共查询到20条相似文献,搜索用时 15 毫秒
1.
Siddhartha Kumar Mishra Neelam Singh Sangwan Rajender Singh Sangwan 《Journal of plant biochemistry and biotechnology.》2014,23(4):379-388
A highly abundant β-glucosidase from petals of Silybum marianum has been purified and characterized for its physico-kinetic properties. The 135 kDa enzyme was a homodimer with subunit molecular mass of 67.6 kDa. The characteristic catalytic properties of the enzyme included acidic pH optimum (5.5), meso-thermostability, and β-linked substrate specificity with preference for gluco-conjugate but a marked (>50 %) activity with D-fuco-conjugates and considerable (~16 %) activity towards D-galacto-conjugates. The enzyme showed high affinity for p-nitrophenyl glucoside (pNPG) with Km and Vmax values of 0.25 mM and 5.35 μkat.mg?1 enzyme protein. Thus, the enzyme had a very high (292,000 M?1.s?1) catalytic efficiency (Kcat/Km). Thermal catalytic optimum of enzyme was 40 °C with activation energy value 8.26 kCal.Mol?1. The enzyme showed significant insensitivity to D-gluconic acid lactone inhibition (57 % at 5 mM) with an apparent Ki 3.8 mM. The transglucosylating ability of enzyme was noticed for glucosylation of geraniol and withaferin-A with pNPG as glucosyl donor but cellobiose did not serve as the glycosyl donor. Partial proteomics of the enzyme revealed two peptide fragment sequences, VTPSNEVH and KRSEESNF. These motifs showed significant matching/sequence conservation with some other glycohydrolases. The novelties of purified enzyme hold potential to expand a library of catalytically characteristic members of the hydrolase family from plants for use in biotransformation applications. 相似文献
2.
Shuhuai Yu Lanjun Zhu Chen Zhou Tao An Tao Zhang Bo Jiang Wanmeng Mu 《Applied microbiology and biotechnology》2014,98(4):1621-1630
The cDNA gene coding for formate dehydrogenase (FDH) from Ogataea parapolymorpha DL-1 was cloned and expressed in Escherichia coli. The recombinant enzyme was purified by nickel affinity chromatography and was characterized as a homodimer composed of two identical subunits with approximately 40 kDa in each monomer. The enzyme showed wide pH optimum of catalytic activity from pH 6.0 to 7.0. It had relatively high optimum temperature at 65 °C and retained 93, 88, 83, and 71 % of its initial activity after 4 h of exposure at 40, 50, 55, and 60 °C, respectively, suggesting that this enzyme had promising thermal stability. In addition, the enzyme was characterized to have significant tolerance ability to organic solvents such as dimethyl sulfoxide, n-butanol, and n-hexane. The Michaelis–Menten constant (K m), turnover number (k cat), and catalytic efficiency (k cat/K m) values of the enzyme for the substrate sodium formate were estimated to be 0.82 mM, 2.32 s?1, and 2.83 mM?1 s?1, respectively. The K m for NAD+ was 83 μM. Due to its wide pH optimum, promising thermostability, and high organic solvent tolerance, O. parapolymorpha FDH may be a good NADH regeneration catalyst candidate. 相似文献
3.
The phytase (PPHY) of Pichia anomala has the requisite properties of thermostability and acidstability, broad substrate spectrum, and protease insensitivity, which make it a suitable candidate as a feed and food additive. The 1,389-bp PPHY gene was amplified from P. anomala genomic DNA, cloned in pPICZαA, and expressed extracellularly in P. pastoris X33. Three copies of PPHY have been detected integrated into the chromosomal DNA of the recombinant P. pastoris. The size exclusion chromatography followed by electrophoresis of the pure rPPHY confirmed that this is a homohexameric glycoprotein of ~420 kDa with a 24.3 % portion as N-linked glycans. The temperature and pH optima of rPPHY are 60 °C and 4.0, similar to the endogenous enzyme. The kinetic characteristics K m, V max, K cat, and K cat/K m of rPPHY are 0.2 ± 0.03 mM, 78.2 ± 1.43 nmol mg?1 s?1, 65,655 ± 10.92 s?1, and 328.3 ± 3.12 μM?1 s?1, respectively. The optimization of medium components led to a 21.8-fold improvement in rPPHY production over the endogenous yeast. The rPPHY titer attained in shake flasks could also be sustained in the laboratory fermenter. The rPPHY accounts for 57.1 % of the total secreted protein into the medium. The enzyme has been found useful in fractionating allergenic protein glycinin from soya protein besides dephytinization. 相似文献
4.
