Efficient solubilization, purification of recombinant extracellular α-amylase from pyrococcus furiosus expressed as inclusion bodies in Escherichia coli

Lisa, W; Qin, Zhou; Chen, Huayou; Chu, Zema; Lu, Junhui; Zhang, Yi; Yang, Sen

doi:10.1007/s10295-006-0185-1

Cited by 20 publications

(22 citation statements)

References 20 publications

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“…2b large aggregates of more than 300 kDa were also observed in native conditions with no heat treatment. It was previously reported that high accumulation of recombinant proteins in plant chloroplasts leads to the formation of inclusion bodies [25]; PFA accumulates in inclusion bodies in E. coli , and requires refolding for activity [7]. Our results suggest that ER-targeted plant-made PFA is correctly folded since aggregates are enzymatically functional, obviating the need for refolding, thus facilitating the process and reducing the cost of PFA production.…”

Section: Resultssupporting

confidence: 54%

“…PFA's advantages over α-amylases from other sources include an optimal temperature of 100 °C, a half-life of 13 h at 98 °C, and Ca 2+ -independent functionality. PFA is very desirable in industrial applications, but its use is hampered by the high production cost in E.coli because it accumulates in the form of insoluble inclusion bodies, and a tedious solubilization procedure is required to produce a soluble functional enzyme [7]. Over the years, there has been interest in exploring new strategies to increase the solubility and yield of recombinant PFA.…”

Section: Introductionmentioning

confidence: 99%

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A hyper-thermostable α-amylase from Pyrococcus furiosus accumulates in Nicotiana tabacum as functional aggregates

2017

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BackgroundAlpha amylase hydrolyzes α-bonds of polysaccharides such as starch and produces malto-oligosaccharides. Its starch saccharification applications make it an essential enzyme in the textile, food and brewing industries. Commercially available α-amylase is mostly produced from Bacillus or Aspergillus. A hyper-thermostable and Ca 2++ independent α-amylase from Pyrococcus furiosus (PFA) expressed in E.coli forms insoluble inclusion bodies and thus is not feasible for industrial applications.ResultsWe expressed PFA in Nicotiana tabacum and found that plant-produced PFA forms functional aggregates with an accumulation level up to 3.4 g/kg FW (fresh weight) in field conditions. The aggregates are functional without requiring refolding and therefore have potential to be applied as homogenized plant tissue without extraction or purification. PFA can also be extracted from plant tissue upon dissolution in a mild reducing buffer containing SDS. Like the enzyme produced in P. furiosus and in E. coli, plant produced PFA preserves hyper-thermophilicity and hyper-thermostability and has a long shelf life when stored in lyophilized leaf tissue. With tobacco’s large biomass and high yield, hyper-thermostable α-amylase was produced at a scale of 42 kg per hectare.ConclusionsTobacco may be a suitable bioreactor for industrial production of active hyperthermostable alpha amylase.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-017-0372-3) contains supplementary material, which is available to authorized users.

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Section: Resultssupporting

confidence: 54%

Section: Introductionmentioning

confidence: 99%

A hyper-thermostable α-amylase from Pyrococcus furiosus accumulates in Nicotiana tabacum as functional aggregates

2017

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“…This added Ca 2ϩ inhibits the activity of glucose isomerase, which is used in later steps for isomerization of glucose to fructose during high-fructose syrup production (29). Calcium oxalate is also produced as a waste product and deposits in the pipes and heat exchangers.…”

Section: Discussionmentioning

confidence: 99%

Novel Maltotriose-Hydrolyzing Thermoacidophilic Type III Pullulan Hydrolase from Thermococcus kodakarensis

Ahmad

Rashid

Haider

et al. 2014

Appl Environ Microbiol

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cA novel thermoacidophilic pullulan-hydrolyzing enzyme (PUL) from hyperthermophilic archaeon Thermococcus kodakarensis (TK-PUL) that efficiently hydrolyzes starch under industrial conditions in the absence of any additional metal ions was cloned and characterized. TK-PUL possessed both pullulanase and ␣-amylase activities. The highest activities were observed at 95 to 100°C. Although the enzyme was active over a broad pH range (3.0 to 8.5), the pH optima for both activities were 3.5 in acetate buffer and 4.2 in citrate buffer. TK-PUL was stable for several hours at 90°C. Its half-life at 100°C was 45 min when incubated either at pH 6.5 or 8.5. The K m value toward pullulan was 2 mg ml ؊1 , with a V max of 109 U mg ؊1 . Metal ions were not required for the activity and stability of recombinant TK-PUL. The enzyme was able to hydrolyze both ␣-1,6 and ␣-1,4 glycosidic linkages in pullulan. The most preferred substrate, after pullulan, was ␥-cyclodextrin, which is a novel feature for this type of enzyme. Additionally, the enzyme hydrolyzed a variety of polysaccharides, including starch, glycogen, dextrin, amylose, amylopectin, and cyclodextrins (␣, ␤, and ␥), mainly into maltose. A unique feature of TK-PUL was the ability to hydrolyze maltotriose into maltose and glucose.

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“…The α-amylase activity was assayed according to Bernfeld and Wang et al (Bernfeld 1955;Wang et al 2007). Briefly, a 10 μl aliquot of suitably diluted enzyme was mixed with 490 μl sodium acetate buffer (50 mM, pH 5.5) containing 1 % soluble starch and incubated at 100°C for 15 min.…”

Section: Amylase Activity Assaymentioning

confidence: 99%

“…Due to the inherent difficulty to cultivate P. furiosus, sufficient production of this enzyme can only be achieved using recombinant expression. Previous studies indicated that recombinant PFA, when expressed at a high level, is always expressed as inclusion bodies (Dong et al 1997;Wang et al 2007). Several studies have tried to express PFA in its soluble form.…”

Section: Introductionmentioning

confidence: 99%

Co-expression of chaperones from P. furiosus enhanced the soluble expression of the recombinant hyperthermophilic α-amylase in E. coli

Peng

Chu

et al. 2016

Cell Stress and Chaperones

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The extracellular α-amylase from the hyperthermophilic archaeum Pyrococcus furiosus (PFA) is extremely thermostable and of an industrial importance and interest. PFA aggregates and accumulates as insoluble inclusion bodies when expressed as a heterologous protein at a high level in Escherichia coli. In the present study, we investigated the roles of chaperones from P. furiosus in the soluble expression of recombinant PFA in E. coli. The results indicate that coexpression of PFA with the molecular chaperone prefoldin alone significantly increased the soluble expression of PFA. Although, co-expression of other main chaperone components from P. furiosus, such as the small heat shock protein (sHSP) or chaperonin (HSP60), was also able to improve the soluble expression of PFA to a certain extent. Co-expression of chaperonin or sHSP in addition to prefoldin did not further increase the soluble expression of PFA. This finding emphasizes the biotechnological potentials of the molecular chaperone prefoldin from P. furiosus, which may facilitate the production of recombinant PFA.

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Efficient solubilization, purification of recombinant extracellular α-amylase from pyrococcus furiosus expressed as inclusion bodies in Escherichia coli

Cited by 20 publications

References 20 publications

A hyper-thermostable α-amylase from Pyrococcus furiosus accumulates in Nicotiana tabacum as functional aggregates

A hyper-thermostable α-amylase from Pyrococcus furiosus accumulates in Nicotiana tabacum as functional aggregates

Novel Maltotriose-Hydrolyzing Thermoacidophilic Type III Pullulan Hydrolase from Thermococcus kodakarensis

Co-expression of chaperones from P. furiosus enhanced the soluble expression of the recombinant hyperthermophilic α-amylase in E. coli

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