共查询到20条相似文献,搜索用时 31 毫秒
1.
Melanie Schmidt Matthias Luff Anne Mollwo Marc Kaminski Maria Mittag Georg Kreimer 《Journal of phycology》2007,43(2):284-294
The eyespot apparatus (EA) of Chlamydomonas reinhardtii P. A. Dang. consists of two layers of carotenoid‐rich lipid globules subtended by thylakoids. The outermost globule layer is additionally associated with the chloroplast envelope membranes and the plasma membrane. In a recent proteomic approach, we identified 202 proteins from isolated EAs of C. reinhardtii via at least two peptides, including, for example, structural components, signalling‐related proteins, and photosynthetic‐related membrane proteins. Here, we have analyzed the proteins of the EA with regard to their topological distribution using thermolysin to find out whether the arrangement of globules and membranes provides protection mechanisms for some of them. From about 230 protein spots separated on two‐dimensional gels, the majority were degraded by thermolysin. Five major protein spots were protected against the action of this protease. These proteins and some that were degradable were identified by mass spectrometry. Surprisingly, the thermolysin‐resistant proteins represented the α and β subunits of the soluble CF1 complex of the chloroplast ATP synthase. Degradable proteins included typical membrane proteins like LHCs, demonstrating that thermolysin is not in general sterically prevented by the EA structure from reaching membrane‐associated proteins. A control experiment showed that the CF1 complex of thylakoids is efficiently degraded by thermolysin. Blue native PAGE of thermolysin‐treated EAs followed by SDS‐PAGE revealed that the α and β subunits are present in conjunction with the γ subunit in a thermolysin‐resistant complex. These results provide strong evidence that a significant proportion of these ATP‐synthase subunits have a specialized localization and function within the EA of C. reinhardtii. 相似文献
2.
Kobchai Pattaragulwanit Daniel C. Brune Hans G. Trüper C. Dahl 《Archives of microbiology》1998,169(5):434-444
Purple sulfur bacteria store sulfur as intracellular globules enclosed by a protein envelope. We cloned the genes sgpA, sgpB, and sgpC, which encode the three different proteins that constitute the sulfur globule envelope of Chromatium vinosum D (DSMZ 180T). Southern hybridization analyses and nucleotide sequencing showed that these three genes are not clustered in the same operon.
All three genes are preceded by sequences resembling σ70-dependent promoters, and hairpin structures typical for rho-independent terminators are found immediately downstream of the
translational stop codons of sgpA, sgpB, and sgpC. Insertional inactivation of sgpA in Chr. vinosum showed that the presence of only one of the homologous proteins SgpA and SgpB suffices for formation of intact sulfur globules.
All three sgp genes encode translation products which – when compared to the isolated proteins – carry amino-terminal extensions. These
extensions meet all requirements for typical signal peptides indicating an extracytoplasmic localization of the sulfur globule
proteins. A fusion of the phoA gene to the sequence encoding the proposed signal peptide of sgpA led to high specific alkaline phosphatase activities in Escherichia coli, further supporting the envisaged targeting process. Together with electron microscopic evidence these results provide strong
indication for an extracytoplasmic localization of the sulfur globules in Chr. vinosum and probably in other Chromatiaceae. Extracytoplasmic formation of stored sulfur could contribute to the transmembranous
Δp that drives ATP synthesis and reverse electron flow in Chr. vinosum.
Received: 1 October 1997 / Accepted: 17 December 1997 相似文献
3.
