 75 °C, respectively. The enzyme was stable from pH 6–9 and retained ~50 % activity after holding at 85 °C for 10 min. Significant activation of Cga-L50 was observed with K+, Ca2+, Co2+, and Na+; whereas, the enzyme activity was inhibited by Sr2+, Mn2+, Fe2+, Cu2+,Cd2+, Mg2+, and EDTA. Cga-L50 is an endo-type λ-carrageenase that hydrolyzes β-1,4-linkages of λ-carrageenan, yielding neo-λ-carrabiose as the main product. This study is the first to present evidence of thermostable λ-carrageenase from hot spring bacteria. 相似文献
11.
Summary
Aspergillus sp NCIM 508 produced 22 U/L of extracellular -mannosidase activity in a medium containing 8 % brewer's yeast cells. The optimum period and pH range for maximum production of the enzyme were 7 days and 4.0–6.0, respectively. The optimum pH and temperature for enzyme activity were 6.0 and 50°C, respectively. The enzyme was stable for 24 h at 28°C, in the pH range 6.0–7.0. The enzyme retained 100 and 65 % of its original activity after heating for 15 min at 45 and 55°C, respectively. The Km and Vmax for p-nitrophenyl--D- mannoside (PNPM) were 71M and 7.5 × 10–2 moles/min/mg, respectively. The enzyme was strongly inhibited by 1 mM Hg++ and Cu++ and partially by Co.++
(NCL Communication No.; 5780) 相似文献
12.
Shujun Fang Jie Chang Yong Seok Lee Weiliang Guo Yong Lark Choi Yongcan Zhou 《World journal of microbiology & biotechnology》2014,30(1):213-223
A β-glucosidase gene bglX was cloned from Lactococcus sp. FSJ4 by the method of shotgun. The bglX open reading frame consisted of 1,437 bp, encoding 478 amino acids. SDS-PAGE showed a recombinant bglX monomer of 54 kDa. Substrate specificity study revealed that the enzyme exhibited multifunctional catalysis activity against pNPG, pNPX and pNPGal. This enzyme shows higher activity against aryl glycosides of xylose than those of glucose or galactose. The enzyme exhibited the maximal activity at 40 °C, and the optimal pH was 6.0 with pNPG and 6.5 with pNPX as the substrates. Molecular modeling and substrate docking showed that there should be one active center responsible for the mutifuntional activity in this enzyme, since the active site pocket was substantially wide to allow the entry of pNPG, pNPX and pNPGal, which elucidated the structure–function relationship in substrate specificities. Substrate docking results indicated that Glu180 and Glu377 were the essential catalytic residues of the enzyme. The CDOCKER_ENERGY values obtained by substrate docking indicated that the enzyme has higher activity against pNPX than those of pNPG and pNPGal. These observations are in conformity with the results obtained from experimental investigation. Therefore, such substrate specificity makes this β-glucosidase of great interest for further study on physiological and catalytic reaction processes. 相似文献
13.
Natsumi Kanzaki Erik J. Ragsdale Matthias Herrmann Ralf J. Sommer 《Journal of nematology》2012,44(1):80-91
Pristionchus fissidentatus n. sp., isolated from soil in Nepal, and P. elegans n. sp., isolated from Phelotrupes auratus (Coleoptera: Scarabaeidae) in Japan, are described. The two new species are recognized as basal within the genus and thus occupy an important position for macroevolutionary studies that center on the model P. pacificus. Pristionchus fissidentatus n. sp. is distinguished by its unique stegostomatal morphology: in the stenostomatous form, the right subventral ridge has three prominent cusps and the left subventral sector has, in addition to a plate with two cusps, a prominent denticle slightly left of ventral; in the eurystomatous form, the right subventral stegostomatal sector shows both a tooth and a ridge with several cusps. Diagnostic of P. elegans n. sp. is the structure of the stenostomatous cheilostom, which bulges medially and is underlain by a large vacuolated ring. No eurystomatous form has been observed in P. elegans n. sp. Reproductive modes of P. fissidentatus n. sp. and P. elegans n. sp. are hermaphroditic and gonochoristic, respectively. The additional isolation of P. fissidentatus n. sp. from soil and two species of scarab beetle on La Réunion Island in the Indian Ocean suggests a broad geographic range for this species. 相似文献
14.