Rui-Rui Su Ao Wang Song-Tao Hou Peng Gao Guo-Ping Zhu Wen Wang 《Molecular biology reports》2014,41(1):497-504
Fumarase is a key enzyme that catalyzes the reversible hydration of fumarate to l-malate in the tricarboxylic acid cycle. This reaction has been extensively utilized for industrial applications in producing l-malate. In this study, a fumarase C gene from Streptomyces lividans TK54 (slFumC) was cloned and expressed as a fused protein (SlFumC) in Escherichia coli. The molecular mass of SlFumC was about 49 kDa determined by SDS-PAGE. Kinetic studies showed that the K m value of SlFumC for l-malate increased by approximately 8.5-fold at pH 6.5 (6.7 ± 0.81 mM) to 8.0 (57.0 ± 1.12 mM), which was higher than some known fumarases. The catalytic efficiency (k cat) and the specific activity increased by about 9.5-fold at pH 6.5 (65 s?1) to 8.0 (620 s?1) and from 79 U/mg at pH 6.5 to 752 U/mg at pH 8.0, respectively. Therefore, SlFumC may acquire strong catalytic ability by increasing pH to partially compensate for the loss of substrate affinity. The enzyme also showed substrate inhibition phenomenon, which is pH-dependent. Specific activity of SlFumC was gradually enhanced with increasing phosphate concentrations. However, no inhibition was observed at high concentration of phosphate ion, which was distinctly different in case of other Class II fumarases. In industrial process, the reaction temperatures for l-malate production are usually set between 40 and 60 °C. The recombinant SlFumC displayed maximal activity at 45 °C and remained over 85 % of original activity after 48 h incubation at 40 °C, which was more thermostable than other fumarases from Streptomyces and make it an efficient enzyme for use in the industrial production of l-malate. 相似文献
5.
María José Torres Sebastián Alejandro Trejo Claudia Luisa Natalucci Laura María Isabel López 《Planta》2013,237(6):1651-1659
The latex from Vasconcellea quercifolia (“oak leaved papaya”), a member of the Caricaceae family, contains at least seven cysteine endopeptidases with high proteolytic activity, which helps to protect these plants against injury. In this study, we isolated and characterized the most basic of these cysteine endopeptidases, named VQ-VII. This new purified enzyme was homogeneous by bidimensional electrophoresis and MALDI-TOF mass spectrometry, and exhibited a molecular mass of 23,984 Da and an isoelectric point >11. The enzymatic activity of VQ-VII was completely inhibited by E-64 and iodoacetic acid, confirming that it belongs to the catalytic group of cysteine endopeptidases. By investigating the cleavage of the oxidized insulin B-chain to establish the hydrolytic specificity of VQ-VII, we found 13 cleavage sites on the substrate, revealing that it is a broad-specificity peptidase. The pH profiles toward p-Glu-Phe-Leu-p-nitroanilide (PFLNA) and casein showed that the optimum pH is about 6.8 for both substrates, and that in casein, it is active over a wide pH range (activity higher than 80 % between pH 6 and 9.5). Kinetic enzymatic assays were performed with the thiol peptidase substrate PFLNA (K m = 0.454 ± 0.046 mM, k cat = 1.57 ± 0.07 s?1, k cat/K m = 3.46 × 103 ± 14 s?1 M?1). The N-terminal sequence (21 amino acids) of VQ-VII showed an identity >70 % with 11 plant cysteine peptidases and the presence of highly conserved residues and motifs shared with the “papain-like” family of peptidases. VQ-VII proved to be a new latex enzyme of broad specificity, which can degrade extensively proteins of different nature in a wide pH range. 相似文献
6.
