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1.
Pinus taeda 《Plant Physiology and Biochemistry》2001,39(12):1037
In germinated loblolly pine (Pinus taeda L.) seeds arginine accumulates in the seedling during its growth immediately following germination. The enzyme arginase (L-arginine amidinohydrolase, EC 3.5.3.1) is responsible for hydrolyzing this arginine into ornithine and urea. Loblolly pine arginase was purified to homogeneity from seedling cotyledons by chromatographic separation on DE-52 cellulose, Matrex Green and arginine-linked Sepharose 4B. The enzyme was purified 148-fold and a single polypeptide band was identified as arginase. The molecular mass was determined to be 140 kDa by FPLC, while the subunit size was shown to be 37 kDa by SDS-PAGE, predicting a homotetramer holoprotein. Removal of manganese from the enzyme abolishes catalytic activity, which can be restored by incubating the protein with Mn2+. Antibodies, raised against the arginase subunit, are able to immunotitrate arginase activity and are monospecific for arginase on immunoblots. 相似文献
2.
Ryohei Sato Toru Matsumoto Norio Hidaka Yasuko Imai Katsumasa Abe Shouji Takahashi Ryo-hei Yamada Yoshio Kera 《Protein expression and purification》2009,64(2):205-212
The gene encoding acetylcholinesterase (AChE) was cloned from common carp muscle tissue. The full-length cDNA was 2368 bp that contains a coding region of 1902 bp, corresponding to a protein of 634 amino acids. The deduced amino acid sequence showed a significant homology with those of ichthyic AChEs and several common features among them, including T peptide encoded by exon T in the C-terminus. Three yeast expression vectors were constructed and introduced into the yeast Pichia pastoris. The transformant harboring carp AChE gene lacking exon T most effectively produced AChE activity extracellularly. The replacement of the native signal sequence with the yeast α-factor prepro signal sequence rather decreased the production. A decrease in cultivation temperature from 30 to 15 °C increased the activity production 32.8-fold. The purified recombinant AChE lacking T peptide, eluted as a single peak with a molecular mass of about 230 kDa on the gel filtration chromatography, exhibited the specific activity of 4970 U/mg. On the SDS–PAGE, three proteins with molecular masses of 73, 54, and 22 kDa were observed. These proteins were N-glycosylated, and their N-terminal sequence showed that the latter two were produced from the former probably by proteolytic cleavage at the C-terminal region. Thus, the recombinant AChE is homotrimer of three identical subunits with 73 kDa. The optimal temperature and pH of the recombinant were comparable to those of the native enzyme purified previously, but the values of kinetic parameters and the sensitivities to substrate inhibition and inhibitors were considerably different between them. 相似文献
3.
N. Sridevi Sameer Srivastava Bashir Mohammad Khan Asmita Ashutosh Prabhune 《Extremophiles : life under extreme conditions》2009,13(2):363-370
A thermophilic microorganism producing bile salt hydrolase was isolated from hot water springs, Pali, Maharashtra, India.
This microorganism was identified as Brevibacillus sp. by 16S rDNA sequencing. Bile salt hydrolase (BSH) was purified to homogeneity from this thermophilic source using Q-sepharose
chromatography and its enzymatic properties were characterized. The subunit molecular mass of the purified enzyme was estimated
to be 28 kDa by SDS-PAGE and, 28.2 kDa by MALDI-TOF analysis. The native molecular mass was estimated to be 56 kDa by gel
filtration chromatography, indicating the protein to be a homodimer. The pH and temperature optimum for the enzyme catalysis
were 9.0 and 60°C, respectively. Even though BSH from Brevibacillus sp. hydrolyzed all of the six major human bile salts, the enzyme preferred glycine conjugated substrates with apparent K
M and k
cat values of 3.08 μM and 6.32 × 102 s−1, respectively, for glycodeoxycholic acid. The NH2-terminal sequence of the purified enzyme was determined and it did not show any homology with other bacterial bile salt hydrolases.
To our knowledge, this is the first report describing the purification of BSH to homogeneity from a thermophilic source.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
4.
