共查询到20条相似文献,搜索用时 15 毫秒
1.
P. Tangngamsakul A. Karnchanatat P. Sihanonth P. Sangvanich 《Applied Biochemistry and Microbiology》2011,47(4):412-418
A strain of endophytic fungus EF6 isolated from Thai medicinal plants was found to produce higher levels of extracellular
glucoamylase. This strain produced glucoamylase of culture filtrate when grown on 1% soluble starch. The enzyme was purified
and characterized. Purification steps involved (NH4)2SO4 precipitation, anion exchange, and gel filtration chromatography. Final purification fold was 14.49 and the yield obtained
was 9.15%. The enzyme is monomeric with a molecular mass of 62.2 kDa as estimated by SDS-PAGE, and with a molecular mass of
62.031 kDa estimated by MALDI-TOF spectrometry. The temperature for maximum activity was 60°C. After 30 min for incubation,
glucoamylase was found to be stable lower than 50°C. The activity decrease rapidly when residual activity was retained about
45% at 55°C. The pH optimum of the enzyme activity was 6.0, and it was stable over a pH range of 4.0–7.0 at 50°C. The activity
of glucoamylase was stimulated by Ca2+, Co2+, Mg2+, Mn2+, glycerol, DMSO, DTT and EDTA, and strongly inhibited by Hg2+. Various types of starch were test, soluble starch proved to be the best substrate for digestion process. The enzyme catalyzes
the hydrolysis of soluble starch and maltose as the substrate, the enzyme had K
m values of 2.63, and 1.88 mg/ml and V
max, values of 1.25, and 2.54 U/min/mg protein, and V
max/K
m values of 0.48 and 1.35, respectively. The internal amino acid sequences of endophytic fungus EF6 glucoamylase; RALAN HKQVV
DSFRS have similarity to the sequence of the glucoamylase purified form Thermomyces lanuginosus. From all results indicated that this enzyme is a glucoamylase (1,4-α-D-glucan glucanohydrolase). 相似文献
2.
Weenink XO Punt PJ van den Hondel CA Ram AF 《Applied microbiology and biotechnology》2006,69(6):711-717
Although filamentous fungi have a unique property of secreting a large amount of homologous extracellular proteins, the use
of filamentous fungi as hosts for the production of heterologous proteins is limited because of the low production levels
that are generally reached. Here, we report a general screening method for the isolation of mutants with increased protein
production levels. The screening method makes use of an Aspergillus niger strain that lacks the two major amylolytic enzymes, glucoamylase (GlaA) and acid amylase (AamA). The double-mutant strain
grows poorly on starch and its growth is restored after reintroducing the catalytic part of the glucoamylase gene (GlaA512). We show that the fusion of a heterologous protein, a laccase from Pleurotus ostreatus (Pox2), to the catalytic part of glucoamylase (GlaA512–Pox2) severely hampers efficient production of the glucoamylase protein, resulting in a slow-growth phenotype on starch.
Laccase-hypersecreting mutants were obtained by isolating mutants that displayed improved growth on starch plates. The mutant
with the highest growth rate on starch displayed the highest laccase activity, indicating that increased glucoamylase protein
levels are correlated with higher laccase production levels. In principle, our method can be applied to any low-produced heterologous
protein that is secreted as a fusion with the glucoamylase protein. 相似文献
3.
Suhas R. Kolhekar Pramod B. Mahajan Sudha S. Ambedkar Prabhakar S. Borkar 《Applied microbiology and biotechnology》1985,22(3):181-186
Summary A higher yielding mutant of Aspergillus candidus Link var. aureus has been isolated using the chemical mutagen N-methyl-N-nitro-N-nitrosoguanidine (MNNG) at 5 g/ml concentration of the mutagen. This mutant produces 88 to 111 U/ml of glucoamylase compared with 40 to 50 U/ml produced by the parent strain. The enzyme from this mutant has been purified to homogeneity using hydrophobic interaction chromatography and a DEAE-cellulose column with over 65% recovery of the enzyme activity and 21-fold purification. The mutant protein is very similar to the enzyme from the parent strain in that it cochromatographs with the parent enzyme on phenylglycyl-Sepharose, norleucyl-Sepharose, Sepharose-CL-6B, Concanavalin A-Sepharose, DEAE-cellulose and DEAE-Sephadex-A-50. The two proteins exhibit similar electrophoretic behavior and have similar molecular weights and amino acid composition. However, the specific activity of the purified mutant protein is 1125 U/mg compared with 560 U/mg for the parent enzyme. Further genetic analysis of the mutant is needed to explain these observations. 相似文献
4.
