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1.
Abrunhosa L Costa M Areias F Venâncio A Proença F 《Journal of industrial microbiology & biotechnology》2007,34(12):787-792
The activity on Aspergillus spp. growth and on ochratoxin A production of two novel chromene dimers (3) was evaluated. The results of the bioassays indicate that the chromene dimer 3a inhibited mycelia growth by approximately 50% (EC50) at 140.1 μmol L−1 for A. niger, 384.2 μmol L−1 for A. carbonarius, 69.1 μmol L−1 for A. alliaceus and 559.1 μmol L−1 for A. ochraceus. When applied at concentrations of 2 mmol L−1, 3a totally inhibited the growth of all Aspergillus spp. tested. Furthermore, ochratoxin A production by A. alliaceus was reduced by about 94% with a 200 μmol L−1 solution of this compound. A moderate inhibitory effect was observed for the analogous structure 3b on ochratoxin A production but not in mycelia growth. No inhibition was registered for compounds 2a and 2b, used as synthetic precursors of the dimeric species 3. 相似文献
2.
Feng B Hu W Ma BP Wang YZ Huang HZ Wang SQ Qian XH 《Applied microbiology and biotechnology》2007,76(6):1329-1338
It has been previously reported that a glucoamylase from Curvularia lunata is able to hydrolyze the terminal 1,2-linked rhamnosyl residues of sugar chains at C-3 position of steroidal saponins. In
this work, the enzyme was isolated and identified after isolation and purification by column chromatography including gel
filtration and ion-exchange chromatography. Analysis of protein fragments by MALDI-TOF/TOF™ proteomics Analyzer indicated
the enzyme to be 1,4-alpha-D-glucan glucohydrolase EC 3.2.1.3, GA and had considerable homology with the glucoamylase from Aspergillus oryzae. We first found that the glucoamylase was produced from C. lunata and was able to hydrolyze the terminal rhamnosyl of steroidal saponins. The enzyme had the general character of glucoamylase,
which hydrolyze starch. It had a molecular mass of 66 kDa and was optimally active at 50°C, pH 4, and specific activity of
12.34 U mg of total protein−1 under the conditions, using diosgenin-3-O-α-L-rhamnopyranosyl(1→4)-[α-L-rhamnopyranosyl (1→2)]-β-D-glucopyranoside (compound II) as the substrate. Furthermore, four kinds of commercial glucoamylases from Aspergillus niger were investigated in this work, and they had the similar activity in hydrolyzing terminal rhamnosyl residues of steroidal
saponin.
This project was supported by the National Natural Science Foundation of China (NSFC; 30572333). 相似文献
3.
Poonperm W Takata G Okada H Morimoto K Granström TB Izumori K 《Applied microbiology and biotechnology》2007,76(6):1297-1307
The l-rhamnose isomerase gene (L
-rhi) encoding for l-rhamnose isomerase (l-RhI) from Bacillus pallidus Y25, a facultative thermophilic bacterium, was cloned and overexpressed in Escherichia coli with a cooperation of the 6×His sequence at a C-terminal of the protein. The open reading frame of L
-rhi consisted of 1,236 nucleotides encoding 412 amino acid residues with a calculated molecular mass of 47,636 Da, showing a
good agreement with the native enzyme. Mass-produced l-RhI was achieved in a large quantity (470 mg/l broth) as a soluble protein. The recombinant enzyme was purified to homogeneity
by a single step purification using a Ni-NTA affinity column chromatography. The purified recombinant l-RhI exhibited maximum activity at 65°C (pH 7.0) under assay conditions, while 90% of the initial enzyme activity could be
retained after incubation at 60°C for 60 min. The apparent affinity (K
m) and catalytic efficiency (k
cat/K
m) for l-rhamnose (at 65°C) were 4.89 mM and 8.36 × 105 M−1 min−1, respectively. The enzyme demonstrated relatively low levels of amino acid sequence similarity (42 and 12%), higher thermostability,
and different substrate specificity to those of E. coli and Pseudomonas stutzeri, respectively. The enzyme has a good catalyzing activity at 50°C, for d-allose, l-mannose, d-ribulose, and l-talose from d-psicose, l-fructose, d-ribose and l-tagatose with a conversion yield of 35, 25, 16 and 10%, respectively, without a contamination of by-products. These findings
indicated that the recombinant l-RhI from B. pallidus is appropriate for use as a new source of rare sugar producing enzyme on a mass scale production. 相似文献
4.