Characterization of salt-tolerant glutaminase from Stenotrophomonas maltophilia NYW-81 and its application in Japanese soy sauce fermentation 总被引:1,自引:0,他引:1
Wakayama M Yamagata T Kamemura A Bootim N Yano S Tachiki T Yoshimune K Moriguchi M 《Journal of industrial microbiology & biotechnology》2005,32(9):383-390
Glutaminase from Stenotrophomonas maltophilia NYW-81 was purified to homogeneity with a final specific activity of 325 U/mg. The molecular mass of the native enzyme was
estimated to be 41 kDa by gel filtration. A subunit molecular mass of 36 kDa was measured with SDS-PAGE, thus indicating that
the native enzyme is a monomer. The N-terminal amino acid sequence of the enzyme was determined to be KEAETQQKLANVVILATGGTIA.
Besides l-glutamine, which was hydrolyzed with the highest specific activity (100%), l-asparagine (74%), d-glutamine (75%), and d-asparagine (67%) were also hydrolyzed. The pH and temperature optima were 9.0 and approximately 60°C, respectively. The enzyme
was most stable at pH 8.0 and was highly stable (relative activities from 60 to 80%) over a wide pH range (5.0–10.0). About
70 and 50% of enzyme activity was retained even after treatment at 60 and 70°C, respectively, for 10 min. The enzyme showed
high activity (86% of the original activity) in the presence of 16% NaCl. These results indicate that this enzyme has a higher
salt tolerance and thermal stability than bacterial glutaminases that have been reported so far. In a model reaction of Japanese
soy sauce fermentation, glutaminase from S. maltophilia exhibited high ability in the production of glutamic acid compared with glutaminases from Aspergillus oryzae, Escherichia coli, Pseudomonas citronellolis, and Micrococcus luteus, indicating that this enzyme is suitable for application in Japanese soy sauce fermentation. 相似文献
5.
Mustapha Hajjou Abdelkrim Smine Fabienne Guerard Yves Le Gal 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》1995,110(4)
A carboxypeptidase B (CPB) has been purified from dogfish (Scyliorhinus canicula) pancreas and partially characterized. The purification procedure included acetone precipitation, ion-exchange chromatography on a CM-cellulose column and gel filtration on Sephadex G-75. The purified enzyme migrates as a single band both on PAGE and SDS-PAGE. Its molecular mass is estimated to be about 32 kDa. The optimum of activity is obtained at pH 7.5–8.2. The enzyme is inhibited by typical metal-chelating agents (EDTA and o-phenanthroline) and by Hg2+. It is activated by Co2+, l-cysteine and by heat treatment at 40° and 50°C. Kinetic parameters, Km and kcat, of native enzyme, Co2+-activated CPB and heat-treated CPB have been determined 相似文献
6.
The presence and localization of a reversible hydrogenase in non-N2-fixing cells of the filamentous cyanobacterium Anabaena variabilis were investigated by in vitro activity measurements, native-PAGE/activity stain, SDS-PAGE/Western immunoblots, and immunogold localization. Reversible hydrogenase activity was induced approximately 100-fold by sparging the cell suspensions with a mixture of 99% argon and 1% CO2 for 20–26 h. Native-PAGE/activity stain demonstrated the presence of an in vitro functional enzyme with an apparent molecular mass of 118 kDa. Native-PAGE/Western immunoblots, using polyclonal antisera directed against purified hydrogenase from the purple sulphur bacterium Thiocapsa roseopersicina, detected two native proteins with molecular masses of 118 and 133 kDa, respectively. SDS-PAGE/Western immunoblots confirmed the presence of a single polypeptide with a molecular mass of approximately 40 kDa in both induced and non-induced cells. Immunocytolocalization experiments using ultrathin sections again demonstrated the presence of hydrogenase in both induced and non-induced cells. A higher specific labeling was associated with the thylakoid regions, which, using an image analyzer, was calculated to be approximately 4 x higher per cell area compared to in the centroplasm. It is suggested that anaerobic incubation induces higher reversible hydrogenase activity, regulated mainly at the level of activating (pre)existing form(s) of inactive enzyme(s)/protein(s), maybe in combination with synthesis of additional subunit(s). 相似文献
7.