Marvin N. Schwalb 《Journal of bacteriology》1971,108(3):1205-1209
During the development of fruit bodies of Schizophyllum commune there is a minimum 10- to 15-fold increase in amylase activity. There is little or no activity in homokaryons or dikaryons. The activity is found early after the onset of morphogenesis and increases until the fruit bodies are mature. Inhibition studies with CO(2) indicate that the activity is directly associated with fruiting, as a change from fruiting to vegetative growth of the dikaryotic mycelium leads to a loss in activity, whereas the already formed fruit bodies show no loss. The activity is unaffected by the level of glucose in the medium. Evidence is presented, based on the mode of starch degradation and on yield and inhibition studies, that the enzyme is a glucoamylase. 相似文献
5.
A mutant of Hordeum vulgare L. (LaPr 85/84) deficient in serine: glyoxylate aminotransferase (EC 2.6.1.45) activity has been isolated. The plant also lacks serine: pyruvate aminotransferase and asparagine: glyoxylate aminotransferase activities. Genetic analysis of the mutation strongly indicates that these three activities are all carried on the same enzyme protein. The mutant is incapable of normal rates of photosynthesis in air but can be maintained at 0.7% CO2. The rate of photosynthesis cannot be restored by supplying hydroxypyruvate, glycerate, glutamate or ammonium sulphate through the xylem stream. This photorespiratory mutant demonstrates convincingly that photorespiration still occurs under conditions in which photosynthesis becomes insensitive to oxygen levels. Two major peaks and one minor peak of serine: glyoxylate aminotransferase activity can be separated in extracts of leaves of wild-type barley by diethylaminoethyl-sephacel chromatography. All three peaks are missing from the mutant, LaPr 85/84. The mutant showed the expected rate (50%) of ammonia release during photorespiration but produced CO2 at twice the wild-type rate when it was fed [14C]glyoxylate. The large accumulation of serine detected in the mutant under photorespiratory conditions shows the importance of the enzyme activity in vivo. The effect of the mutation on transient changes in chlorophyll a fluorescence initiated by changing the atmospheric CO2 concentration are presented and the role of the enzyme activity under nonphotorespiratory conditions is discussed.Abbreviations DEAE
diethylaminoethyl
- PFR
photon fluence rate
- SGAT
serine:glyoxylate aminotransferase 相似文献
6.
Glucoamylase, which catalyses the hydrolysis of the α-1,4 glycosidic bonds of starch, is an important industrial enzyme used in starch enzymatic saccharification. In this study, a glucoamylase gene from Aspergillus awamori, under the control of the promoter of seed storage protein Gt1, was introduced into rice by Agrobacterium-mediated transformation. Significant glucoamylase activity was detected specifically in the seeds but not other tissues of the transgenic rice lines. The highest enzymatic activity was found in the transgenic line Bg17-2, which was estimated to have about 500 units per gram of seeds (one unit is defined as the amount of enzyme that produces 1 μmol of reducing sugar in 1 min at 60 °C using soluble starch as substrate). The optimum pH for the activity of the rice produced enzyme is 5.0–5.5, and the optimum temperature is around 60 °C. One part of this transgenic glucoamylase rice seed flour fully converted 25 parts of corn starch pre-liquefied by an α-amylase also produced by a transgenic rice into glucose in 16 h incubation. This study suggests that this hydrolysis enzyme may substitute commercial fermentation enzymes for industrial starch conversion. 相似文献
7.
Sueo Urushizaki Yasuo Ota Tetsuya Haruyama Hiromasa Kano Kuniomi Matsumoto Minoru Haga 《Bioscience, biotechnology, and biochemistry》2013,77(12):3159-3161
To find amino acid residues which are required for glucoamylase activity, mutant glucoamylase genes were constructed by in vitro mutations of GLU1 DNA encoding Saccharomycopsis fibuligera glucoamylase and introduced into Saccharomyces cerevisiae, and the resulting mutant proteins were assayed for enzymatic activities. Eighteen mutant proteins were obtained by random insertions of a BamHX linker DNA. Six out of 7 proteins with mutations in conserved regions among divergent glucoamylases showed no activities, while 8 out of 11 proteins with mutations in unconserved regions had similar specific activities to a wild-type value, suggesting that the conserved regions are important to the activity. A series of amino-terminal deletion mutants were also constructed. A mutant protein with a deletion of only two amino acid residues from the amino terminus had a significant reduction in the activity, suggesting an essential role for the amino-terminal peptide. Ten mutant proteins with single amino acid replacements were produced by site-directed mutagenesis. Analyses for thermal stability and temperature dependency of these mutant proteins revealed that Ala81, Asp89, Trp94, Arg96, Asp97, and Trp166 are required for wild-type levels of activities, and that at least Ala81 and Asp89 are not essential to catalytic activities, but act in thermal stability. 相似文献
8.
