One-Step Production of Immobilized α-Amylase in Recombinant Escherichia coli

ABSTRACT Industrial enzymes are often immobilized via chemical cross-linking onto solid supports to enhance stability and facilitate repeated use in bioreactors. For starch-degrading enzymes, immobilization usually places constraints on enzymatic conversion due to the limited diffusion of the macromolecular substrate through available supports. This study describes the one-step immobilization of a highly thermostable α-amylase (BLA) from Bacillus licheniformis and its functional display on the surface of polyester beads inside engineered Escherichia coli. An optimized BLA variant (Termamyl) was N-terminally fused to the polyester granule-forming enzyme PhaC of Cupriavidus necator. The fusion protein lacking the signal sequence mediated formation of stable polyester beads exhibiting α-amylase activity. The α-amylase beads were assessed with respect to α-amylase activity, which was demonstrated qualitatively and quantitatively. The immobilized α-amylase showed Michaelis-Menten enzyme kinetics exerting a V max of about 506 mU/mg of bead protein with a Km of about 5 μM, consistent with that of free α-amylase. The stability of the enzyme at 85°C and the capacity for repeated usage in a starch liquefaction process were also demonstrated. In addition, structural integrity and functionality of the beads at extremes of pH and temperature, demonstrating their suitability for industrial use, were confirmed by electron microscopy and protein/enzyme analysis. This study proposes a novel, cost-effective method for the production of immobilized α-amylase in a single step by using the polyester granules forming protein PhaC as a fusion partner in engineered E. coli.

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Rapid Golden Gate assembly of exons from genomic DNA for protein expression in Escherichia coli and Pichia pastoris

BioTechniques ◽

10.2144/btn-2021-0039 ◽

2021 ◽

Author(s):

Junhao Cheng ◽

Mingkun Wu ◽

Ren Zhong ◽

Dayong Si ◽

Geng Meng ◽

...

Keyword(s):

Escherichia Coli ◽

Pichia Pastoris ◽

Genomic Dna ◽

Low Cost ◽

Cost Effective ◽

Single Step ◽

Golden Gate ◽

Cloning Efficiency ◽

Expression In Escherichia Coli ◽

Expression Plasmids

The development of a quick, single-step cloning system for generation of multiexon gene expression constructs is presented. The system allows efficient and cost-effective assembly of multiple exons of interest genes into different expression plasmids in both Escherichia coli and Pichia pastoris. The high cloning efficiency and low cost of the system make it ideal for a novel workflow for the assembly of intron-bearing genes for expression in two different expression hosts.

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The ability to increase the base of support and recover stability is limited in its generalisation for different balance perturbation tasks

European Review of Aging and Physical Activity ◽

10.1186/s11556-021-00274-w ◽

2021 ◽

Vol 18 (1) ◽

Author(s):

Jil Bosquée ◽

Julian Werth ◽

Gaspar Epro ◽

Thorben Hülsdünker ◽

Wolfgang Potthast ◽

...

Keyword(s):

Dynamic Stability ◽

Single Step ◽

Stability Performance ◽

Rate Of Increase ◽

One Step ◽

Recovery Performance ◽

The Stability ◽

Base Of Support ◽

Age Range ◽

Induced Trip

Abstract Background The assessment of stability recovery performance following perturbations contributes to the determination of fall resisting skills. This study investigated the association between stability recovery performances in two perturbation tasks (lean-and-release versus tripping). Methods Healthy adults (12 young: 24 ± 3 years; 21 middle-aged: 53 ± 5 years; 11 old: 72 ± 5 years) were suddenly released from a forward-inclined position attempting to recover stability with a single step. In a second task, all participants experienced a mechanically induced trip during treadmill walking. To assess dynamic stability performance, the antero-posterior margin of stability (MoS), the base of support (BoS), and the rate of increase in BoS were determined at each foot touchdown (TD) for both tasks. Results Only weak to moderate correlations in dynamic stability performance parameters were found between the two tasks (0.568 > r > 0.305, 0.001 < p < 0.04). A separation of participants according to the number of steps required to regain stability in the lean-and-release task revealed that multiple- (more than one step) compared to single-steppers showed a significantly lower MoS at TD (p = 0.003; g = 1.151), lower BoS at TD (p = 0.019; g = 0.888) and lower rate of increase in BoS until TD (p = 0.002; g = 1.212) after release. Despite these profound subgroup differences in the lean-and-release task, no differences between multiple- and single-steppers were observed in the stability recovery performance during tripping. Conclusion The results provide evidence that the ability to effectively control dynamic stability following a sudden balance disturbance in adults across a wide age range is limited in its generalisation for different perturbation tasks.