Phototactic responses of four marine dinoflagellates with different types of eyespot and chloroplast 总被引:1,自引:0,他引:1
Takeo Horiguchi Hiroshi Kawai Mamoru Kubota Tetsuo Takahashi Masakatsu Watanabe 《Phycological Research》1999,47(2):101-107
Great structural variety is seen in the eyespot of dinoflagellates, a structure involved in phototaxis. Although there are several works on the phototactic responses in some species of dinoflagellates, none of the dinoflagellates used in these studies possessed an eyespot and, therefore, we have no knowledge of the relationship between eyespot type and phototactic response. In this study, we determined wavelength dependency curves for phototaxis in four marine dinoflagellates that possess a different type of either eyespot or chloroplast. These include: (i) a dinoflagellate possessing a peridinin-containing ohioroplast with an eyespot (Scrippsiella hexapraecingula Horiguchi et Chihara); (ii) a dinoflagellate containing a diatom endosymbiont and with the type B eyespot sensu Dodge (1984; (Peridinium foli-aceum (Stein) Biecheler); (iii) a dinoflagellate with peri-dinin-containing chloroplasts, but lacking an eyespot (Atexandrium hiranoi Kita et Fukuyo); and (iv) a dinoflagellate with fucoxanthin, 19′-hexanoyloxyfucoxanthin and 19′-butanoyloxyfucoxanthin, but lacking an eyespot (Gymnodinium mikimotoi Miyabe et Kominami ex Oda), Regardless of the eyespot or the chloroplast type, all four dinoflagellates showed similar wavelength dependency curves for phototaxis, with sensitivity between 380 and 520 nm, the highest peak at approximately 440 or 460 nm and smaller peaks or shoulders at 400–420 nm and 480–500 nm. Substantial peaks have also been noted in the ultraviolet range (260–280 nm). The ultrastructural study of the eye-spot of Scrippsiella hexapraecingula revealed that the eyespot consists of two layers of lipid globules and probably acts as a quarter-wave stack antenna. 相似文献
4.
To elucidate the graviperception of the unicellular fungus, Phycomycesblakesleeanus, sporangiophores were inspected for intracellular structures which relocate with respect to gravity. Two structures, paracrystalline
proteins (so-called octahedral crystals) and an aggregate of lipid globules, were identified which showed redistribution upon
reorientation of the sporangiophore. Octahedral crystals occur throughout the sporangiophore, including the apical growing
zone, and are localized inside vacuoles in which they reside singly or in clusters of up to 40 loosely associated individuals.
Upon a 90° reorientation of sporangiophores, crystal clusters sedimented in approximately 50–200 s from the upper to the lower
side, corresponding to a speed of 0.5–2 μm s−1. Stage-4 sporangiophores (with sporangium) of three mutants which lack the crystals displayed anormal kinetics of gravitropism
and substantially reduced bending angles in comparison to sporangiophores of the wild type. While horizontally placed wild-type
sporangiophores reached the vertical position after 10–12 h, the crystal-lacking mutants bent maximally 40°–50° upward. In
stage-1 sporangiophores a conspicuous aggregate of lipid globules is positioned about 50 μm below the apex. The globules floated
upwards when the sporangiophore was placed horizontally forming in this way a cap-like aggregate. It is proposed that both
the sedimenting protein crystals and the upward-floating globules are involved in gravisensing.
Received: 23 March 1999 / Accepted: 27 May 1999 相似文献
5.
6.
Hong SH Gohya M Ono H Murakami H Yamashita M Hirayama N Murooka Y 《Applied microbiology and biotechnology》2000,54(1):84-89
Genes for dimeric and tetrameric human metallothionein (hMT) were designed and successfully overexpressed in Escherichia coli to generate functional oligomeric hMTs. An hMT synthesized with prokaryotic codons, a linker encoding a gly-gly-gly tripeptide,
and Met-deficient hMT-II was ligated to create a dimeric hMT, from which a tetrameric hMT was then constructed. The increased
molecular size of the constructs resulted in improved stability and productivity in E. coli. The oligomeric proteins formed inclusion bodies which were dissolved with dithiothreitol, and the purified apo-metallothioneins were reconstituted with Cd or Zn ions in a reducing condition. The oligomeric hMT proteins incubated with
Cd ions showed a typical Cd-thiolate absorbance peak at 245–255 nm. The dimeric and tetrameric hMT proteins exhibited both
Cd and Zn binding activities that were respectively two and four times higher than those of the hMT-II monomer protein. These
novel oligomeric hMTs may be useful in bioremediation for heavy metals.
