Isolation of a RecombinantBacillus sp. TS-23 α-Amylase by Adsorption-Elution on Raw Starch

Lin, Long-Liu; Lo, Huei-Fen; Chen, Jen‐Ni; Ku, Kow-Loung; Hsu, Wen-Hwei

doi:10.1002/1521-379x(200208)54:8<338::aid-star338>3.0.co;2-3

Cited by 10 publications

(5 citation statements)

References 18 publications

(14 reference statements)

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“…Furthermore, amylase activity was increased 34% by PMSF (3 mM) whereas urea diminished the enzyme activity to 43% 26 have reported an activity of 97% when using 10 mM PMSF, during incubation with Bacillus sp. TS‐23 α‐amylase produced by recombinant Escherichia coli .…”

Section: Resultsmentioning

confidence: 98%

Novel extracellular hyper acidophil and thermostable α‐amylase from Micrococcus sp.NS 211

2011

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Among several bacteria examined in this study, a hyper acidophil and thermostable Micrococcus sp.NS 211 designated as M.Amy NS 211 was selected for production of amylase using starch agar plates with following incubation at 85°C. Identification by 16SrRNA on selected bacterium disclosed the highest similarity for protean regions of this gene, 27 F and 1492R as Micrococcus sp.NS 211. Although activity of M.Amy NS 211 was established at temperatures between 70 and 110°C and pH ranges 1.2–8.0, the optimum temperature and pH was achieved at 85°C and 3.5 in sodium citrate buffer system respectively. Two‐step chromatography was performed using (CM Bio‐Gel A) and (Bio‐Gel A‐150) columns to purify 84 kDa hyper acidophil and thermostable α‐amylase. SDS‐PAGE analysis showed molecular mass and amylolytic activity as single band. Enhancement of enzyme activity was obtained in presence of 5 mM MnCl2 (298%), CaCl2 (347%), FeCl2 (211%), MgCl2 (253%), ZnCl2 (146%), NiCl (142%), NaCl (141%), Na‐sulfate (153%) while inhibition was observed with (5 mM) EDTA, PMSF (3 mM), urea (8 M), and SDS (1%) at 143, 134, 43, and 119%, respectively. M.Amy NS 211 can be applied in laundry detergents, textile, and modern relevant industrial processes at extreme temperatures and under acidic conditions.

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Section: Resultsmentioning

confidence: 98%

Novel extracellular hyper acidophil and thermostable α‐amylase from Micrococcus sp.NS 211

2011

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“…During the elution stage, the addition of d ‐glucose in the elution buffer enabled further precipitation by complexing with glucoamylase. This could be due to the presence specific binding sites on the enzyme for both the substrate (insoluble), as well as the soluble ligands . The purification strategy of starch affinity chromatography was employed by many researchers .…”

Section: Discussionmentioning

confidence: 99%

“…The bacterial glucoamylase in the present study was purified by starch adsorption and gel filtration chromatography which revealed 40.02% yield with 2.73-fold and specific activity of 2300.5 (U/mg). During the elution stage, the addition of D-glucose in the elution buffer enabled further [29]. The purification strategy of starch affinity chromatography was employed by many researchers [30][31][32][33].…”

Section: Discussionmentioning

confidence: 99%

Purification and biochemical characterization of extracellular glucoamylase from Paenibacillus amylolyticus strain

Lincoln

More²,

2019

J Basic Microbiol

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In the present study, glucoamylase produced from a soil bacterium Paenibacillus amylolyticus NEO03 was cultured under submerged fermentation conditions. The extracellular enzyme was purified by starch adsorption chromatography and further by gel filtration, with 2.73‐fold and recovery of 40.02%. The protein exhibited molecular mass of ∼66,000 Da as estimated by SDS–PAGE and depicted to be a monomer. The enzyme demonstrated optimum activity at pH range 6.0–7.0 and temperature range 30–40 °C. Glucoamylase was mostly activated by Mn2+ metal ions and depicted no dependency on Ca2+ ions. The enzyme preferentially hydrolyzed all the starch substrates. High substrate specificity was demonstrated towards soluble starch and kinetic values Km and Vmax were 2.84 mg/ml and 239.2 U/ml, respectively. The products of hydrolysis of soluble starch were detected by thin layer chromatography which showed only D‐glucose, indicating a true glucoamylase. The secreted glucoamylase from P. amylolyticus strain possesses properties suitable for saccharification processes such as biofuel production.

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“…We previously demonstrated that recombinant a-amylase of Bacillus sp. strain TS-23 can be recovered from crude E. coli extract by adsorption to starch granules followed by gentle elution with a Tris-HCl buffer [11]. The conditions for recovery of chimeric proteins from starch, however, remain to be optimized.…”

Section: Adsorption Of Lapsbd To Raw Starchmentioning

confidence: 99%

Fusion of Bacillus stearothermophilus leucine aminopeptidase II with the raw-starch-binding domain of Bacillus sp. strain TS-23 α-amylase generates a chimeric enzyme with enhanced thermostability and catalytic activity

Hua

Chi

et al. 2004

J IND MICROBIOL BIOTECHNOL

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Bacillus stearothermophilus leucine aminopeptidase II (LAPII) was fused at its C-terminal end with the raw-starch-binding domain of Bacillus sp. strain TS-23 alpha-amylase. The chimeric enzyme (LAPsbd), with an apparent molecular mass of approximately 61 kDa, was overexpressed in IPTG-induced Escherichia coli cells and purified to homogeneity by nickel-chelate chromatography. The purified enzyme retained LAP activity and adsorbed raw starch. LAPsbd was stable at 70 degrees C for 10 min, while the activity of wild-type enzyme was completely abolished under the same environmental condition. Compared with the wild-type enzyme, the twofold increase in the catalytic efficiency for LAPsbd was due to a 218% increase in the k(cat) value.

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Isolation of a RecombinantBacillus sp. TS-23 α-Amylase by Adsorption-Elution on Raw Starch

Cited by 10 publications

References 18 publications

Novel extracellular hyper acidophil and thermostable α‐amylase from Micrococcus sp.NS 211

Novel extracellular hyper acidophil and thermostable α‐amylase from Micrococcus sp.NS 211

Purification and biochemical characterization of extracellular glucoamylase from Paenibacillus amylolyticus strain

Fusion of Bacillus stearothermophilus leucine aminopeptidase II with the raw-starch-binding domain of Bacillus sp. strain TS-23 α-amylase generates a chimeric enzyme with enhanced thermostability and catalytic activity

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