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Characteristics of some single-step mutants to chloramphenicol resistance in Escherichia coli K12 and their interactions with R-factor genes

Genetics Research ◽

10.1017/s0016672300009708 ◽

1966 ◽

Vol 7 (2) ◽

pp. 281-286 ◽

Cited By ~ 11

Author(s):

E. C. R. Reeve

Keyword(s):

Escherichia Coli ◽

Single Step ◽

Chloramphenicol Resistance ◽

R Factor ◽

Resistance Patterns ◽

One Step ◽

Increased Sensitivity ◽

Resistant Mutants ◽

High Level ◽

R Factors

Six one-step Chloramphenicol (Cm)-resistant mutants of Escherichia coli K12 were graded for resistance to Cm, Tetracycline (Tc) and Puromyein (Pm) by streaking on minimal agar plates containing antibiotic. They fell into at least three distinct groups on the basis of their resistance patterns. One mutant showed increased sensitivity to Pm. Most of the mutants expressed their effect on resistance to Cm and Tc in the presence of R-factors carrying resistance genes for these antibiotics, but one mutant with a relatively high level of resistance to Cm had its resistance effect completely masked in the presence of R-mediated resistance. Similar cases were found among mutants selected for Cm-resistance in another strain of K12.

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Single-step affinity and cost-effective purification of recombinant proteins using the Sepharose-binding lectin-tag from the mushroom Laetiporus sulphureus as fusion partner

Protein Expression and Purification ◽

10.1016/j.pep.2015.11.004 ◽

2016 ◽

Vol 119 ◽

pp. 51-56 ◽

Cited By ~ 7

Author(s):

Xiao-jing Li ◽

Jin-ling Liu ◽

Dong-sheng Gao ◽

Wen-yan Wan ◽

Xia Yang ◽

...

Keyword(s):

Recombinant Proteins ◽

Cost Effective ◽

Single Step ◽

Fusion Partner ◽

Laetiporus Sulphureus ◽

Binding Lectin

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Faculty Opinions recommendation of The Fh8 tag: a fusion partner for simple and cost-effective protein purification in Escherichia coli.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718130710.793488152 ◽

2013 ◽

Author(s):

Mario Lebendiker

Keyword(s):

Escherichia Coli ◽

Protein Purification ◽

Cost Effective ◽

Fusion Partner

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Design and Optimization of Short DNA Sequences That Can Be Used as 5′ Fusion Partners for High-Level Expression of Heterologous Genes in Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.01676-13 ◽

2013 ◽

Vol 79 (21) ◽

pp. 6655-6664 ◽

Cited By ~ 8

Author(s):

Veronika Kucharova ◽

Jørgen Skancke ◽

Trygve Brautaset ◽

Svein Valla

Keyword(s):

Escherichia Coli ◽

Dna Sequences ◽

Protein Production ◽

Signal Sequence ◽

Protein Product ◽

Suitable Model ◽

Fusion Partner ◽

Content Type ◽

Protein Levels

ABSTRACTThe 5′ terminal nucleotide sequence of a gene is often a bottleneck in recombinant protein production. Theifn-α2bSgene is poorly expressed inEscherichia coliunless a translocation signal sequence (pelB) is fused to the 5′ end of the gene. A combinedin silicoandin vivoanalysis reported here further indicates that theifn-α2bS5′ coding sequence is suboptimal for efficient gene expression.ifn-α2bStherefore presents a suitable model gene for describing properties of 5′ fusions promoting expression. We show that short DNA sequences corresponding to the 5′ end of the highly expressedcelBgene, whose protein product is cytosolic, can functionally replacepelBas a 5′ fusion partner for efficientifn-α2bSexpression.celBfusions of various lengths (corresponding to a minimum of 8 codons) led to more than 7- and 60-fold stimulation of expression at the transcript and protein levels, respectively. Moreover, the presence of acelB-based fusion partner was found to moderately reduce the decay rate of the corresponding transcript. The 5′ fusions thus appear to act by enhancing translation, and bound ribosomes may accordingly contribute to increased mRNA stability and reduced mRNA decay. However, other effects, such as altered protein stability, cannot be excluded. We also developed an experimental protocol that enabled us to identify improved variants of thecelBfusion, and one of these (celBD11) could be used to additionally increaseifn-α2bSexpression more than 4-fold at the protein level. Interestingly,celBD11also stimulated greater protein production of three other medically important human genes than the wild-typecelBfragment.

