Purification and characterization of intracellular and extracellular <i>α</i>‐glucosidases from <i>Geobacillus toebii</i> strain E134

Cihan, Arzu Çöleri; Benli, Mehlika; Çökmüş, Cumhur

doi:10.1002/cbf.1820

Cited by 7 publications

(6 citation statements)

References 45 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The results obtained for corn α-glucosidase were in correlation with the previous reports. Most of α-glucosidases have an optimum temperature between 35-65ºC [7,14,33,36,37]. Similar results have been reported by Iwata et al [6].…”

Section: Effect Of Temperature On Enzyme Activity and Stabilitysupporting

confidence: 70%

“…Various temperature-stability results of α-glucosidases have been obtained depending the source of enzyme, temperature, incubation time and reaction conditions. Cihan et al [37] have found that both intracellular and extracellular α-glucosidases from G. toebii strain were very stable between temperature ranges of 35-70ºC. Marin et al [38] have also found that α-glucosidase from X. dendrorhous was very stable to thermal treatment up to 50ºC.…”

Section: Effect Of Temperature On Enzyme Activity and Stabilitymentioning

confidence: 99%

“…The K M value of the enzyme is within the range of the majority of other α-glucosidases. The results obtained by several other researchers showed that the kinetic constants had varying values depending on the source of the enzyme and used substrate [1,7,37]. For instance, the K M values of α-glucosidases from palm weevil [40], Apis cerena japonica [41] and N. aenescens [42] were found as 0.3, 1.0 and 0.54 mM, respectively.…”

Section: Kinetics Of α-Glucosidasementioning

confidence: 99%

See 2 more Smart Citations

Partial Purification and Biochemical Characterization of ?-Glucosidase from Corn by Three-Phase Partitioning

Bilen¹,

Bayraktar²,

Önal

2018

HJBC

View full text Add to dashboard Cite

Glukozidaz önce mısırdan üçlü-faz ayırma (TPP) tekniği ile saflaştırıldı ve ardından karakterize edildi. Üç faz elde etmek için amonyum sülfat ve t-butanol kullanıldı. Mısır α-glukozidazının yüksek saflaştırma katı ve aktivite geri kazanımı ile etkin bir şekilde saflaştırılması için çeşitli proses parametrelerinin örneğin; amonyum sülfat konsantrasyonu, enzim: t-butanol oranı ve pH etkisi çalışıldı. TPP sisteminin optimum saflaştırma koşulları %50(w/v) amonyum sülfat saturasyonu, 1.0:0.75 (v/v) ham ekstrakt:t-butanol oranı ve pH 4.5 olarak belirlendi. Optimize koşullar altında α-glukozidaz %60 aktivite verimi ile 3.8 kat saflaştırıldı. α-Glukozidazın molekül kütlesi sodyum dodesil sülfat poliakrilamid jel elektroforezi (SDS-PAGE) ile yaklaşık 29 kDa olarak belirlendi. Karakterizasyon çalışmaları, enzimin optimum pH ve sıcaklığının pH 4.5 ve 55°C olduğunu gösterdi. Saflaştırılan enzimin 25-60°C sıcaklık aralığında ve pH 3.5-5.5 aralığında oldukça kararlı olduğu bulundu. Kinetik sabitler (K M ve V max ) Lineweaver-Burk diyagramından 0.64 mM ve 0.45 U olarak hesaplandı. α-Glukozidaz aktivitesi 10 mM CaCl2 konsantrasyonunda oldukça arttı (%130). TPP α-glukozidazın uygulamaları için konsantre edilmesi ve saflaştırılması için kullanışlı bir stratejidir. Anahtar KelimelerÜçlü-faz ayırma (TPP), α-glukozidaz, mısır, saflaştırma.

show abstract

Section: Effect Of Temperature On Enzyme Activity and Stabilitysupporting

confidence: 70%

Section: Effect Of Temperature On Enzyme Activity and Stabilitymentioning

confidence: 99%

Section: Kinetics Of α-Glucosidasementioning

confidence: 99%

See 1 more Smart Citation

Partial Purification and Biochemical Characterization of ?-Glucosidase from Corn by Three-Phase Partitioning

Bilen¹,

Bayraktar²,

Önal

2018

HJBC

View full text Add to dashboard Cite

show abstract

“…Furthermore, the optimum temperature for α-glucosidase activity of the r Aci HBGase II-(His) 6 enzyme was at 45°C (with 80–90% residual activity at 40–55°C), which is much higher than that previously reported by Wu et al [32], and higher than the physiological temperature of the bees. However, within extreme thermophiles, Nashiru et al [33] reported a novel HBGase from Thermus caldophilus GK24 with an optimal temperature of 90°C, whilst the intracellular and extracellular HBGases from G. toebii strain E134 showed an optimum activity of 65°C and 70°C, respectively [34]. …”