The starch-degrading yeastCandida tsukubaensis CBS 6389 secreted amylase at high activity when grown in a medium containing soluble starch. The extracellular α-amylase
activity was very low. The major amylase component was purified by DEAE-Sephadex A-50 chromatography and Ultrogel AcA 44 gel
filtration and characterized as a glucoamylase. The enzyme proved to be a glycoprotein with a molecular weight of 56000. The
glucoamylase had a temperature optimum at 55°C and displayed highest activity in a pH range of 2.4–4.8. Acarbose strongly
inhibited the purified glucoamylase. Debranching activity was present as demonstrated by the release of glucose from pullulan. 相似文献
9.
10.
D. F. Day 《Current microbiology》1978,1(3):181-184
A novel exocellular glucoamylase produced by a thermophilic fungus,Cephalosporium eichhorniae, was purified by a combination of membrane filtration and Sephadex chromatography. The enzyme was a glycoprotein, 28% carbohydrate
by weight. It was composed of a single polypeptide chain with a molecular weight of 26,850. The enzyme was thermostable with
optimum activity between 45 and 62°C. It had a substrate preference of amylose>amylopectin. Analysis by thin-layer and gas-liquid
chromatography showed the major hydrolytic product of starch was glucose, classifying this enzyme as a thermophilic glucoamylase. 相似文献
11.
Chitinase,β-1,3-glucanase, cellulase, xylanase and protease activity were detected in a crude enzyme preparation obtained from a slime
mold (Badhamia utricularis) which was grown on autoclaved mycelia ofPholiota nameko in a petri dish. The optimal pH of the enzyme preparation for lytic activity against fruit bodies ofLentinus edodes was 4.0, and those ofβ-1,3-glucanase and cellulase were the same. On the other hand, chitinase and protease showed optimal activity at pH 5.0 and
8.0, respectively. The lytic activity was stable below 40°C but completely inactivated at 70°C, and was most stable at pH
5.0. The studies of the optimal pH, thermal stability, and pH stability, and isoelectric focusing analysis of the enzyme preparation
suggest that chitinase,β-1,3-glucanase and cellulase activities may be responsible for lysis of fruit bodies of some mushrooms. The crude enzyme preparation
from the slime mold lysed fruit bodies of several mushrooms more efficiently than did commercial lytic enzymes preparations
(Driselase and Usukizyme). 相似文献
12.
An intracellular glucoamylase (E.C. 3.2.1.3) was purified
to homogeneity from Lactobacillus amylovorus on a Fast Protein Liquid
Chromatography System (FPLC) with a Mono Q ion-exchanger and two Superose 12
gel filtration columns arranged in series. The enzyme activity was quantified
with a specific, chromogenic substrate, p-nitrophenyl-β-maltoside.
Preparative gel electrophoresis was then used to further purify active enzyme
fractions. Native polyacrylamide gel electrophoresis (Native-PAGE) and sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified
enzyme showed a single protein band of molecular weight 47 kDa. Glucoamylase
activity of the purified protein was confirmed by its ability to degrade
starch on a 0.025% starch-polyacrylamide gel stained with
I2/KI. Glucoamylase exhibited optimum catalytic activity at pH 6.0
and 45°C, and the enzyme had an isoelectric point near 4.39. The
glucoamylase contained high levels of hydrophilic amino acids, comparable to
fungal glucoamylases.
Received: 12 July 1996 / Accepted: 10 September 1996 相似文献
13.
Summary An intracellular glucoamylase, purified from Lactobacillus amylovorus, reacted selectively with polysaccharides. Kinetic studies indicated low affinity for maltose and maltotriose (Km 58 g/ml and 178 g/ml) and higher affinity for starch and dextrin (Km 0.01 g/ml and 0.02 g/ml). Glucoamylase was inhibited almost 50% by 10 mM glucose. Cu2+ and Pb2+ inhibited glucoamylase at 1.0 mM but EDTA and other metal chelators had no effect on the enzyme activity. Acarbose and Tris inhibited the enzyme by 84% and 98%, respectively at 1 mM, while iodoacetate and p-chloromecuribenzoic acid inhibited activity by 98% and 78%, respectively at 10 mM. The purified enzyme was thermolabile at temperatures greater than 55°C and thus has potential for application in the brewing industry. 相似文献
14.