Eight of eleven ochratoxigenic isolates of Penicillium nordicum and Penicillium verrucosum produced guttation droplets when grown on Czapek yeast extract (CYA) agar for 10–14 days at 25°C. Parallel cultivation of
one strain each of P. nordicum and P. verrucosum on malt extract agar demonstrated that higher volumes of exudate are produced on this agar. However, HPLC analyses revealed
higher concentrations of ochratoxin A (OTA) and B (OTB) in droplets originating from cultures on CYA. For quantitative determination
of the mycotoxin contents, triplicates of three isolates each of P. nordicum and P. verrucosum were grown as single spot cultures on CYA for up to 14 days at 25°C. Guttation droplets were carefully collected between
day 11 and 14 with a microliter syringe from each culture. Extracts from exudates and corresponding mycelia as well as fungal
free agar were analyzed by HPLC for the occurrence of ochratoxin A (OTA) and B (OTB). Mean concentrations ranging between
92.7–8667.0 ng OTA and 159.7–2943.3 ng OTB per ml were detected in the guttation fluids. Considerably lower toxin levels were
found in corresponding samples of the underlying mycelia (9.0–819.3 ng OTA and 4.5–409.7 ng OTB/g) and fungal free agar (15.3–417.0 ng
OTA and 12.7–151.3 ng OTB/g). This is the first report which shows that high amounts of mycotoxins could be excreted from
toxigenic Penicillium isolates into guttation droplets. 相似文献
5.
Dihydroorotase was purified to homogeneity fromPseudomonas putida. The relative molecular mass of the native enzyme was 82 kDa and the enzyme consisted of two identical subunits with a relative
molecular mass of 41 kDa. The enzyme only hydrolyzed dihydro-l-orotate and its methyl ester, and the reactions were reversible. The apparentK
m andV
max values for dihydro-l-orotate hydrolysis (at pH 7.4) were 0.081 mM and 18 μmol min−1 mg−1, respectively; and those forN-carbamoyl-dl-aspartate (at pH 6.0) were 2.2 mM and 68 μmol min−1 mg−1, respectively. The enzyme was inhibited by metal ion chelators and activated by Zn2+. However, excessive Zn2+ was inhibitory. The enzyme was inhibited by sulfhydryl reagents, and competitively inhibited byN-carbamoylamino acids such asN-carbamoylglycine, with aK
i value of 2.7 mM. The enzyme was also inhibited noncompetitively by pyrimidine-metabolism intermediates such as dihydrouracil
and orotate, with aK
i value of 3.4 and 0.75 mM, respectively, suggesting that the enzyme activity is regulated by pyrimidine-metabolism intermediates
and that dihydroorotase plays a role in the control of pyrimidine biosynthesis. 相似文献
6.
Hsiang-Ling Chen Chen-Tien Chang Long-Liu Lin Tein-Yu Li Huei-Fen Lo 《World journal of microbiology & biotechnology》2009,25(2):323-330
To express Escherichia coli novablue dipeptidyl carboxypeptidase (EcDCP), the gene was amplified by PCR and cloned into the expression plasmid pQE-31 to yield pQE-EcDCP. His6-tagged EcDCP (His6-EcDCP) was over-expressed in E. coli M15 (pQE-EcDCP) as a soluble and active form under 0.05 mM IPTG induction at 26°C for 12 h. The recombinant enzyme was purified to homogeneity
by Ni2+-NTA resin and had a molecular mass of approximately 75 kDa. The temperature and pH optima for His6-EcDCP were 37°C and 7.0, respectively. In the presence of 200 mM NaCl, His6-EcDCP was stimulated by 1.5 fold. The K
M and k
cat values of the enzyme for N-benzoyl-l-glycyl-l-histidyl-l-leucine were 1.83 mM and 168.3 s−1, respectively. His6-EcDCP activity was dramatically inhibited by 10 mM EDTA, 0.25 mM 1.10-phenanthroline, and 2.5 mM DEPC, but it was not affected
by Ser, Asp, Lys, and Trp protease inhibitors. Analysis of His6-EcDCP by circular dichroism revealed that the secondary structures of the enzyme in 30 mM universal buffer (pH 7.0) were 17%
α-helix, 35% β-sheet and 47% random coil. Mid point of thermal transition was calculated to be 55°C for the recombinant enzyme. 相似文献
7.