B. S. Sandeep T. C. Kishor Mohan K. Murugan 《Journal of plant biochemistry and biotechnology.》2014,23(2):221-224
Polygalacturonase-3 was isolated and purified to homogeneity from palmyrah palm (Borassus flabellifer L.) fruit using Con A-Sepharose affinity column. The purified enzyme migrated as a single band on native and SDS–polyacrylamide gel electrophoresis. The molecular mass of the purified enzyme was estimated to be 66 kDa by size elution chromatography. Optimum polygalacturonase activity as a function of pH and temperature was determined using polygalacturonic acid as substrate. Optimum pH and temperature values ranged between the pH?4.0–5.0 and temperature 30–40 °C. At the optimum pH and temperature, the Km and Vmax values were determined by Lineweaver–Burk method. The value Km (0.33 mM) reveals that polygalacturonase has significant reactivity towards polygalacturonic acid. The enzyme showed varied responses towards divalent and monovalent metal ions. Ca2+ activated the polygalacturonase-3 enzyme protein. Both teepol and cetyltrimethylammonium bromide inhibited polygalacturonase-3 activity by 44 %, while 2-mercaptoethanol stimulated the enzyme marginally. 相似文献
7.
Siddhartha K. Mishra Neelam S. Sangwan Rajender S. Sangwan 《Acta Physiologiae Plantarum》2013,35(5):1439-1451
β-glucosidase from Withania somnifera (Solanaceae) leaf has been purified to homogeneity and characterized for its physico-kinetic properties. The enzyme purification was achieved through a sequence of gel filtration and ion-exchange column chromatography, and PAGE revealed the homogeneity purification status of the enzyme. The properties of the enzyme included an acidic pH optima (4.8), alkaline pI (8.7), meso-thermostabity, monomeric structure with subunit molecular weight of about 50 kDa, high affinity for substrate (K m) for pNPG (0.19 mM) and high (105,263 M?1 s?1) catalytic efficiency (K cat/K m). The mesostable enzyme had a stringent substrate specificity restricted only to β-linked gluco-conjugate. The enzyme is optimally active at 40 °C with 12.4 kcal Mol?1 activation energy, and was highly sensitive to d-gluconic acid lactone inhibition (94 % at 1 mM) with an apparent K i 0.21 mM. The enzyme could catalyze transglucosylation of geraniol with pNPG as glucosyl donor, but not with cellobiose. Some of the physico-kinetic properties were noted to be novel when comprehensively compared with its counterparts from plant, animal and microbial counterparts. Nevertheless, the catalytic and other features of the enzyme were relatively closer to Oryza sativa among plants and Talaromyces thermophillus among fungi. Significance of building-up of a library of novel plant β-glucosidases for structural investigation to understand naturally evolved mechanistics of catalysis has been indicated. 相似文献
8.
Carboxylesterases (CEs) are enzymes responsible for the detoxification of insecticides in insects. In the Cydia pomonella, CEs are involved in synthetic pyrethroid, neonicotinoid, carbamate, and organophosphate detoxification. However, functional overexpression of CEs proteins in Escherichia coli systems often results in insoluble proteins. In this study, we expressed the fusion protein CpCE-1 in E. coli BL21 (DE3). This recombinant protein was overexpressed as inclusion bodies at 37 °C whereas it produced a higher percentage of soluble protein at lower growth temperatures. Production of soluble proteins and enzyme activity increased in the presence of sorbitol in the growth medium. The fusion protein was purified from the lysate supernatant using a Ni2+-NTA agarose gel column. The enzyme exhibited a higher affinity and substrate specificity for α-naphthyl acetate (α-NA), with k cat/K m of 100 s?1 μM?1 for α-NA, and the value is 29.78 s?1 μM?1 for β-naphthyl acetate. The V max and K m were also determined to be 12.9 μmol/min/mg protein and 13.4 μM using substrate α-NA. The optimum pH was 7.0 and temperature was 25 °C. An enzyme inhibition assay shows that PMSF and DEPC strongly inhibit the enzyme activity, while the metal ions Cu2+ and Mg2+ significantly activated the activity. More importantly, cypermethrin, methomyl, and acephate were found to suppress enzyme activity. The data demonstrated here provide information for heterologous expression of soluble protein and further study on insecticide metabolism in C. pomonella in vitro. This is the first report of the characterization of CEs protein from C. pomonella. 相似文献
9.