Lorena Latorre-García Ana Cristina Adam Julio Polaina 《World journal of microbiology & biotechnology》2008,24(12):2957-2963
Production of glucoamylase encoded by the Saccharomyces cerevisiae (var. diastaticus) STA1 gene has been assayed in laboratory S. cerevisiae strains of different ploidy and in different industrial Saccharomyces strains, in which STA1 was expressed under control of an inducible promoter. Highest enzyme activity was achieved with a tetraploid strain constructed
by crossing preselected parental strains. Maximal glucoamylase production correlated with heterogeneity in enzyme mass, likely
due to incomplete glycosylation, suggesting that the secretion-glycosylation process is the limiting step in the production
of the STA-encoded glucoamylase by Saccharomyces. Industrial strains showed quite different capacity to produce glucoamylase. High production was achieved with a S. pastorianus brewer’s strain. Overall, our results allowed the selection of strains capable of yielding a high level of glucoamylase and
suggest specific approaches for further enhancing this capability. 相似文献
15.
Four silica supports differing in pore dimensions were activated by treatment with SiCl4 and then with ethylenediamine to obtain alkylamine groups on the silica surface. Three enzymes, peroxidase from cabbage, glucoamylase from Aspergillus niger C and urease from soybean were immobilized on these supports using glutaraldehyde as coupling agent. It was found that the protein content, the retained enzymatic activity and the storage stability of the silica supported enzymes were considerably affected by support pore size and enzyme molecular weight, the factors which are supposed to alter protein distribution inside the support pores. The highest activity was found for peroxidase and glucoamylase attached to the silica with the widest pores, but their loss in activity during storage was considerable. The urease retained less activity after immobilization, but its storage stability was excellent. 相似文献
16.
【目的】建立对糖化酶生产菌种黑曲霉随机突变文库进行筛选的方法,以获得糖化酶酶活提高的突变菌株。【方法】以一株可产糖化酶的黑曲霉菌株Aspergillus niger X1为出发菌株,经硫酸二乙酯诱变获得突变文库,采用葡萄糖的结构类似物——2-脱氧葡萄糖进行筛选,并在筛选过程中逐渐提高2-脱氧葡萄糖浓度,定向选育具有2-脱氧葡萄糖抗性、高产糖化酶的突变株。【结果】获得的高产突变菌株DG36摇瓶发酵糖化酶产量比出发菌株A.niger X1提高22.2%–33.8%,经工业水平50 m~3罐发酵测试,突变株DG36发酵128 h糖化酶活可达49094 U/m L,在相同发酵时间内,其酶活较出发菌株A.niger X1提高32.8%,发酵时间缩短16.9%。【结论】本研究开发了一种以2-脱氧葡萄糖为抗性标记选育高产糖化酶突变株的方法,所得突变株DG36遗传性状稳定,与出发菌相比具有菌丝粗壮、产酶期提前、糖化酶活高、发酵时间短、有利于发酵后处理的优点。 相似文献
17.
Tony Marcio da Silva Alexandre Maller André Ricardo de Lima Damásio Michele Michelin Richard John Ward Izaura Yoshico Hirata João Atilio Jorge Héctor Francisco Terenzi Maria Lourdes T. M. de Polizeli 《Journal of industrial microbiology & biotechnology》2009,36(12):1439-1446
A glucoamylase from Aspergillus niveus was produced by submerged fermentation in Khanna medium, initial pH 6.5 for 72 h, at 40°C. The enzyme was purified by DEAE-Fractogel
and Concanavalin A-Sepharose chromatography. The enzyme showed 11% carbohydrate content, an isoelectric point of 3.8 and a
molecular mass of 77 and 76 kDa estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or Bio-Sil-Sec-400
gel filtration, respectively. The pH optimum was 5.0–5.5, and the enzyme remained stable for at least 2 h in the pH range
of 4.0–9.5. The temperature optimum was 65°C and retained 100% activity after 240 min at 60°C. The glucoamylase remained completely
active in the presence of 10% methanol and acetone. After 120 min hydrolysis of starch, glucose was the unique product formed,
confirming that the enzyme was a glucoamylase (1,4-alpha-d-glucan glucohydrolase). The K
m was calculated as 0.32 mg ml−1. Circular dichroism spectroscopy estimated a secondary structure content of 33% α-helix, 17% β-sheet and 50% random structure,
which is similar to that observed in the crystal structures of glucoamylases from other Aspergillus species. The tryptic peptide sequence analysis showed similarity with glucoamylases from A. niger, A. kawachi,
A. ficcum, A. terreus, A. awamori and A. shirousami. We conclude that the reported properties, such as solvent, pH and temperature stabilities, make A. niveus glucoamylase a potentially attractive enzyme for biotechnological applications. 相似文献
18.