Jin Zhou Ju Chu Yong-Hong Wang Si-Liang Zhang Ying-Ping Zhuang Zhong-Yi Yuan 《World journal of microbiology & biotechnology》2008,24(6):789-796
An intracellular S-adenosylmethionine synthetase (SAM-s) was purified from the fermentation broth of Pichia pastoris GS115 by a sequence chromatography column. It was purified to apparent homogeneity by (NH4)2SO4 fractionation (30–60%), anion exchange, hydrophobic interaction, anion exchange and gel filtration chromatography. HPLC showed
the purity of purified SAM-s was 91.2%. The enzyme was purified up to 49.5-fold with a final yield of 20.3%. The molecular
weight of the homogeneous enzyme was 43.6 KDa, as determined by electro-spray ionization mass spectrometry (ESI-MS). Its isoelectric
point was approximately 4.7, indicating an acidic character. The optimum pH and temperature for the enzyme reaction were 8.5
and 35 °C, respectively. The enzyme was stable at pH 7.0–9.0 and was easy to inactivate in acid solution (pH ≤ 5.0). The temperature
stability was up to 45 °C. Metal ions, such as, Mn2+ and K+ at the concentration of 5 mM had a slight activation effect on the enzyme activity and the Mg2+ activated the enzyme significantly. The enzyme activity was strongly inhibited by heavy metal ions (Cu2+ and Ag2+) and EDTA. The purified enzyme from the transformed Pichia pastoris synthesized S-adenosylmethionine (SAM) from ATP and l-methionine in vitro with a K
m of 120 and 330 μM and V
max of 8.1 and 23.2 μmol/mg/min for l-methionine and ATP, respectively. 相似文献
8.
Extracellular lipase of <Emphasis Type="Italic">Aspergillus niger</Emphasis> NRRL3; production,partial purification and properties 总被引:1,自引:0,他引:1
Four strains of Aspergillus niger were screened for lipase production. Each was cultivated on four different media differing in their contents of mineral components
and sources of carbon and nitrogen. Aspergillus niger NRRL3 produced maximal activity (325U/ml) when grown in 3% peptone, 0.05% MgSO4.7H2O, 0.05% KCl, 0.2% K2HPO4 and 1% olive oil:glucose (0.5:0.5). A. niger NRRL3 lipase was partially purified by ammonium sulphate precipitation. The majority of lipase activity (48%) was located
in fraction IV precipitated at 50–60% of saturation with a 18-fold enzyme purification. The optimal pH of the partial purified
lipase preparation for the hydrolysis of emulsified olive oil was 7.2 and the optimum temperature was 60°C. At 70°C, the enzyme
retained more than 90% of its activity. Enzyme activity was inhibited by Hg2+ and K+, whereas Ca2+ and Mn2+ greatly stimulated its activity. Additionally, the formed lipase was stored for one month without any loss in the activity. 相似文献
9.