The catalytic amino acid residues of the β-d-xylosidase (EC 3.2.1.37; GH43), from Thermobifida fusca TM51 (TfBXyl43), were investigated by direct chemical modifications. The pH dependence curves of the kinetic parameters (kcat and kcat/KM) gave pK values for the free enzyme (5.55 ± 0.19; 6.44 ± 0.19), and pK values of for the enzyme–substrate complex (4.85 ± 0.23; 7.60 ± 0.28) respectively, by using an artificial substrate p-nitrophenyl-β-d-xylopyranoside (pNP-Xyl). The detailed inhibition studies demonstrated well the hydrophobic character of the glycon binding site. Carbodiimide-mediated chemical modifications of the enzyme with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) in the presence of glycine methyl ester supports the conclusion that a carboxylate residue can be fundamental in the catalytic process. We have also synthesized and tested N-bromoacetyl-β-d-xylopyranosylamine (NBAXA) for TfBXyl43 as an affinity label, which also inactivated the enzyme irreversible. The pH dependence studies in both cases of inactivation revealed that the modified group is the catalytic proton donor (NBAXA pKA = 6.68 ± 0,1; EDAC pKA = 7.42 ± 0.22) which displays its essential role in the hydrolytic process. The β-d-xylopyranosylazide as competitive inhibitor protected the enzyme in all cases against the inactivation, suggesting that the chemical modification which has an impact on the activity took place in the active center. Changing of the enzyme conformation was followed by CD spectroscopy, as a result of the NBAXA inactivation. Our study is important because to our knowledge no similar investigations were made in the case of an inverting β-d-xylosidase. 相似文献
10.
Priscila?Innocenti?Justo Juliana?Mo?o?Corrêa Alexandre?Maller Marina?Kimiko?Kadowaki José?Luis?da?Concei??o-Silva Rinaldo?Ferreira?Gandra Rita de?Cássia?Garcia?Sim?o
The Caulobacter crescentus (NA1000) xynB5 gene (CCNA_03149) encodes a predicted β-glucosidase-β-xylosidase enzyme that was amplified by polymerase chain reaction; the product was cloned into the blunt ends of the pJet1.2 plasmid. Analysis of the protein sequence indicated the presence of conserved glycosyl hydrolase 3 (GH3), β-glucosidase-related glycosidase (BglX) and fibronectin type III-like domains. After verifying its identity by DNA sequencing, the xynB5 gene was linked to an amino-terminal His-tag using the pTrcHisA vector. A recombinant protein (95 kDa) was successfully overexpressed from the xynB5 gene in E. coli Top 10 and purified using pre-packed nickel-Sepharose columns. The purified protein (BglX-V-Ara) demonstrated multifunctional activities in the presence of different substrates for β-glucosidase (pNPG: p-nitrophenyl-β-D-glucoside) β-xylosidase (pNPX: p-nitrophenyl-β-D-xyloside) and α-arabinosidase (pNPA: p-nitrophenyl-α-L-arabinosidase). BglX-V-Ara presented an optimal pH of 6 for all substrates and optimal temperature of 50 °C for β-glucosidase and α-l-arabinosidase and 60 °C for β-xylosidase. BglX-V-Ara predominantly presented β-glucosidase activity, with the highest affinity for its substrate and catalytic efficiency (Km 0.24 ± 0.0005 mM, Vmax 0.041 ± 0.002 µmol min?1 mg?1 and Kcat/Km 0.27 mM?1 s?1), followed by β-xylosidase (Km 0.64 ± 0.032 mM, Vmax 0.055 ± 0.002 µmol min?1 mg?1 and Kcat/Km 0.14 mM?1s?1) and finally α-l-arabinosidase (Km 1.45 ± 0.05 mM, Vmax 0.091 ± 0.0004 µmol min?1 mg?1 and Kcat/Km 0.1 mM?1 s?1). To date, this is the first report to demonstrate the characterization of a GH3-BglX family member in C. crescentus that may have applications in biotechnological processes (i.e., the simultaneous saccharification process) because the multifunctional enzyme could play an important role in bacterial hemicellulose degradation. 相似文献
11.