Keinosuke Oda Haruyuki Atomi Mitsuyoshi Ueda Jun Kondo Yutaka Teranishi Atsuo Tanaka 《Archives of microbiology》1991,156(6):439-443
The genomic DNA of peroxisomal isocitrate lyase (ICL) isolated from an n-alkane-assimilating yeast, Candida tropicalis, was truncated to utilize the original open reading frame under the control of the GAL7 promoter and was expressed in Saccharomyces cerevisiae. The recombinant ICL was synthesized as a functionally active enzyme with a specific activity similar to the enzyme purified from C. tropicalis, and was accounted for approximately 30% of the total extractable proteins in the yeast cells. This recombinant enzyme was easily purified to homogeneity. N-Terminal amino acid sequence, molecular masses of native form and subunit, amino acid composition, peptide maps, and kinetic parameters of the recombinant ICL were essentially the same as those of ICL purified from C. tropicalis. From these facts, S. cerevisiae was suggested to be an excellent microorganism to highly express the genes encoding peroxisomal proteins of C. tropicalis.Abbreviations ICL
isocitrate lyase
- SDS-PAGE
sodium dodecylsulfate-polyacrylamide gel electrophoresis 相似文献
8.
Manavalan Arulmani Kalaichelvan Aparanjini Kalyanasundaram Vasanthi Perumal Arumugam Manavalan Arivuchelvi P. Thangavelu Kalaichelvan 《World journal of microbiology & biotechnology》2007,23(4):475-481
An extracellular thermostable alkaline protease isolated from Bacillus
laterosporus-AK1 was purified by sephadex G-200 gel filtration and DEAE cellulose ion-exchange chromatography techniques. The purified
protease showed a maximum relative activity of 100% on casein substrate and appeared as a single band on SDS-PAGE with the
molecular mass of 86.29 kDa. The protease was purified to 11.1-folds with a yield of 34.3%. Gelatin zymogram also revealed
a clear hydrolytic zone due to proteolytic activity, which corresponded to the band obtained with SDS-PAGE. The protease enzyme
had on optimum pH of 9.0 and exhibited highest activity at 75°C. The enzyme activity was highly susceptible to the specific
serine protease inhibitor PMSF, suggesting the presence of serine residues at the active sites. Enzyme activity strongly enhanced
by the metal ions Ca2+ and Mg2+ and this enzyme compatible with aril detergent stability retained 75% even 1-h incubation. The purified protease remove bloodstain
completely when used with Wheel detergent. 相似文献
9.
A single-strand specific nuclease was identified during a particular stage of a defined cellular differentiation pathway characteristic of xylem development. Using a hormone-inducible system in which cultured mesophyll cells of Zinnia elegans differentiated to xylem cells in synchrony, the enzymatic activity on single-stranded (ss) DNA was highest during the maturation phase of differentiation. Nondifferentiating cells contained little of this activity throughout a similar course of culture. After electrophoresis of extracts from differentiating cells, a 43-kilodalton (kDa) polypeptide was detected by its activity in the gels containing either ssDNA or RNA. Lectins specific for mannose residues on glycoproteins bound to the 43-kDa nuclease and were used to distinguish it from several ribonucleases. The nuclease was purified by a two-step chromatographic procedure: a lectin-affinity column followed by a phosphocellulose column. The purified protein was determined to be a single polypeptide with a relative molecular mass of 43000 by the analysis of its mobility during sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel filtration of the native enzyme. A sensitive detection system using biotinylated-concanavalin A and avidin was demonstrated to be specific as a probe for the nuclease protein. An N-terminal amino-acid sequence was derived from 5 pmol of the enzyme. The nuclease was most active on ssDNA at pH 5.5 in the presence of Zn2+ and dithiothreitol. The purified preparation hydrolyzed RNA and to a lesser extent, native DNA. It digested closed circular duplex DNA by introducing a single endonucleolytic cleavage followed by random hydrolysis. During the induced pathway of synchronous differentiation in Zinnia the 43-kDa nuclease rapidly increased just prior to the onset of visibly differentiated features, and developed to a maximum level during xylem cell maturation. At this time a similar but slightly smaller nuclease appeared and became dominant as differentiation continued, and subsequently both enzymes decayed. After autolysis, a nuclease of about 37 kDa was found together with the 43-kDa enzyme in the culture medium. Complementing these analyses was the examination of the tissue distribution of the 43-kDa enzyme in Zinnia and other dicotyledonous plants, which also indicated an invivo role of the nuclease in autolysis, the terminal stage of vascular differentiation in plants. The Zinnia nuclease is therefore a potential marker for xylogenesis.Abbreviations Con A
Canavalia ensiformis (concanavalin) agglutinin
- DNase
deoxyribonuclease
- DTT
dithiothreitol
- EDTA
ethylenediaminetetraacetic acid
- kDa
kilodalton
- Mr
relative molecular mass
- RNase
ribonuclease
- ss
single-stranded
- SDS-PAGE
sodium dodecyl sulfate-polyacrylamide gel electrophoresis 相似文献
10.