Kemel Jellouli Ali Bougatef Laila Manni Rym Agrebi Rayda Siala Islem Younes Moncef Nasri 《Journal of industrial microbiology & biotechnology》2009,36(7):939-948
A protease-producing bacterium was isolated from an alkaline wastewater of the soap industry and identified as Vibrio metschnikovii J1 on the basis of the 16S rRNA gene sequencing and biochemical properties. The strain was found to over-produce proteases
when it was grown at 30°C in media containing casein as carbon source (14,000 U ml−1). J1 enzyme, the major protease produced by V. metschnikovii J1, was purified by a three-step procedure, with a 2.1-fold increase in specific activity and 33.3% recovery. The molecular
weight of the purified protease was estimated to be 30 kDa by SDS-PAGE and gel filtration. The N-terminal amino acid sequence
of the first 20 amino acids of the purified J1 protease was AQQTPYGIRMVQADQLSDVY. The enzyme was highly active over a wide
range of pH from 9.0 to 12.0, with an optimum at pH 11.0. The optimum temperature for the purified enzyme was 60°C. The activity
of the enzyme was totally lost in the presence of PMSF, suggesting that the purified enzyme is a serine protease. The kinetic
constants K
m and K
cat of the purified enzyme using N-succinyl-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide were 0.158 mM and 1.14 × 105 min−1, respectively. The catalytic efficiency (K
cat
/K
m) was 7.23 × 108 min−1 M−1. The enzyme showed extreme stability toward non-ionic surfactants and oxidizing agents. In addition, it showed high stability
and compatibility with some commercial liquid and solid detergents. The aprJ1 gene, which encodes the alkaline protease from V. metschnikovii J1, was isolated, and its DNA sequence was determined. The deduced amino acid sequence of the preproenzyme differs from that
of V. metschnikovii RH530 detergent-stable protease by 12 amino acids, 7 located in the propeptide and 5 in the mature enzyme. 相似文献
10.
An N-acetylglucosaminidase produced by Streptomyces cerradoensis was partially purified giving, by SDS-PAGE analysis, two main protein bands with Mr of 58.9 and 56.4 kDa. The Km and Vmax values for the enzyme using p-nitrophenyl-β-N-acetylglucosaminide as substrate were of 0.13 mM and 1.95 U mg−1 protein, respectively. The enzyme was optimally activity at pH 5.5 and at 50 °C when assayed over 10 min. Enzyme activity
was strongly inhibited by Cu2+ and Hg2+ at 10 mM, and was specific to substrates containing acetamide groups such as p-nitrophenyl-β-N-acetylglucosaminide and p-nitrophenyl-β-D-N,N′-diacetylchitobiose. 相似文献
11.
Canakci S Belduz AO Saha BC Yasar A Ayaz FA Yayli N 《Applied microbiology and biotechnology》2007,75(4):813-820
The gene encoding an α-l-arabinofuranosidase from Geobacillus caldoxylolyticus TK4, AbfATK4, was isolated, cloned, and sequenced. The deduced protein had a molecular mass of about 58 kDa, and analysis
of its amino acid sequence revealed significant homology and conservation of different catalytic residues with α-l-arabinofuranosidases belonging to family 51 of the glycoside hydrolases. A histidine tag was introduced at the N-terminal
end of AbfATK4, and the recombinant protein was expressed in Escherichia coli BL21, under control of isopropyl-β-D-thiogalactopyranoside-inducible T7 promoter. The enzyme was purified by nickel affinity
chromatography. The molecular mass of the native protein, as determined by gel filtration, was about 236 kDa, suggesting a
homotetrameric structure. AbfATK4 was active at a broad pH range (pH 5.0–10.0) and at a broad temperature range (40–85°C),
and it had an optimum pH of 6.0 and an optimum temperature of 75–80°C. The enzyme was more thermostable than previously described
arabinofuranosidases and did not lose any activity after 48 h incubation at 70°C. The protein exhibited a high level of activity
with p-nitrophenyl-α-l-arabinofuranoside, with apparent K
m and V
max values of 0.17 mM and 588.2 U/mg, respectively. AbfATK4 also exhibited a low level of activity with p-nitrophenyl-β-d-xylopyranoside, with apparent K
m and V
max values of 1.57 mM and 151.5 U/mg, respectively. AbfATK4 released l-arabinose only from arabinan and arabinooligosaccharides. No endoarabinanase activity was detected. These findings suggest
that AbfATK4 is an exo-acting enzyme. 相似文献
12.
Studies on the <Emphasis Type="Italic">Pycnoporus sanguineus</Emphasis> CCT-4518 laccase purified by hydrophobic interaction chromatography 总被引:1,自引:0,他引:1
A laccase from Pycnoporus sanguineus was purified by two steps using phenyl-Sepharose columm. A typical procedure provided 54.1-fold purification, with a yield
of 8.37%, using syringaldazine as substrate. The molecular weight of the purified laccase was 69 and 68 kDa as estimated by
12% (w/v) SDS-PAGE gel and by gel filtration, respectively. The K
m values for the substrates ABTS, syringaldazine, and guaiacol were 58, 8.3, and 370 μM, respectively. The enzyme’s pH optimum
for syringaldazine was 4.2 and optimal activity was 50°C. The enzyme showed to be thermostable because when kept at 50°C for
24 and 48 h it retained 93 and 76% activity. This laccase was inhibited by l-cysteine, β-mercaptoethanol, NaN3, NaF, and HgCl2. 相似文献
13.