A new fungal strain that was isolated from our library was identified as an Aspergillus oryzae and noted to produce a novel proly endopeptidase. The enzyme was isolated, purified, and characterized. The molecular mass of the prolyl endopeptidase was estimated to be 60 kDa by using SDS-PAGE. Further biochemical characterization assays revealed that the enzyme attained optimal activity at pH 4.0 with acid pH stability from 3.0 to 5.0. Its optimum temperature was 30 °C and residual activity after 30 min incubation at 55 °C was higher than 80 %. The enzyme was activated and stabilized by Ca2+ but inhibited by EDTA (10 mM) and Cu2+. The K m and k cat values of the purified enzyme for different length substrates were also evaluated, and the results imply that the enzyme from A. oryzae possesses higher affinity for the larger substrates. Furthermore, this paper demonstrates for the first time that a prolyl endopeptidase purified from A. oryzae is able to hydrolyze intact casein. 相似文献
12.
Juliana L. França Marcelo R. Pinto Malson N. Lucena Daniela P. Garçon Wagner C. Valenti John C. McNamara Francisco A. Leone 《The Journal of membrane biology》2013,246(7):529-543
The stimulation by Mg2+, Na+, K+, NH4 +, and ATP of (Na+, K+)-ATPase activity in a gill microsomal fraction from the freshwater prawn Macrobrachium rosenbergii was examined. Immunofluorescence labeling revealed that the (Na+, K+)-ATPase α-subunit is distributed predominantly within the intralamellar septum, while Western blotting revealed a single α-subunit isoform of about 108 kDa M r. Under saturating Mg2+, Na+, and K+ concentrations, the enzyme hydrolyzed ATP, obeying cooperative kinetics with V M = 115.0 ± 2.3 U mg?1, K 0.5 = 0.10 ± 0.01 mmol L?1. Stimulation by Na+ (V M = 110.0 ± 3.3 U mg?1, K 0.5 = 1.30 ± 0.03 mmol L?1), Mg2+ (V M = 115.0 ± 4.6 U mg?1, K 0.5 = 0.96 ± 0.03 mmol L?1), NH4 + (V M = 141.0 ± 5.6 U mg?1, K 0.5 = 1.90 ± 0.04 mmol L?1), and K+ (V M = 120.0 ± 2.4 U mg?1, K M = 2.74 ± 0.08 mmol L?1) followed single saturation curves and, except for K+, exhibited site–site interaction kinetics. Ouabain inhibited ATPase activity by around 73 % with K I = 12.4 ± 1.3 mol L?1. Complementary inhibition studies suggest the presence of F0F1–, Na+-, or K+-ATPases, but not V(H+)- or Ca2+-ATPases, in the gill microsomal preparation. K+ and NH4 + synergistically stimulated enzyme activity (≈25 %), suggesting that these ions bind to different sites on the molecule. We propose a mechanism for the stimulation by both NH4 +, and K+ of the gill enzyme. 相似文献
13.
Muhammad Aamer Mehmood Xiang Xiao Fauzia Yusuf Hafeez Yingbao Gai Fengping Wang 《World journal of microbiology & biotechnology》2009,25(11):1955-1961
A chitinase gene from Serratia proteamaculans 18A1 was cloned, sequenced, and expressed in Escherichia coli M15. Recombinant enzyme (ChiA) was purified by Ni-NTA affinity column chromatography. The ChiA gene contains an open reading frame (ORF), encoding an endochitinase with a deduced molecular weight 60 kDa and predicted isoelectric point of 6.35. Comparison of ChiA with other chitinases revealed a modular structure containing an N-terminal PKD-domain, a family 18 catalytic domain and a C-terminal putative chitin-binding domain. Turn over rate (K cat) of the enzyme was determined using colloidal chitin (49.71 ± 1.15 S?1) and crystalline β-chitin (17.20 ± 0.83 S?1) as substrates. The purified enzyme was active over a broad range of pH (pH 4.5–9.0) and temperature (4–70°C) with a peak activity at pH 5.5 and 55°C. However, enzyme activity was found to be stable up to 45°C for longer incubation periods. Purified enzyme was shown to inhibit fungal spore germination and hyphal growth of pathogenic fungi Fusarium oxysporum and Aspergillus niger. 相似文献
14.