Received: 18 October 1999 / Received last revision: 21 January 2000 / Accepted: 13 February 2000 相似文献
7.
Shota Kato Kazunari Ozasa Mizuo Maeda Yuri Tanno Shun Tamaki Mieko Higuchi‐Takeuchi Keiji Numata Yutaka Kodama Mayuko Sato Kiminori Toyooka Tomoko Shinomura 《The Plant journal : for cell and molecular biology》2020,101(5):1091-1102
Carotenoids are the most universal and most widespread pigments in nature. They have played pivotal roles in the evolution of photosensing mechanisms in microbes and of vision in animals. Several groups of phytoflagellates developed a photoreceptive organelle called the eyespot apparatus (EA) consisting of two separable components: the eyespot, a cluster of carotenoid‐rich globules that acts as a reflector device, and actual photoreceptors for photobehaviors. Unlike other algal eyespots, the eyespot of Euglenophyta lacks reflective properties and is generally considered to act as a shading device for the photoreceptor (paraflagellar body, PFB) for major photomovements. However, the function of the eyespot of Euglenophyta has not yet been fully proven. Here, we report that the blocking carotenoid biosynthesis in Euglena gracilis by suppressing the phytoene synthase gene (crtB) caused a defect in eyespot function resulting in a loss of phototaxis. Raman spectroscopy and transmission electron microscopy suggested that EgcrtB‐suppressed cells formed eyespot globules but had a defect in the accumulation of carotenoids in those packets. Motion analysis revealed the loss of phototaxis in EgcrtB‐suppressed cells: a defect in the initiation of turning movements immediately after a change in light direction, rather than a defect in the termination of cell turning at the appropriate position due to a loss of the shading effect on the PFB. This study revealed that carotenoids are essential for light perception by the EA for the initiation of phototactic movement by E. gracilis, suggesting one possible photosensory role of carotenoids in the EA for the phototaxis. 相似文献
8.
The aim of the present investigation was to evaluate microemulsion as a vehicle for dermal drug delivery and to develop microemulsion-based
gel of terbinafine for the treatment of onychomycosis. D-optimal mixture experimental design was adopted to optimize the amount
of oil (X
1), Smix (mixture of surfactant and cosurfactant; X
2) and water (X
3) in the microemulsion. The formulations were assessed for globule size (in nanometers; Y
1) and solubility of drug in microemulsion (in milligrams per milliliter; Y
2). The microemulsion containing 5.75% oil, 53.75% surfactant–cosurfactant mixture and 40.5% water was selected as the optimized
batch. The globule size and solubility of the optimized batch were 18.14 nm and 43.71 mg/ml, respectively. Transmission electron
microscopy showed that globules were spherical in shape. Drug containing microemulsion was converted into gel employing 0.75%
w/w carbopol 934P. The optimized gel showed better penetration and retention in the human cadaver skin as compared to the commercial
cream. The cumulative amount of terbinafine permeated after 12 h was 244.65 ± 18.43 μg cm−2 which was three times more than the selected commercial cream. Terbinafine microemulsion in the gel form showed better activity
against Candida albicans and Trichophyton rubrum than the commercial cream. It was concluded that drug-loaded gel could be a promising formulation for effective treatment
of onychomycosis. 相似文献
9.
A M Belles T J Montville B P Wasserman 《Journal of industrial microbiology & biotechnology》2000,24(1):71-74
Alcohol-extractable, hydrophobic zein proteins contaminate starch granule surfaces and can be removed by enzymatic digestion
with thermolysin. The goal of this research was to find practical alternatives to thermolysin that might be used during the
corn wet-milling process. All of the commercial thermostable alkaline proteases studied (SP 709, Neutrase, and Spezyme FAN)
removed the zein proteins from various types of cornstarch, as demonstrated by the lack of protein bands below 30 kDa under
the reducing conditions of SDS-PAGE gel. Each enzyme removed the zein proteins as effectively as thermolysin removed them.