Verónica Beatriz Rajal Alicia Graciela Cid Guillermo Ellenrieder Carlos Mario Cuevas 《World journal of microbiology & biotechnology》2009,25(6):1025-1033
Penicillium ulaiense is a post-harvest pathogenic fungus that attacks citrus fruits. The objective of this work was to study this microorganism
as an α-l-rhamnosidase producer and to characterize it from P. ulaiense. The enzyme under study is used for different applications in food and beverage industries. α-l-Rhamnosidase was produced in a stirred-batch reactor using rhamnose as the main carbon source. The kinetic parameters for
the growth of the fungi and for the enzyme production were calculated from the experimental values. A method for partial purification,
including (NH4)2SO4 precipitation, incubation at pH 12 and DEAE-sepharose chromatography yielded an enzyme with very low β-glucosidase activity.
The pH and temperature optima were 5.0 and 60°C, respectively. The Michaelis–Menten constants for the hydrolysis of p-nitrophenyl-α-l-rhamnoside were V
max = 26 ± 4 IU ml−1 and K
m
= 11 ± 2 mM. The enzyme showed good thermostability up to 60°C and good operational stability in white wine. Co2+ affected positively the activity; EDTA, Mn2+, Mg2+, dithiotreitol and Cu2+ reduced the activity by different amounts, and Hg2+ completely inhibited the enzyme. The enzyme showed more activity on p-nitrophenyl-α-l-rhamnoside than on naringin. According to these results, this enzyme has potential for use in the food and pharmacy industries
since P. ulaiense does not produce mycotoxins. 相似文献
14.
Michelin M Ruller R Ward RJ Moraes LA Jorge JA Terenzi HF Polizeli Mde L 《Journal of industrial microbiology & biotechnology》2008,35(1):17-25
An extracellular glucoamylase produced by Paecilomyces variotii was purified using DEAE-cellulose ion exchange chromatography and Sephadex G-100 gel filtration. The purified protein migrated
as a single band in 7% PAGE and 8% SDS-PAGE. The estimated molecular mass was 86.5 kDa (SDS-PAGE). Optima of temperature and
pH were 55 °C and 5.0, respectively. In the absence of substrate the purified glucoamylase was stable for 1 h at 50 and 55 °C,
with a t
50 of 45 min at 60 °C. The substrate contributed to protect the enzyme against thermal denaturation. The enzyme was mainly activated
by manganese metal ions. The glucoamylase produced by P. variotii preferentially hydrolyzed amylopectin, glycogen and starch, and to a lesser extent malto-oligossacarides and amylose. Sucrose,
p-nitrophenyl α-d-maltoside, methyl-α-d-glucopyranoside, pullulan, α- and β-cyclodextrin, and trehalose were not hydrolyzed. After 24 h, the products of starch hydrolysis,
analyzed by thin layer chromatography, showed only glucose. The circular dichroism spectrum showed a protein rich in α-helix.
The sequence of amino acids of the purified enzyme VVTDSFR appears similar to glucoamylases purified from Talaromyces emersonii and with the precursor of the glucoamylase from Aspergillus oryzae. These results suggested the character of the enzyme studied as a glucoamylase (1,4-α-d-glucan glucohydrolase). 相似文献
15.