The subunit MW of Dioscorea bulbifera polyphenol oxidase (MW 115 000 ± 2000) determined by SDS-PAGE is ca. 31 000 indicating that the enzyme is an oligomeric protein with four subunits. Ki values of various inhibitors and their modes of inhibition have been determined with catechol and pyrogallol as substrates. p-Nitrophenol, p-cresol, quinoline and resorcinol are competitive inhibitors of catechol binding while only orcinol and p-nitrophenol behave in the same way towards pyrogallol as substrate. From the effect of pH on Vmax, groups with pK values ca. 4.7 and 6.8 have been identified to be involved in catalytic activity. The Arrhenius activation energy (Ea) at pH 4.0 is 8.9 kcal/mol between 40–65°. At pH 7.0, the value is 22.1 kcal/mol between 40 and 60°. The enthalpies (ΔH) at pH 4.0 and pH 7.0 are 2.3 kcal/mol and 32.4 kcal/mol respectively. The results are discussed considering the conformational changes of the enzyme during substrate binding. 相似文献
15.
《Inorganica chimica acta》1986,120(2):131-134
The equilibrium, kinetics and mechanism of the reaction of chromium(III) with pentane-2,4-dione (Hpd) have been investigated in aqueous solution at 55°C and ionic strength 0.5 mol dm−3 NaClO4. The equilibrium constant (log β1) is 10.08(±0.01) while the pK of Hpd is 8.69(±0.01). The kinetics are consistent with a mechanism in which [Cr(H20)6]3+ and [Cr(H20)5(OH)]2+ react with the enol tautomer of Hpd with rate constants of 1.05(±0.26) × 10−2 and 2.78(±0.08) × 10−1 dm3 mol−1 s−1 respectively. These rate constants are considerably more rapid than those predicted by the Eigen-Wilkins mechanism. These data are compared with literature values. 相似文献
16.
Jianfang Li Xihuan Wei Cunduo Tang Junqing Wang Mei Zhao Qingfeng Pang Minchen Wu 《Journal of industrial microbiology & biotechnology》2014,41(4):693-700
β-Mannanases (EC 3.2.1.78) can catalyze the cleavage of internal β-1,4-d-mannosidic linkages of mannan backbones, and they have found applications in food, feed, pulp and paper, oil, pharmaceutical and textile industries. Suitable amino acid substitution can promote access to the substrate-binding groove and maintain the substrate therein, which probably improves the substrate affinity and, thus, increases catalytic efficiency of the enzyme. In this study, to improve the substrate affinity of AuMan5A, a glycoside hydrolase (GH) family 5 β-mannanase from Aspergillus usamii, had its directed modification conducted by in silico design, and followed by site-directed mutagenesis. The mutant genes, Auman5A Y111F and Auman5A Y115F, were constructed by megaprimer PCR, respectively. Then, Auman5A and its mutant genes were expressed in Pichia pastoris GS115 successfully. The specific activities of purified recombinant β-mannanases (reAuMan5A, reAuMan5AY111F and reAuMan5AY115F) towards locust bean gum were 152.5, 199.6 and 218.9 U mg?1, respectively. The two mutants were found to be similar to reAuMan5A regarding temperature and pH characteristics. Nevertheless, the K m values of reAuMan5AY111F and reAuMan5AY115F, towards guar gum, decreased to 2.95 ± 0.22 and 2.39 ± 0.33 mg ml?1 from 4.49 ± 0.07 mg ml?1 of reAuMan5A, which would make reAuMan5AY111F and reAuMan5AY115F promising candidates for industrial processes. Structural analysis showed that the two mutants increased their affinity by decreasing the steric conflicts with those more complicated substrates. The results suggested that subtle conformational modification in the substrate-binding groove could substantially alter the substrate affinity of AuMan5A. This study laid a solid foundation for the directed modification of substrate affinities of β-mannanases and other enzymes. 相似文献
17.
Claudia Zubiolo Rafaela Cristiane Andrade Santos Nayara Bezerra Carvalho Cleide Mara Faria Soares Alvaro Silva Lima Luciana Cristina Lins de Aquino Santana 《Bioprocess and biosystems engineering》2014,37(9):1781-1788
Lipase from Aspergillus niger was obtained from the solid-state fermentation of a novel agroindustrial residue, pumpkin seed flour. The partially purified enzyme was encapsulated in a sol–gel matrix, resulting in an immobilization yield of 71.4 %. The optimum pH levels of the free and encapsulated enzymes were 4.0 and 3.0, respectively. The encapsulated enzyme showed greater thermal stability at temperatures of 45 and 60 °C than the free enzyme. The positive influence of the encapsulation process was observed on the thermal stability of the enzyme, since a longer half-life t 1/2 and lower deactivation constant were obtained with the encapsulated lipase when compared with the free lipase. Kinetic parameters were found to follow the Michaelis–Menten equation. The K m values indicated that the encapsulation process reduced enzyme–substrate affinity and the V max was about 31.3 % lower than that obtained with the free lipase. The operational stability was investigated, showing 50 % relative activity up to six cycles of reuse at pH 3.0 at 37 °C. Nevertheless, the production of lipase from agroindustrial residue associated with an efficient immobilization method, which promotes good catalytic properties of the enzyme, makes the process economically viable for future industrial applications. 相似文献
18.
