Aspergillus oryzae S2 AmyA amylase expression in Pichia pastoris: production, purification and novel properties

Trabelsi, Sahar; Sahnoun, Mouna; Elgharbi, Fatma; Ameri, Rihab; Mabrouk, Sameh; Mezghani, Monia; Hmida-Sayari, Aïda; Béjar, Samír

doi:10.1007/s11033-018-4548-2

Cited by 11 publications

(8 citation statements)

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“…Strain EGB 14,000NMNMNMNM[54] Pontibacillus sp. ZY (AmyZ1)14,81512,621905651582623This studyFungi Aspergillus penicillioides 118NMNMNMNM[43] Aspergillus oryzae cmc132NMNMNMNM[44] Aspergillus oryzae cmc1 mutant M-100(6)2461NMNMNMNM[44] Aspergillus oryzae strain S2 (rAmyA)5150NMNMNMNM[45] Rhizopus oryzae (RoAmy)11231044144914941089[13] Thermomyces dupontii (TdAmyA)28142339270924171315[14]Yeast Cryptococcus flavus (rAmy1)4165 a NMNMNMNM[46] Cryptococcus flavus 843 b NMNMNMNM[47] Cryptococcus sp. S-22539…”

Section: Resultsmentioning

confidence: 99%

AmyZ1: a novel α-amylase from marine bacterium Pontibacillus sp. ZY with high activity toward raw starches

Fang

Xue

Deng

et al. 2019

Biotechnol Biofuels

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Background Starch is an inexpensive and renewable raw material for numerous industrial applications. However, most starch-based products are not cost-efficient due to high-energy input needed in traditional enzymatic starch conversion processes. Therefore, α-amylase with high efficiency to directly hydrolyze high concentration raw starches at a relatively lower temperature will have a profound impact on the efficient application of starch. Results A novel raw starch digesting α-amylase (named AmyZ1) was screened and cloned from a deep-sea bacterium Pontibacillus sp. ZY. Phylogenetic analysis showed that AmyZ1 was a member of subfamily 5 of glycoside hydrolase family 13. When expressed in Escherichia coli , the recombinant AmyZ1 showed high activity at pH 6.0–7.5 and 25–50 °C. Its optimal pH and temperature were 7.0 and 35 °C, respectively. Similar to most α-amylases, AmyZ1 activity was enhanced (2.4-fold) by 1.0 mM Ca 2+ . Its half-life time at 35 °C was also extended from about 10 min to 100 min. In comparison, AmyZ1 showed a broad substrate specificity toward raw starches, including those derived from rice, corn, and wheat. The specific activity of AmyZ1 towards raw rice starch was 12,621 ± 196 U/mg, much higher than other reported raw starch hydrolases. When used in raw starch hydrolyzing process, AmyZ1 hydrolyzed 52%, 47% and 38% of 30% (w/v) rice, corn, and wheat starch after 4 h incubation. It can also hydrolyze marine raw starch derived from Chlorella pyrenoidosa , resulting in 50.9 mg/g DW (dry weight of the biomass) of reducing sugars after 4 h incubation at 35 °C. Furthermore, when hydrolyzing raw corn starch using the combination of AmyZ1 and commercial glucoamylase, the hydrolysis rate reached 75% after 4.5 h reaction, notably higher than that obtained in existing starch-processing industries. Conclusions As a novel raw starch-digesting α-amylase with high specific activity, AmyZ1 efficiently hydrolyzed raw starches derived from both terrestrial and marine environments at near ambient temperature, suggesting its application potential in starch-based industrial processes. Electronic supplementary material The online version of this article (10.1186/s13068-019-1432-9) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

AmyZ1: a novel α-amylase from marine bacterium Pontibacillus sp. ZY with high activity toward raw starches