Soo-Jin Yeom Nam-Hee Kim Ran-Young Yoon Hyun-Jung Kwon Chang-Su Park Deok-Kun Oh 《Biotechnology letters》2009,31(8):1273-1278
A recombinant mannose-6-phosphate isomerase from Geobacillus thermodenitrificans (GTMpi) isomerizes aldose substrates possessing hydroxyl groups oriented in the same direction at the C2 and C3 positions
such as the d- and l-forms of ribose, lyxose, talose, mannose, and allose. The activity of GTMpi for d-lyxose isomerization was optimal at pH 7.0, 70°C and 1 mM Co2+. Under these conditions, the k
cat and K
m values were 74,300 s−1 and 390 mM for d-lyxose and 28,800 s−1 and 470 mM for l-ribose, respectively. The half-lives of the enzyme at 60, 65, and 70°C were 388, 73, and 27 h, respectively. GTMpi catalyzed
the conversion of d-lyxose to d-xylulose with a 38% conversion yield after 3 h, and converted l-ribose to l-ribulose with a 29% conversion yield. 相似文献
16.
Lahjouji K Storms R Xiao Z Joung KB Zheng Y Powlowski J Tsang A Varin L 《Applied microbiology and biotechnology》2007,75(2):337-346
A cellobiohydrolase-encoding cDNA, Tvcel7a, from Trametes versicolor has been cloned and expressed in Aspergillus niger. The deduced amino acid sequence shows that Tvcel7a encodes a 456-amino acid polypeptide belonging to glycosyl hydrolase family 7. TvCel7a possesses a 19-amino acid secretion
signal but does not possess a linker region nor a carbohydrate-binding domain. Two peaks of activity were obtained after TvCel7a
was purified to apparent homogeneity by gel-filtration followed by anion-exchange chromatography. Mass spectrometry performed
on the purified proteins confirmed that both peaks corresponded to the predicted sequence of the T. versicolor cellulase. The biochemical properties of the purified TvCel7a obtained from both peaks were studied in detail. The pH and
temperature optima were 5.0 and 40°C, respectively. The enzyme was stable over a pH range extending from pH 3.0 to 9.0 and
at temperatures lower than 50°C. The kinetic parameters with the substrate p-nitrophenyl β-d-cellobioside (pNPC) were 0.58 mM and 1.0 μmol/min/mg protein for the purified TvCel7a found in both peaks 1 and 2. TvCel7a
catalyzes the hydrolysis of pNPC, filter paper, β-glucan, and avicel to varying extents, but no detectable hydrolysis was
observed when using the substrates carboxymethylcellulose, laminarin and pNPG. 相似文献
17.
Canakci S Kacagan M Inan K Belduz AO Saha BC 《Applied microbiology and biotechnology》2008,81(1):61-68
The gene, AbfAC26Sari, encoding an α-l-arabinofuranosidase from Anoxybacillus kestanbolensis AC26Sari, was isolated, cloned, sequenced, and characterizated. On the basis of amino acid sequence similarities, this 57-kDa
enzyme could be assigned to family 51 of the glycosyl hydrolase classification system. Characterization of the purified recombinant
α-l-arabinofuranosidase produced in Escherichia coli BL21 revealed that it is active at a broad pH range (pH 4.5 to 9.0) and at a broad temperature range (45–85°C) and it has
an optimum pH of 5.5 and an optimum temperature of 65°C. Kinetic experiment at 65°C with p-nitrophenyl α-l-arabinofuranoside as a substrate gave a V
max and K
m values of 1,019 U/mg and 0.139 mM, respectively. The enzyme had no apparent requirement of metal ions for activity, and its
activity was strongly inhibited by 1 mM Cu2+ and Hg2+. The recombinant arabinofuranosidase released l-arabinose from arabinan, arabinoxylan, oat spelt xylan, arabinobiose, arabinotriose, arabinotetraose, and arabinopentaose.
Endoarabinanase activity was not detected. These findings suggest that AbfAC26Sari is an exo-acting enzyme. 相似文献
18.
Employing the techniques of (NH4)2SO4 fractionation, ion exchange chromatography on DEAE cellulose and gel filtration through Sephadex G-100, pectinmethylesterase
(EC 3.1.1.11) was purified from guava (Psidium guajava L.) fruits var. Hisar Safeda harvested at turning stage of maturity to 129-fold with 28% recovery. Molecular weight as determined
by gel filtration was found to be 51 kDa and the enzyme preparation exhibited the same molecular weight under native (Native-PAGE)
and denaturating conditions (SDS-PAGE) indicating that the enzyme was a monomer. With pectin as the substrate, it exhibited
the Michaelis Menten kinetics with K
m value of 3.1 g l−1. The enzyme was found to be stimulated by Ca++ and Na+ and inhibited competitively by d-galacturonic acid with K
i
value of 1.97 mM. The enzyme was completely inactivated by iodine while with diethyl pyrocarbonate and N-acetylimidazole, the enzyme was inhibited up to the extent of 56 and 45%, respectively. However, DTNB had no inhibitory effect
whatsoever precluding the participation of any –SH group in the active centre. It is tentatively proposed that the enzyme
has tyrosine and histidine residues at its active centre. 相似文献
19.