Li Cui Ming Sheng Dong Xiao Hong Chen Mei Jiang Xin Lv Guijun Yan 《World journal of microbiology & biotechnology》2008,24(4):483-489
A novel fibrinolytic enzyme from Cordyceps militaris was purified and partially characterized for the first time, which was designated C. militaris fibrinolytic enzyme (CMase). This extracellular enzyme from C. militaris was isolated by ammonium sulphate fraction, and purified to electrophoretic homogeneity using gel filtration chromatography.
The apparent molecular mass of the purified enzyme was estimated to be 27.3 kDa by SDS-PAGE. The optimum pH and temperature
for the enzyme activity were pH 6.0 and 25 °C, respectively. In the presence of metal ions such as Mg2+ and Fe2+ ions the activity of the enzyme increased, whereas EDTA and Cu2+ ion inhibited the enzyme activity. Interestingly the N-terminal amino acid sequences of the enzyme is extremely similar to
those of the trypsin proteinases from insects, and has no significant homology with those of the fibrinolytic enzyme from
other medicinal mushroom. In conclusion, C. militaris produces a strong fibrinolytic enzyme CMase and may be considered as a new source for thrombolytic agents. 相似文献
11.
Henrik U. Stotz James J. A. Contos Ann L. T. Powell Alan B. Bennett John M. Labavitch 《Plant molecular biology》1994,25(4):607-617
A polygalacturonase inhibitor protein (PGIP) was characterized from tomato fruit. Differential glycosylation of a single polypeptide accounted for heterogeneity in concanavalin A binding and in molecular mass. Tomato PGIP had a native molecular mass of 35 to 41 kDa, a native isoelectric point of 9.0, and a chemically deglycosylated molecular mass of 34 kDa, suggesting shared structural similarities with pear fruit PGIP. When purified PGIPs from pear and tomato were compared, tomato PGIP was approximately twenty-fold less effective an inhibitor of polygalacturonase activity isolated from cultures of Botrytis cinerea. Based on partial amino acid sequence, polymerase chain reaction products and genomic clones were isolated and used to demonstrate the presence of PGIP mRNA in both immature and ripening fruit as well as cell suspension cultures. Nucleotide sequence analysis indicates that the gene, uninterrupted by introns, encodes a predicted 36.5 kDa polypeptide containing amino acid sequences determined from the purified protein and sharing 68% and 50% amino acid sequence identity with pear and bean PGIPs, respectively. Analysis of the PGIP sequences also revealed that they belong to a class of proteins which contain leucine-rich tandem repeats. Because these sequence domains have been associated with protein-protein interactions, it is possible that they contribute to the interaction between PGIP and fungal polygalacturonases. 相似文献
12.