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One-step purification of histidine-tagged cytochrome bo3 from Escherichia coli and demonstration that associated quinone is not required for the structural integrity of the oxidase

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology ◽

10.1016/s0167-4838(97)00036-8 ◽

1997 ◽

Vol 1340 (1) ◽

pp. 131-142 ◽

Cited By ~ 40

Author(s):

Jon N. Rumbley ◽

Elizabeth Furlong Nickels ◽

Robert B. Gennis

Keyword(s):

Escherichia Coli ◽

Structural Integrity ◽

Cytochrome Bo3 ◽

One Step

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Complete signal peptide of Tk1884, an α-amylase from Thermococcus kodakarensis, is not necessary for extracellular secretion of the enzyme by Escherichia coli

Amylase ◽

10.1515/amylase-2017-0007 ◽

2017 ◽

Vol 1 (1) ◽

Cited By ~ 1

Author(s):

Majida A. Muhammad ◽

Samia Falak ◽

Naeem Rashid ◽

Nasir Ahmed ◽

Qurra-Tul-Ann A. Gardner ◽

...

Keyword(s):

Escherichia Coli ◽

Recombinant Protein ◽

Signal Peptide ◽

Amylase Activity ◽

Signal Sequence ◽

Ionization Mass ◽

Extracellular Medium ◽

E Coli ◽

Thermococcus Kodakarensis ◽

Extracellular Secretion

AbstractIn order to elucidate if Escherichia coli secretion system recognizes the N-terminally truncated signal sequence of an archaeal α-amylase from Thermococcus kodakarensis (Tk1884) and secretes the recombinant protein to the extracellular medium, we have cloned Tk1884 with the deletion of the sixteen N-terminal amino acids and produced the recombinant protein Tk1884Δ16 in E. coli. Analysis of the intracellular, membranous and extracellular fractions demonstrated the presence of Tk1884Δ16 in all the three fractions. The intracellular α-amylase activity, similar to the membranous fraction, increased with the passage of time till 8 h of induction and then decreased. In contrast, the extracellular α-amylase activity slowly increased with the passage of time after induction. The extracellular amylase activity was purified and determination of the molecular mass by electrospray ionization mass spectrometry demonstrated that Tk1884Δ16 was secreted to the extracellular medium without cleavage of the signal peptide. To the best of our knowledge, this is the first report on recognition of N-terminally truncated signal peptide of archaeal origin by E. coli.

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Biogenic synthesis of bimetallic nanoparticles and their applications

Reviews in Inorganic Chemistry ◽

10.1515/revic-2020-0024 ◽

2021 ◽

Vol 0 (0) ◽

Author(s):

Krishnan Sundarrajan Sasireka ◽

Pottail Lalitha

Keyword(s):

Bimetallic Nanoparticles ◽

Synergistic Effects ◽

Cost Effective ◽

Industrial Applications ◽

Facile Synthesis ◽

Actual Mechanism ◽

One Step ◽

Physicochemical Methods ◽

The Stability ◽

Future Direction

Abstract The current advancements in nanotechnology suggest a sustainable development in the green synthesis of bimetallic nanoparticles (BMNPs) through green approaches. Though challenging, nano phyto technology has versatile methods to achieve desired unique properties like optic, electronic, magnetic, therapeutic, and catalytic efficiencies. Bio-inspired, facile synthesis of bifunctional BMNPs is possible using abundant, readily available natural plant sources, bio-mass wastes and microorganisms. Synergistic effects of two different metals on mixing, bring new insight for the vast applications, which is not achievable in using monometallic NPs. By adopting bio-inspired greener approaches for synthesizing NPs, the risk of environmental toxicity caused by conventional physicochemical methods become negligible. This article hopes to provide the significance of cost-effective, one-step, eco-friendly and facile synthesis of noble/transition bimetallic NPs. This review article endows an overview of the bio-mediated synthesis of bimetallic NPs, classifications of BMNPs, current characterization techniques, possible mechanistic aspects for reducing metal ions, and the stability of formed NPs and bio-medical/industrial applications of fabricated NPs. The review also highlights the prospective future direction to improve reliability, reproducibility of biosynthesis methods, its actual mechanism in research works and extensive application of biogenic bimetallic NPs.

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