However, the removal of the zein protein did not reduce the quantity of free fatty acids associated with the starch. Journal of Industrial Microbiology & Biotechnology (2000) 24, 71–74.
Received 27 May 1999/ Accepted in revised form 01 October 1999 相似文献
10.
Stéphane Helleboid Guy Bauw Lionel Belingheri Jacques Vasseur Jean-Louis Hilbert 《Planta》1998,205(1):56-63
In leaf tissues of the Cichorium hybrid clone `474' (C. intybus L. var. sativum × C. endivia L. var. latifolia), the acquisition and expression of embryogenic competence was characterised by the appearance of 15 polypeptides (Boyer
et al., 1993, Plant Sci 93: 41–53). The 38-kDa proteins were found to be abundantly present in conditioned embryogenic medium
after the first division of the induced cells. These proteins seemed to be glycosylated as indicated by general carbohydrate
detection methods. Internal amino-acid sequences obtained after microsequencing tryptic peptides appeared to be 36–57% homologous
with plant β-1,3-endoglucanases. In addition, these 38-kDa proteins were recognised by antibodies raised against the pathogenesis-related
tobacco glucanase PR2a and their β-1,3-glucanase activity was demonstrated by direct detection in polyacrylamide gels after
electrophoresis. These results strongly suggested that the 38-kDa somatic-embryogenesis-related (SER) polypeptides are β-1,3-glucanases.
Moreover, the level of glucanase activity was nearly three times higher in the medium of the embryogenic `474' line than in
the medium of a non-embryogenic line. The possible involvement of the extracellular 38-kDa proteins in callose degradation
during somatic embryogenesis is discussed.
Received: 5 May 1997 / Accepted: 25 September 1997 相似文献
11.
Kim HS Yoon BD Choung DH Oh HM Katsuragi T Tani Y 《Applied microbiology and biotechnology》1999,52(5):713-721
One yeast strain, SY16, was selected as a potential producer of a biosurfactant, and identified as a Candida species. A biosurfactant produced from Candida sp. SY16 was purified and confirmed to be a glycolipid. This glycolipid-type biosurfactant lowered the surface tension of
water to 29 dyne/cm at critical micelle concentration of 10 mg/l (1.5 × 10−5 M), and the minimum interfacial tension was 0.1 dyne/cm against kerosene. Thin-layer and high-pressure liquid chromatography
studies demonstrated that the glycolipid contained mannosylerythritol as a hydrophilic moiety. The hydrophilic sugar moiety
of the biosurfactant was determined to be β-d-mannopyranosyl-(1 → 4)-O-meso-erythritol by nuclear magnetic resonance (NMR) and fast atom bombardment mass–spectroscopy analyses. The hydrophobic moiety,
fatty acids, of the biosurfactant was determined to be hexanoic, dodecanoic, tetradecanoic, and tetradecenoic acid by gas
chromatography–mass spectroscopy. The structure of the native biosurfactant was determined to be 6-O-acetyl-2,3- di-O-alkanoyl-β-d-mannopyranosyl-(1 → 4)-O-meso-erythritol by NMR analyses. We newly determined that an acetyl group was linked to the C-6 position of the d-mannose unit in the hydrophilic sugar moiety.
Received: 18 December 1999 / Received last revision: 2 June 1999 / Accepted: 4 June 1999 相似文献
12.