Section: Discussionmentioning

confidence: 99%

Expression of a secretory α-glucosidase II from Apis cerana indica in Pichia pastoris and its characterization

et al. 2013

View full text Add to dashboard Cite

Backgroundα–glucosidase (HBGase) plays a key role in hydrolyzing α-glucosidic linkages. In Apis mellifera, three isoforms of HBGase (I, II and III) have been reported, which differ in their nucleotide composition, encoding amino acid sequences and enzyme kinetics. Recombinant (r)HBGase II from A. cerana indica (rAciHBGase II) was focused upon here due to the fact it is a native and economic honeybee species in Thailand. The data is compared to the two other isoforms, AciHBGase I and III from the same bee species and to the three isoforms (HBGase I, II and III) in different bee species where available.ResultsThe highest transcript expression level of AciHBGase II was found in larvae and pupae, with lower levels in the eggs of A. cerana indica but it was not found in foragers. The full-length AciHBGase II cDNA, and the predicted amino acid sequence it encodes were 1,740 bp and 579 residues, respectively. The cDNA sequence was 90% identical to that from the HBGase II from the closely related A. cerana japonica (GenBank accession # NM_FJ752630.1). The full length cDNA was directionally cloned into the pPICZαA expression vector in frame with a (His)6 encoding C terminal tag using EcoRI and KpnI compatible ends, and transformed into Pichia pastoris. Maximal expression of the rAciHBGase II–(His)6 protein was induced by 0.5% (v/v) methanol for 96 h and secreted into the culture media. The partially purified enzyme was found to have optimal α-glucosidase activity at pH 3.5 and 45°C, with > 80% activity between pH 3.5–5.0 and 40–55°C, and was stabile (> 80% activity) at pH 4–8 and at < 25–65°C. The optimal substrate was sucrose.ConclusionsLike in A. mellifera, there are three isoforms of AciHBGase (I, II and III) that differ in their transcript expression pattern, nucleotide sequences and optimal enzyme conditions and kinetics.

show abstract

“…Industrial application of these enzymes requires stability at high temperatures as well as toward common denaturant agents, and therefore, enzymes isolated from thermophiles have gained attention over the past decade [ 7 ]. There are many thermostable α-glucosidases from different thermophilic and hyperthermophilic microorganisms such as Sulfolobus tokodaii [ 7 ], Geobacillus toebii [ 19 ], Thermus caldophilus [ 20 ], Thermoplasma acidophilum [ 21 ], Bacillus stearothermophilus [ 22 ] and Thermus thermophilus [ 23 , 24 ] have been discovered and characterized, and several mesophilic α-glucosidases have been engineered by mutagenesis to enhance enzyme thermostability [ 25 , 26 ].…”

Section: Introductionmentioning

confidence: 99%

Evaluation and directed evolution for thermostability improvement of a GH 13 thermostable α-glucosidase from Thermus thermophilus TC11

Zhou

Xue

2015

BMC Biotechnol

View full text Add to dashboard Cite

BackgroundThermal stable α-glucosidases with transglycosylation activity could be applied to the industrial production of oligosaccharides as well as conjugation of sugars to biologically useful materials. Therefore, α-glucosidases isolated from thermophiles have gained attention over the past decade. In this study, the characterization of a highly thermostable α-glucosidase and its thermostability improved mutant from newly isolated strain Thermus thermophilus TC11 were investigated.ResultsThe recombinant α-glucosidase (TtAG) from Thermus thermophilus TC11 was expressed in Escherichia coli BL21 (DE3) and purified. The purified enzyme had a molecular mass of 184 kDa and consisted of 59-kDa subunits; it showed hydrolytic activity for pNP-α-d-glucopyranoside (pNPG), sucrose, trehalose, panose, and isomaltooligosaccharides and very low activity for maltose. The highest specific activity of 288.96 U/mg was observed for pNPG at 90 °C and pH 5.0; Pb2+ provided a 20 % activity increase. TtAG was stable at 70 °C for more than 7 h and had a half-life of 195 min at 80 °C and 130 min at 90 °C. Transglycosylation activity was also observed with sucrose and trehalose as substrates. TtAG showed differences on substrate specificity, transglycosylation, multimerization, effects of metal ions and optimal pH from other reported Thermus α-glucosidases. One single-substitution TtAG mutant Q10Y with improved thermostability was also obtained from random mutagenesis library. The site-saturation mutagenesis and structural modelling analysis indicated that Q10Y substitution stabilized TtAG structure via additional hydrogen bonding and hydrophobic interactions.ConclusionOur findings indicate that TtAG is a highly thermostable and more acidic α-glucosidase distinct from other reported Thermus α-glucosidases. And this work also provides new insights into the catalytic and thermal tolerance mechanisms of α-glucosidases, which may guide molecular engineering of α-glucosidase and other thermostable enzymes for industrial application.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-015-0197-x) contains supplementary material, which is available to authorized users.

show abstract

Purification and characterization of intracellular and extracellular α‐glucosidases from Geobacillus toebii strain E134

Cited by 7 publications

References 45 publications

Partial Purification and Biochemical Characterization of ?-Glucosidase from Corn by Three-Phase Partitioning

Partial Purification and Biochemical Characterization of ?-Glucosidase from Corn by Three-Phase Partitioning

Expression of a secretory α-glucosidase II from Apis cerana indica in Pichia pastoris and its characterization

Evaluation and directed evolution for thermostability improvement of a GH 13 thermostable α-glucosidase from Thermus thermophilus TC11

Contact Info

Product

Resources

About