A multifunctional Ca2+/calmodulin dependent protein kinase was purified approximately 650 fold from cytosolic extract of Candida albicans. The purified preparation gave a single band of 69 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis with its native molecular mass of 71 kDa suggesting that the enzyme is monomeric. Its activity was dependent on calcium, calmodulin and ATP when measured at saturating histone IIs concentration. The purified Ca2+/CaMPK was found to be autophosphorylated at serine residue(s) in the presence of Ca2+/calmodulin and enzyme stimulation was strongly inhibited by W-7 (CaM antagonist) and KN-62 (Ca2+/CaM dependent PK inhibitor). These results confirm that the purified enzyme is Ca2+/CaM dependent protein kinase of Candida albicans. The enzyme phosphorylated a number of exogenous and endogenous substrates in a Ca2+/calmodulin dependent manner suggesting that the enzyme is a multifunctional Ca2+/calmodulin-dependent protein kinase of Candida albicans. 相似文献
13.
Digitalis lanata 《Plant Physiology and Biochemistry》2000,38(12):929
A cardenolide-hydrolysing β-D-glucosidase was isolated from young leaves of Digitalis lanata. Since this enzyme differs from the cardenolide glucohydrolase (CGH) described and characterised previously, it was termed cardenolide glucohydrolase II (CGH II). CGH II was detected in various Digitalis tissue cultures as well as in young leaves of D. lanata. The latter source was used as the starting material for the isolation and purification of CGH II. The specific enzyme activity reached about 15 pkat·mg–1 protein in buffered leaf extracts. Optimal CGH II activity was seen at around pH 6.0 and 50 °C. CGH II was purified about 600-fold by anion exchange chromatography, size exclusion chromatography and hydroxyapatite chromatography. The apparent molecular mass of CGH II was 65 kDa as determined by SDS-PAGE. CGH II exhibited a high substrate specificity towards cardenolide disaccharides, especially to those with a 1-4-β-linked glucose-digitoxose moiety such as glucoevatromonoside. The Km- and Vmax-values for this particular substrate were calculated to be 101 μM and 19.8 nkat·mg–1 protein, respectively. 相似文献
14.
Sung-Il Kang Young-Boo Jang Yeung-Joon Choi Jai-Yul Kong 《Biotechnology and Bioprocess Engineering》2005,10(6):593-598
A collagenolytic enzyme, produced byVibrio vulnificus CYK279H, was purified by ultrafiltration, dialysis, Q-Sepharose ion exchange and Superdex-200 gel chromatography. The enzyme
from the supernatant was purified 13.2 fold, with a yield of 11.4%. The molecular weight of the purified enzyme was estimated
by SDS-PAGE to be approximately 35.0 kDa. The N-terminal sequence of the enzyme was determined as Gly-Asp-Pro-Cys-Met-Pro-Ile-Ile-Asn.
The optimum temperature and pH for the enzyme activity were 35°C and 7.5, respectively. The enzyme activity was stable within
the pH and temperature ranges 6.8∼8.0 and 20∼35°C, respectively. The purified enzyme was strongly activated by Zn2+, Li2+, and Ca2+, but inhibited by Cu2+. In addition, the enzyme was strongly inhibited by 1, 10-phenanthroline and EDTA. The purified enzyme was suggested to be
a neutral metalloprotease. 相似文献
15.
L-asparaginase EC 3.5.1.1 was purified to homogeneity from Thermus thermophilus. The apparent molecular mass of L-asparaginase by SDS-PAGE was found to be 33 kDa, whereas by its mobility on Sephacryl S-300 superfine column was around 200 kDa, indicating that the enzyme at the native stage acts as hexamer. The purified enzyme showed a single band on acrylamide gel electrophoresis with pI = 6.0. The optimum pH was 9.2 and the Km for L-asparagine was 2.8 mM. It is a thermostable enzyme and it follows linear kinetics even at 77°C. Chemical modification experiments implied the existence of histidyl, arginyl and a carboxylic residues located at or near active site while serine and mainly cysteine seems to be necessary for active form. 相似文献
16.