Thermomonospora curvata produced a thermostable β-xylosidase during growth on birch xylan. The enzyme, extracted by sonication of early stationary
phase mycelia, was purified by isoelectric focusing and size exclusion HPLC. The isoelectric point was pH 4.8. The molecular
weight was estimated to be 102 000 by size exclusion HPLC and 112 000 by SDS-PAGE. Maximal activity occurred at pH 6–7 and
60–68°C. K
m values for xylobiose and p-nitrophenyl-β -D-xylopyranoside were 4.0 M and 0.6 M respectively. The enzyme was sensitive to low levels of Hg2+ (50% inhibition at 0.2 μM), but was stimulated by Co2+ and Pb2+. Addition of the xylosidase to a xylanase reaction mixture increased the liberation of xylose equivalents from xylan and
decreased the proportion of xylobiose in the hydrolysate.
Received 14 April 1997/ Accepted in revised form 21 October 1997 相似文献
13.
Identification and partial characterization of proteins and proteoglycans encrusting the secondary cell walls of flax fibres 总被引:3,自引:0,他引:3
Four proteins were isolated from depectinised elementary fibres of flax (Linum usitatissimum L.), using either alkali or cellulase digestion treatments. All the four proteins were characterized by a deficiency or low
contents of hydroxyproline and by high levels of glutamic acid/glutamine and/or aspartic acid/asparagine. The two proteoglycans
solubilized with cellulase strongly reacted with β-glucosyl Yariv reagent but not with α-glucosyl Yariv reagent and contained
appreciable amounts of alanine, glycine, serine and threonine, suggesting a relationship with cell wall hydroxyproline-deficient
arabinogalactan-proteins. The two alkali-extracted proteins did not show any reaction with β-glucosyl Yariv dye. Due to the
harsh treatment, they might only partially represent the original proteins. Due to its high level of glycine (41%), one of
these proteins might be classified as a glycine-rich protein. The latter polypeptide, of low molecular molar mass, contained
14.6% leucine and might consist of a domain related to leucine-rich proteins. The data show that these proteins and arabinogalactan-protein-like
proteoglycans were strongly associated with the secondary walls of flax fibres. Their presence in small amounts (0.1–0.4%),
raises the problem of their putative structural role.
Received: 22 October 1999 / Accepted: 17 January 2000 相似文献
14.
S Chakraborti R K Sani U C Banerjee R C Sobti 《Journal of industrial microbiology & biotechnology》2000,24(1):58-63
An extracellular β-galactosidase which catalyzed the production of galacto-oligosaccharide from lactose was harvested from
the late stationary-phase of Bacillus sp MTCC 3088. The enzyme was purified 36.2-fold by ZnCl2 precipitation, ion exchange, hydrophobic interaction and gel filtration chromatography with an overall recovery of 12.7%.
The molecular mass of the purified enzyme was estimated to be about 484 kDa by gel filtration on a Sephadex G-200 packed column
and the molecular masses of the subunits were estimated to be 115, 86.5, 72.5, 45.7 and 41.2 kDa by sodium dodecyl sulfate-polyacrylamide
gel electrophoresis. The isoelectric point of the native enzyme, determined by polyacrylamide gel electrofocusing, was 6.2.
The optimum pH and temperature were 8 and 60°C, respectively. The Michaelis–Menten constants determined with respect to o-NO2-phenyl-β-D-galactopyranoside and lactose were 6.34 and 6.18 mM, respectively. The enzyme activity was strongly inhibited (68%) by galactose,
the end product of lactose hydrolysis reaction. The β-galactosidase was specific for β-D anomeric linkages. Enzyme activity was significantly inhibited by metal ions (Hg2+, Cu2+ and Ag+) in the 1–2.5 mM range. Mg2+ was a good activator. Catalytic activity was not affected by the chelating agent EDTA. Journal of Industrial Microbiology & Biotechnology (2000) 24, 58–63.
Received 09 February 1999/ Accepted in revised form 24 September 1999 相似文献
15.