The present investigation was aimed at producing a thermostable and neutral glucoamylase (amyloglucosidase, EC 3.2.1.3) by
a thermophilic mould, Thermomucor indicae-seudaticae in submerged cultivation and testing its applicability in starch saccharification. Parametric optimization resulted in the
secretion of 30,000 U/l of glucoamylase in a synthetic medium (5% soluble starch, 0.1% yeast extract, 0.05% K2HPO4 and 0.01% MgSO4· 7H2O) using 5 × 106 spores/50 ml of a 3-day-old inoculum at 40 °C and 250 rev/min in shake flasks in 48 h. The enzyme secretion was not affected
to any significant extent by the tested additives and detergents. A 1.7-fold increase in glucoamylase secretion was attained
when T. indicae-seudaticae was grown in a laboratory fermenter. The enzyme alone catalysed the hydrolysis of soluble starch to an extent of 65%. A prior
treatment of starch with thermostable α-amylase and amylopullulanase, followed by glucoamylase, resulted in a greater extent of hydrolysis, 79 and 91%, respectively. 相似文献
19.
Rubinder K Chadha BS Singh N Saini HS Singh S 《Journal of industrial microbiology & biotechnology》2002,29(2):70-74
Thermomyces lanuginosus was subjected to three cycles of mutagenesis (UV/NTG) and a selection procedure to develop amylase-hyperproducing, catabolite-repression-resistant
and partially constitutive strains. One of the selected derepressed mutant strain III51, produced ∼7- and 3-fold higher specific activity of α-amylase (190 U/mg protein) and glucoamylase (105 U/mg protein), respectively,
compared to a wild-type parental strain. Further, the effect of production parameters on mutant strain III51 was studied using a Box–Behnken design. The regression models computed showed significantly high R
2 values of 96 and 97% for α-amylase and glucoamylase activities, respectively, indicating that they are appropriate for predicting
relationships between corn flour, soybean meal and pH with α-amylase and glucoamylase production. Journal of Industrial Microbiology & Biotechnology (2002) 29, 70–74 doi:10.1038/sj.jim.7000270
Received 05 July 2001/ Accepted in revised form 16 April 2002 相似文献
20.
Bratislaw Stanković Shunnosuke Abe Kishu Azama Koichi Shibata Yoko Ito Stanislaw Weidner Eric Davies 《Acta Physiologiae Plantarum》1999,21(4):383-389
Maize endosperm was homogenized in a cytoskeleton-stabilizing buffer, filtered and layered on gradients of 20–80% sucrose
and analyzed by monitoring their UV absorbance. A major peak of UV-light absorbing material was detected on the gradient,
at about 60–65% sucrose (density of approximately 1.3 g·ml−1). Biochemical, fluorescence microscopic, and immunoblot analyses of this peak showed that it consisted of protein bodies
associated with actin, membranes, and RNA (ribosomes). Seeds of wild type and opaque-2 mutant were then homogenized, the homogenate was modified using detergents and/or cytoskeleton-disrupting agents, and centrifuged
on sucrose gradients. In wild type maize endosperm, detergent treatment caused the major peak (protein bodies) to increase
in density so that they sediment further down the gradient. However, in opaque-2 the protein bodies formed a broader, but smaller peak which, upon treatment with detergent, generated protein bodies which
pelleted to the bottom of the gradient. Analysis of gradient fractions by gel electrophoresis and immuno-blotting showed that
both the wild type and the mutant had cytoskeleton proteins in the upper regions (soluble, non-polymerized microfilaments
and microtubules) as well as in the peak regions. Comparisons of both the UV-absorbance profiles and the immunoblot data suggest
that the protein bodies from the two maize types associate differently with the membranes and the cytoskeleton. 相似文献