Anna Fricke Felix Bast Agustín Moreira-Saporiti Giovanni Martins Bussanello Flower E. Msuya Mirta Teichberg 《Journal of phycology》2024,60(2):554-573
Algal blooms are increasing worldwide, driven by elevated nutrient inputs. However, it is still unknown how tropical benthic algae will respond to heatwaves, which are expected to be more frequent under global warming. In the present study, a multifactorial experiment was carried out to investigate the potential synergistic effects of increased ammonium inputs (25 μM, control at 2.5 μM) and a heatwave (31°C, control at 25°C) on the growth and physiology (e.g., ammonium uptake, nutrient assimilation, photosynthetic performance, and pigment concentrations) of two bloom-forming algal species, Cladophoropsis sp. and Laurencia sp. Both algae positively responded to elevated ammonium concentrations with higher growth and chlorophyll a and lutein concentrations. Increased temperature was generally a less important driver, interacting with elevated ammonium by decreasing the algaes' %N content and N:P ratios. Interestingly, this stress response was not captured by the photosynthetic yield (Fv/Fm) nor by the carbon assimilation (%C), which increased for both algae at higher temperatures. The negative effects of higher temperature were, however, buffered by nutrient inputs, showing an antagonistic response in the combined treatment for the concentration of VAZ (violaxanthin, antheraxanthin, zeaxanthin) and thalli growth. Ammonium uptake was initially higher for Cladophoropsis sp. and increased for Laurencia sp. over experimental time, showing an acclimation capacity even in a short time interval. This experiment shows that both algae benefited from increased ammonium pulses and were able to overcome the otherwise detrimental stress of increasingly emerging temperature anomalies, which provide them a strong competitive advantage and might support their further expansions in tropical marine systems. 相似文献
15.
Purification and characterization of a liquefying α-amylase from alkalophilic thermophilic Bacillus sp. AAH-31 总被引:1,自引:0,他引:1
DH Kim N Morimoto W Saburi A Mukai K Imoto T Takehana S Koike H Mori H Matsui 《Bioscience, biotechnology, and biochemistry》2012,76(7):1378-1383
α-Amylase (EC 3.2.1.1) hydrolyzes an internal α-1,4-glucosidic linkage of starch and related glucans. Alkalophilic liquefying enzymes from Bacillus species are utilized as additives in dishwashing and laundry detergents. In this study, we found that Bacillus sp. AAH-31, isolated from soil, produced an alkalophilic liquefying α-amylase with high thermostability. Extracellular α-amylase from Bacillus sp. AAH-31 (AmyL) was purified in seven steps. The purified enzyme showed a single band of 91 kDa on SDS-PAGE. Its specific activity of hydrolysis of 0.5% soluble starch was 16.7 U/mg. Its optimum pH and temperature were 8.5 and 70 °C respectively. It was stable in a pH range of 6.4-10.3 and below 60 °C. The calcium ion did not affect its thermostability, unlike typical α-amylases. It showed 84.9% of residual activity after incubation in the presence of 0.1% w/v of EDTA at 60 °C for 1 h. Other chelating reagents (nitrilotriacetic acid and tripolyphosphate) did not affect the activity at all. AmyL was fully stable in 1% w/v of Tween 20, Tween 80, and Triton X-100, and 0.1% w/v of SDS and commercial detergents. It showed higher activity towards amylose than towards amylopectin or glycogen. Its hydrolytic activity towards γ-cyclodextin was as high as towards short-chain amylose. Maltotriose was its minimum substrate, and maltose and maltotriose accumulated in the hydrolysis of maltooligosaccharides longer than maltotriose and soluble starch. 相似文献
16.
Xia Li Tongcheng Xu Hongbo Lu Xiaohang Ma Lei Kai Kangping Guo Yuhua Zhao 《Protein expression and purification》2010,69(1):16-20
Purification of a cis-epoxysuccinic acid hydrolase was achieved by ammonium sulfate precipitation, ionic exchange chromatography, hydrophobic interaction chromatography followed by size-exclusion chromatography. The enzyme was purified 177-fold with a yield of 14.4%. The apparent molecular mass of the enzyme was determined to be 33 kDa under denaturing conditions. The optimum pH for enzyme activity was 7.0, and the enzyme exhibited maximum activity at about 45 °C in 50 mM sodium phosphate buffer (pH 7.5). EDTA and o-phenanthrolin inhibited the enzyme activity remarkably, suggesting that the enzyme needs some metal cation to maintain its activity. Results of inductively coupled plasma mass spectrometry analysis indicated that the cis-epoxysuccinic acid hydrolase needs Zn2+ as a cofactor. Eight amino acids sequenced from the N-terminal region of the cis-epoxysuccinic acid hydrolase showed the same sequence as the N-terminal region of the beta subunit of the cis-epoxysuccinic acid hydrolase obtained from Alcaligenes sp. 相似文献
17.