S Hayashi S Sako H Yokoi Y Takasaki K Imada 《Journal of industrial microbiology & biotechnology》1999,22(3):160-163
β-Glucosidase hydrolyzing cellobiose was extracted from Aureobasidium sp ATCC 20524 and purified to homogeneity. The molecular mass was estimated to be about 331 kDa. The enzyme contained 26.5%
(w/w) carbohydrate. The optimum pH and temperature for the enzyme reaction were pH 4 and 80°C, respectively. The enzyme was
stable at a wide range of pH, 2.2–9.8, after 3 h and at 75°C for 15 min. The kinetic parameters were determined. The enzyme
was relatively stable against typical organic enzyme inhibitors. The enzyme also hydrolyzed gentiobiose, p-nitrophenyl-β-glucoside and salicin.
Received 05 November 1998/ Accepted in revised form 14 February 1999 相似文献
16.
Embryonic, larval, and juvenile development of two cyprinid species belonging to the Zacco temminckii species' group, Z. temminckii (Temminck and Schlegel) and Zacco sp. (type A), are described and compared with each other from laboratory-reared and wild specimens. The eggs of both species
were closely similar except in diameter [1.92–2.20 mm in Z. temminckii vs. 1.60–1.75 mm in Z. sp. (type A)], being demersal, almost spherical in shape, transparent and unpigmented, with a pale yellow yolk, and no oil
globule. Hatching occurred 40–53 h after fertilization in Z. temminckii and after 47–60 h in Z. sp. (type A). The newly hatched larvae of both species [4.9–5.3 mm in body length (BL) in Z. temminckii and 3.5–4.8 mm BL in Z. sp. (type A)] also resembled each other, having a large transparent pear-shaped yolk and lacking body pigmentation. Myomere
counts of Z. temminckii and Z. sp. (type A) larvae and juveniles were 24–27 + 14–17 = 41–42 and 23–27 + 14–17 = 40–41, respectively. The yolk was completely
absorbed at 8.3 mm BL in Z. temminckii and at 6.6 mm BL in Z. sp. (type A). Notochord flexion was initiated and completed at 7.8 mm BL and 8.2 mm BL in Z. temminckii and at 6.3 mm BL and 6.6 mm BL in Z. sp. (type A), respectively. Aggregate numbers of all fin rays were completed at 17 mm BL in Z. temminckii and 13 mm BL in Z. sp. (type A). Although the morphology of larvae and juveniles of both species was very similar, differences in body length
of each developmental stage, the duration and process of disappearance of the adipose finfold, the anal fin ray counts, and
pigmentation on the lateral body surface were clearly recognized.
Received: August 10, 2001 / Revised: March 14, 2002 / Accepted: March 27, 2002 相似文献
17.
Methyl ent-17-hydroxy-16β-kauran-19-oate was fed to a 2-day-old culture of the fungus Rhizopus stolonifer, fermenting at room temperature (25 °C) in an orbital shaker (2 l). After 11 days, both broth and mycelia were extracted
with ethyl acetate. Two novel compounds were isolated from this experiment: methyl ent-9α,17-dihydroxy-16β-kauran-19-oate and methyl ent-7α,17-dihydroxy-16β-kauran-19-oate. Their structures were fully confirmed by spectroscopic methods.
Received: 22 July 1999 / Received revision: 2 November 1999 / Accepted: 12 November 1999 相似文献
18.
Fabiano de A. Ximenes Fabiane Quirino de Paula Silveira Edivaldo Ximenes F. Filho 《Current microbiology》1996,33(2):71-77
Nine Trichoderma harzianum strains were screened for β-xylosidase activity when grown in solid-state cultures on media containing wheat bran as the
carbon source. All strains produced β-xylosidase activity, the most active being in extracts of cultures of T. harzianum strain 4. A β-xylosidase was purified by ammonium sulfate precipitation, ultrafiltration, gel filtration, and ion exchange
chromatography from solid-state cultures of T. harzianum strain C. Enzyme preparations yielded a single band when stained for protein following eletrophoresis. The molecular weight
value, calculated following SDS-PAGE, was determined to be 60 kDa. β-Xylosidase was most active at pH 4.0–4.5 and 70°C. This
enzyme had a K
m
value of 0.053 mM. The phenol-sulfuric acid method detected the presence of a small amount of carbohydrate in the purified
enzyme preparation. β-Xylosidase was active against some p-nitrophenylglycosides. The enzyme was inactive against xylan and PNPG. β-xylosidase activity was inhibited by xylose and
SDS. Iodoacetamide, dithiothreitol, gluconolactone, glucose, and mercuric chloride failed to inactivate this enzyme's activity.