The major albumin, a polypeptide of 21 kilodaltons (kDa), from the seeds of cocoa (Theobroma cacao L.), has been identified and partially purified by preparative gel electrophoresis. Some N-terminal sequence was obtained, permitting the construction of an oligonucleotide probe. This probe was used to isolate the corresponding copy DNA (cDNA) clone from a library made from poly(A)+ RNA from immature cocoa beans. The cDNA sequence has a single major open reading frame, that translates to give a 221-amino-acid polypeptide of Mr 24003. The existence of a precursor to the 21-kDa polypeptide of this size was confirmed by immunoprecipitation from total poly(A)+ RNA translation products. The polypeptide has a hydrophobic signal sequence of 26 amino acids before the mature start, and the mature polypeptide would have an Mr of 21223. The protein sequence is homologous with sequences of the Kunitz protease and -amylase inhibitor family, and the protein probably functions to defend the seed's protein reserves from the digestive enzymes of invading pests. However because the protein comprises 25–30% of the total seed protein it may itself also function as a storage protein. Electron micrographs of immunogold-labelled embryo sections show that the protein is located in membrane-enclosed organelles.Abbreviations cDNA
copy DNA
- IgG
immunoglobulin G
- kb
kilobase pairs
- kDa
kilodaltons
- Mr
relative molecular mass
- SDS-PAGE
sodium dodecyl sulphate-polyacylamide gel electrophoresis
The authors are very grateful to Dr R. Jennings of the Virology Department, Sheffield University Medical School, for help in raising antibodies, and to Dr G. Cope, of the Biological Sciences Electron Microscopy Unit, Sheffield University, for taking the electron micrographs.To whom correspondence should be addressed. 相似文献
17.
Mello GC Oliva ML Sumikawa JT Machado OL Marangoni S Novello JC Macedo ML 《Journal of Protein Chemistry》2001,20(8):625-632
A second trypsin inhibitor (DMTI-II) was purified from the seed of Dimorphandra mollis (Leguminosae-Mimosoideae) by ammonium sulfate precipitation (30–60%), gel filtration, and ion-exchange and affinity chromatography. A molecular weight of 23 kDa was estimated by gel filtration on a Superdex 75 column SDS-PAGE under reduced conditions showed that DMTI-II consisted of a single polypeptide chain, although isoelectric focusing revealed the presence of three isoforms. The dissociation constant of 1.7 × 10–9 M with bovine trypsin indicated a high affinity between the inhibitor and this enzyme. The inhibitory activity was stable over a wide pH range and in the presence of DTT. The N-terminal sequence of DMTI-II showed a high degree of homology with other Kunitz-type inhibitors. 相似文献
18.
A potent fibrinolytic enzyme-producing bacterium was isolated from the traditional Korean condiment Chungkook-jang and identified
as Bacillus vallismortis Ace02. The extracellular fibrinolytic enzyme was purified with a 18% recovery of activity from supernatant cultures using
CM-Sepharose column chromatography and Sephacryl S-200 gel filtration. The specific activity of the purified enzyme was 757 kFU mg−1. Its molecular mass was about 28 kDa and the initial amino acids of the N-terminal sequence were AQSVPYGVSQ. The full amino acid sequence of fibrinolytic enzyme Ace02 corresponded with bacteriolytic
enzyme, L27, from Bacillus licheniformis, which has strong lytic activity against Streptococcus mutans, a major causative strain of dental caries. This suggests that the purified enzyme should be used for prevention of dental
caries as well as being an effective thrombolytic agent. 相似文献
19.
Mohamed TM 《Experimental & applied acarology》2006,40(2):101-111
Adenosine deaminase is involved in purine metabolism and is a key enzyme for the control of the cellular levels of adenosine. Adenosine deaminase activity showed significant changes during embryogenesis of the camel tick Hyalomma dromedarii. From the elution profile of chromatography on DEAE-sepharose, three forms of enzyme (ADAI, ADAII and ADAIII) were separated. ADAII was purified to homogeneity after chromatography on Sephacryl S-200. The molecular mass of adenosine deaminase ADAII was 42 kDa for the native enzyme and represented a monomer of 42 kDa by SDS-PAGE. The enzyme had a pH optimum at 7.5 and temperature optimum at 40°C with heat stability up to 40°C. ADAII had a K
m of 0.5 mM adenosine with higher affinity toward deoxyadenosine and adenosine than other purines. Ni2+, Ba2+, Zn2+, Li2+, Hg2+ and Mg2+ partially inhibited the ADAII. Mg2+ was the strongest inhibitor by 91% of the enzyme's activity. 相似文献
20.
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