Two new Ulvella species, U. elegans R. Nielsen & K. Gunnarsson and U. islandica R. Nielsen & K. Gunnarsson are described. These microfilamentous marine green algae were found in the sublittoral zone in northern Iceland, epiphytic on Euthora cristata and associated with a calcareous polychaete tube, respectively. Unialgal cultures were established from field-collected material for morphological observations. In culture, Ulvella elegans was characterized by rosettes of monostromatic pseudoparenchyma consisting of radiating filaments with a margin of mutually free filaments. Each cell had one pyrenoid. Hairs were not observed. Ulvella islandica had a heterotrichous morphology, consisting of dense tufts of upright broad branches and much narrower, rhizoid-like branches. Acrochaete-type hairs occurred; these are hyaline non-septate merocytic extensions from a more or less bulbous base, which may be separated from the vegetative cell below. Most cells had one pyrenoid except for a few broad cells which had two or three. In a phylogenetic reconstruction based on the chloroplast-encoded tufA gene, the sequences for the two species were clearly distinct from any other Ulvella sequence available for this gene. Ulvella islandica was placed in a clade together with U. lens, U. wittrockii, U. reticulata and U. pseudorepens. Ulvella elegans occupied a branch deep in the phylogeny but the position was poorly supported. 相似文献
18.
Toshiyuki Suzuki Takeshi Uozumi Teruhiko Beppu 《Bioscience, biotechnology, and biochemistry》2013,77(10):2939-2947
NADH-dependent soluble l-α-hydroxyglutarate dehydrogenase (l-2-hydroxyglutarate: NAD+ 2-oxidoreductase) was found in a bacterium belonging to the genus Alcaligenes obtained from soil by citrate enrichment culture. A mutant with about 2.5-fold higher activity of the enzyme was derived from the bacterium and used as the enzyme source. High level of the enzyme was produced at the late stage of cultivation in the presence of citrate and with limited aeration. The enzyme was purified from the cells to homogeneity to give crystals, and its enzymatic properties were studied. The enzyme strongly reduced α-ketoglutarate to stereochemically pure l-α-hydroxyglutarate with NADH as a coenzyme, but it oxidized d-α-hydroxyglutarate with about 1/10 of the rate for l-form oxidation. 相似文献
19.
《Bioscience, biotechnology, and biochemistry》2013,77(7):1378-1383
α-Amylase (EC 3.2.1.1) hydrolyzes an internal α-1,4-glucosidic linkage of starch and related glucans. Alkalophilic liquefying enzymes from Bacillus species are utilized as additives in dishwashing and laundry detergents. In this study, we found that Bacillus sp. AAH-31, isolated from soil, produced an alkalophilic liquefying α-amylase with high thermostability. Extracellular α-amylase from Bacillus sp. AAH-31 (AmyL) was purified in seven steps. The purified enzyme showed a single band of 91 kDa on SDS–PAGE. Its specific activity of hydrolysis of 0.5% soluble starch was 16.7 U/mg. Its optimum pH and temperature were 8.5 and 70 °C respectively. It was stable in a pH range of 6.4–10.3 and below 60 °C. The calcium ion did not affect its thermostability, unlike typical α-amylases. It showed 84.9% of residual activity after incubation in the presence of 0.1% w/v of EDTA at 60 °C for 1 h. Other chelating reagents (nitrilotriacetic acid and tripolyphosphate) did not affect the activity at all. AmyL was fully stable in 1% w/v of Tween 20, Tween 80, and Triton X-100, and 0.1% w/v of SDS and commercial detergents. It showed higher activity towards amylose than towards amylopectin or glycogen. Its hydrolytic activity towards γ-cyclodextin was as high as towards short-chain amylose. Maltotriose was its minimum substrate, and maltose and maltotriose accumulated in the hydrolysis of maltooligosaccharides longer than maltotriose and soluble starch. 相似文献
20.
-Mannanase produced by Bacillus sp. W-2, isolated from decayed commercial konjak cake, was purified from the culture supernatant by (NH4)2 SO4 precipitation, adsorption to konjak gel, and column chromatography with DEAE-cellulose, Sephadex G-100 and Sephacryl S-200. Its molecular size was estimated by SDS-PAGE as 40 kDa, and by gel filtration as 36 kDa. The enzyme was most active at pH 7 and 70°C and was stable for at least 1 h between pH 5 and 10 and below 60°C. Its activity was completely inhibited by Hg2+. The enzyme hydrolysed galactomannan better than glucomannan and mainly produced mannose and mannobiose.The authors are with the Department of Bioproductive Science, Faculty of Agriculture, Utsunomiya University. Utsunomiya, Tochigi 321, Japan 相似文献