Apiradee Pothipongsa Saowarath Jantaro Tiina A. Salminen Aran Incharoensakdi 《World journal of microbiology & biotechnology》2017,33(4):72
Spermidine synthase (Spds) catalyzes the formation of spermidine by transferring the aminopropyl group from decarboxylated S-adenosylmethionine (dcSAM) to putrescine. The Synechococcus spds gene encoding Spds was expressed in Escherichia coli. The purified recombinant enzyme had a molecular mass of 33 kDa and showed optimal activity at pH 7.5, 37?°C. The enzyme had higher affinity for dcSAM (K m, 20 µM) than for putrescine (K m, 111 µM) and was highly specific towards the diamine putrescine with no activity observed towards longer chain diamines. The three-dimensional structural model for Synechococcus Spds revealed that most of the ligand binding residues in Spds from Synechococcus sp. PCC 7942 are identical to those of human and parasite Spds. Based on the model, the highly conserved acidic residues, Asp89, Asp159 and Asp162, are involved in the binding of substrates putrescine and dcSAM and Pro166 seems to confer substrate specificity towards putrescine. 相似文献
19.
Raheleh Jahanbani Mahmood Ghaffari Kourosh Vahdati Maryam Salami Mohammadreza Khalesi Nader Sheibani Ali Akbar Moosavi-Movahedi 《International journal of peptide research and therapeutics》2018,24(1):77-85
Bioactive peptides are defined as protein-based components having nutritional value and have proved roles important for the human health. In this study inhibition of angiotensin converting enzyme (ACE) by protein-based hydrolysate extracted from walnut (Juglanse regia. L.) seeds was evaluated. The peptide fraction obtained by enzymatic hydrolysis with trypsin showed higher ACE-inhibitory and lower IC50 value (0.39?±?0.05 mg/mL) than obtained by hydrolysis with chymotrypsin and proteinase K. The study of kinetics showed that by increasing the concentration of the trypsin hydrolysate from 0.01–0.5 mg/mL, Km increased, while Vmax decreased. Also the value of Ki was found to be 0.17?±?0.01 mg/mL, which means that binding affinity for the substrate decreased in the presence of inhibitor. The structural studies of ACE demonstrated that, in comparison with a commercial antihypertension drug (enalapril), the trypsin hydrolysate had no effect on secondary structure and less tertiary structure changes of protein was observed. 相似文献
20.
Alteration in properties of thymidylate synthetase from pyrimethamine-resistant smodium chabaudi. International Journal for Parasitology16: 483–490. Thymidylate synthetase from cloned strains of pyrimethamine-sensitive and resistant P. chubaudi were partially purified and characterized. The enzyme from both strains have equal mol. wt of 120,000 as estimated by Sephadex G-200 column chromatography. The enzyme from drug-sensitive parasites has an optimum pH of 6.5–7.5 and is stable at pH 4–11 while that from drug-resistant strain has an pH optimum of 7.0–8.0 and is stable at pH 5–10. The Km for methylenetetrahydrofolate are 206 ± 6 and 495 ± 5 μm for the enzyme from drug-resistant and sensitive parasites, respectively. The Km for dUMP of the enzyme from drug-resistant and sensitive parasites are 42 ± 1 and 49 ± 6 μm, respectively. Inhibition of the enzyme from both strains by FdUMP are competitive with dUMP; however,the Kis for the enzyme from drug-resistant strain (0.043 ± 0.005 μm) is less than that from drug-sensitive strain (0.11 ± 0.007 μm) by a factor of 2.5. The Kii for methotrexate with respect to methylenetetrahydrofolate of the enzyme from drug-resistant parasites (58 ± 3 μm) is 3 times larger than that from drug-sensitive parasites (17 ± 1 μm). 相似文献