A synergistic effect was observed when β-xylosidase from T. harzianum strain C and β-xylanase from Aspergillus fumigatus were incubated with pretreated arabinoxylan.
Received: 1 December 1995 / Accepted: 11 December 1995 相似文献
19.
A number of nutritional factors influencing growth and glucose oxidase (EC 1.1.3.4) production by a newly isolated strain
of Penicillium pinophilum were investigated. The most important factors for glucose oxidase production were the use of sucrose as the carbon source,
and growth of the fungus at non-optimal pH 6.5. The enzyme was purified to apparent homogeneity with a yield of 74%, including
an efficient extraction step of the mycelium mass at pH 3.0, cation-exchange chromatography and gel filtration. The relative
molecular mass (M
r) of native glucose oxidase was determined to be 154 700 ± 4970, and 77 700 for the denatured subunit. Electron-microscopic
examinations revealed a sandwich-shaped dimeric molecule with subunit dimensions of 5.0 × 8.0 nm. Glucose oxidase is a glycoprotein
that contains tightly bound FAD with an estimated stoichiometry of 1.76 mol/mol enzyme. The enzyme is specific for d-glucose, for which a K
m value of 6.2 mM was determined. The pH optimum was determined in the range pH 4.0–6.0. Glucose oxidase showed high stability
on storage in sodium citrate (pH 5.0) and in potassium phosphate (pH 6.0), each 100 mM. The half-life of the activity was
considerably more than 305 days at 4 °C and 30 °C, and 213 days at 40 °C. The enzyme was unstable at temperatures above 40 °C
in the range pH 2.0–4.0 and at a pH above 7.0.
Received: 18 November 1996 / Received revision: 3 March 1997 / Accepted: 7 March 1997 相似文献
20.
Purification and properties of a novel raw starch degrading cyclomaltodextrin glucanotransferase from Bacillus firmus 总被引:3,自引:0,他引:3
B. N. Gawande A. Goel A. Y. Patkar S. N. Nene 《Applied microbiology and biotechnology》1999,51(4):504-509
A novel raw starch degrading cyclomaltodextrin glucanotransferase (CGTase; E.C. 2.4.1.19), produced by Bacillus firmus, was purified to homogeneity by ultrafiltration, affinity and gel filtration chromatography. The molecular weight of the
pure protein was estimated to be 78 000 and 82 000 Da, by SDS-PAGE and gel filtration, respectively. The pure enzyme had a
pH optimum in the range 5.5–8.5. It was stable over the pH range 7–11 at 10 °C, and at pH 7.0 at 60 °C. The optimum temperature
for enzyme activity was 65 °C. In the absence of substrate, the enzyme rapidly lost its activity above 30 °C. K
m and k
cat for the pure enzyme were 1.21 mg/ml and 145.17 μM/mg per minute respectively, with soluble starch as the substrate. For cyclodextrin
production, tapioca starch was the best substrate used when gelatinized, while wheat starch was the best substrate used when
raw. This CGTase could degrade raw wheat starch very efficiently; up to 50% conversion to cyclodextrins was obtained from
150 g/l starch without using any additives. The enzyme produced α-, β- and γ-cyclodextrins in the ratio of 0.2:9.2:0.6 and
0.2:8.6:1.2 from gelatinized tapioca starch and raw wheat starch with 150 g/l concentration respectively, after 18 h incubation.
Received: 25 September 1998 / Received revision: 15 December 1998 / Accepted: 21 December 1998 相似文献