Liu F Tachibana S Taira T Ishihara M Yasuda M 《Journal of industrial microbiology & biotechnology》2004,31(1):23-28
Carboxypeptidase produced by Monascus purpureus IFO 4478 was purified to homogeneity. The purified enzyme is a heterodimer with a molecular mass of 132 kDa and consists of two subunits of 64 and 67 kDa. It is an acidic glycoprotein with an isoelectric point of 3.67 and 17.0% carbohydrate content. The optimum pH and temperature were 4.0 and 40 °C, respectively. The enzyme was stable between pH 2.0 and 8.0 at 37 °C for 1 h, and up to 50 °C at pH 5.0 for 15 min. The enzyme was strongly inhibited by piperastatin A, diisopropylfluoride phosphate (DFP), phenylmethylsulfonylfluoride (PMSF), and chymostatin, suggesting that it is a chymotrypsin-like serine carboxypeptidase. Monascus purpureus carboxypeptidase was also strongly inhibited by p-chloromercuribenzoic acid (PCMB) but not by ethylenediaminetetraacetic acid (EDTA) and 1,10-phenanthroline, indicating that it requires cysteine residue but not metal ions for activity. Benzyloxycarbonyl-l-tyrosyl-l-glutamic acid (Z-Tyr-Glu), among the substrates tested, was the best substrate of the enzyme. The Km, Vmax, Kcat, and Kcat/Km values of the enzyme for Z-Tyr-Glu at pH 4.0 and 37 °C were 0.86 mM, 0.917 mM min–1, 291 s–1, and 339 mM–1 s–1, respectively. 相似文献
20.
Galactinol: raffinose-6-galactosyltransferase (EC 2.4.1.67), a stachyose synthase, was extracted from mature leaves of Cucumis melo cv. Ranjadew and was purified to homogeneity by (NH4)2SO4 precipitation, ion-exchange chromatography, gel-filtration and non-denaturing polyacrylamide gel electrophoresis. A specific activity of 516 kat · mg-1 and a 160-fold purification was achieved. The pH optimum of the enzyme reaction was found to be 6.8 in sodium-phosphate buffer, and the temperature optimum 32° C. The purified enzyme was very sensitive towards SH-poisons but its reaction was hardly affected by changes in the ion composition of the assay medium. The two-substrate enzyme was specific for galactinol and raffmose; uridine-diphosphate galactose and p-nitrophenyl--d-galactoside as well as melibiose were not accepted by the purified enzyme. Stachyose synthesis was competitively inhibited by concentrations >4 mM raffinose as well as 2.5 mM galactinol. The K
m values determined under non-saturating conditions were 3.3 mM for raffinose and 7.7 mM for galactinol. Myoinositol was a strong competitive inhibitor with a K
i of 1.8mM. Galactinol was hydrolyzed in the absence of raffinose with a K
m of 0.8 mM. The pure enzyme is a protein with a molecular weight of at least 95 kDa and an isoelectric point of 5.1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the presence of two subunits of 45 and 50 kDa. Polyclonal antibodies from rabbit were obtained which were specific for the native enzyme but cross-reacted with other proteins separated under denaturing conditions.Abbreviations DEAE
diethylaminoethyl
- DTT
dithiothreitol
- FPLC
fast protein liquid chromatography
- HPLC
high-performance liquid chromatography
- PAGE
polyacrylamide gel electrophoresis
- SDS
sodium dodecyl sulfate
This work was supported by Deutsche Forschungsgemeinschaft. The gift of galactinol by Dr. T. Schweizer (Nestlé, Switzerland) is gratefully acknowledged. 相似文献