Fang

Xue

Deng

et al. 2019

Biotechnol Biofuels

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“…Most industrial important enzymes especially -amylases are preferably isolated from microbial sources encompassing mainly the bacteria, yeasts and fungi due to the ease in genetic manipulations and bulk productions ( Lim, Oslan & Oslan, 2020 ). Therefore, native and recombinant -amylases have frequently been produced by or cloned into various microbial expression hosts including Bacillus subtilis ( Trabelsi et al, 2019a ), Enterococcus faecalis ( Meruvu, 2019 ), Aspergillus clavatus ( Shruthi, Achur & Boramuthi, 2020 ), Tepidimonas fonticaldi ( Allala et al, 2019 ), Komatagaella phaffii ( Trabelsi et al, 2019b ; Wang et al, 2019 ) and Meyerozyma guilliermondii ( Nasir et al, 2020 ).…”

Section: Survey Methodologymentioning

confidence: 99%

Native to designed: microbial α-amylases for industrial applications

Lim

Oslan

2021

PeerJ

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Background α-amylases catalyze the endo-hydrolysis of α-1,4-D-glycosidic bonds in starch into smaller moieties. While industrial processes are usually performed at harsh conditions, α-amylases from mainly the bacteria, fungi and yeasts are preferred for their stabilities (thermal, pH and oxidative) and specificities (substrate and product). Microbial α-amylases can be purified and characterized for industrial applications. While exploring novel enzymes with these properties in the nature is time-costly, the advancements in protein engineering techniques including rational design, directed evolution and others have privileged their modifications to exhibit industrially ideal traits. However, the commentary on the strategies and preferably mutated residues are lacking, hindering the design of new mutants especially for enhanced substrate specificity and oxidative stability. Thus, our review ensures wider accessibility of the previously reported experimental findings to facilitate the future engineering work. Survey methodology and objectives A traditional review approach was taken to focus on the engineering of microbial α-amylases to enhance industrially favoured characteristics. The action mechanisms of α- and β-amylases were compared to avoid any bias in the research background. This review aimed to discuss the advances in modifying microbial α-amylases via protein engineering to achieve longer half-life in high temperature, improved resistance (acidic, alkaline and oxidative) and enhanced specificities (substrate and product). Captivating results were discussed in depth, including the extended half-life at 100 °C, pH 3.5 and 10, 1.8 M hydrogen peroxide as well as enhanced substrate (65.3%) and product (42.4%) specificities. These shed light to the future microbial α-amylase engineering in achieving paramount biochemical traits ameliorations to apt in the industries. Conclusions Microbial α-amylases can be tailored for specific industrial applications through protein engineering (rational design and directed evolution). While the critical mutation points are dependent on respective enzymes, formation of disulfide bridge between cysteine residues after mutations is crucial for elevated thermostability. Amino acids conversion to basic residues was reported for enhanced acidic resistance while hydrophobic interaction resulted from mutated hydrophobic residues in carbohydrate-binding module or surface-binding sites is pivotal for improved substrate specificity. Substitution of oxidation-prone methionine residues with non-polar residues increases the enzyme oxidative stability. Hence, this review provides conceptual advances for the future microbial α-amylases designs to exhibit industrially significant characteristics. However, more attention is needed to enhance substrate specificity and oxidative stability since they are least reported.

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“…The ompA signal sequence has been recently used to translocate a recombinant peptide to the periplasm for secretion into the cultivation medium. Translocation was enhanced by the co-expression of two secretion factors along with an increase in the level of the recombinant peptide in the cultivation medium [ 114 , 115 ]. Recombinant proteins probably exit the periplasm passively via destabilized membrane structures, either upon cellular aging or a change in the culture conditions.…”

Section: Location Of Expressionmentioning

confidence: 99%

Strategies for Optimizing the Production of Proteins and Peptides with Multiple Disulfide Bonds

Lee

Park

2020

Antibiotics

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Bacteria can produce recombinant proteins quickly and cost effectively. However, their physiological properties limit their use for the production of proteins in their native form, especially polypeptides that are subjected to major post-translational modifications. Proteins that rely on disulfide bridges for their stability are difficult to produce in Escherichia coli. The bacterium offers the least costly, simplest, and fastest method for protein production. However, it is difficult to produce proteins with a very large size. Saccharomyces cerevisiae and Pichia pastoris are the most commonly used yeast species for protein production. At a low expense, yeasts can offer high protein yields, generate proteins with a molecular weight greater than 50 kDa, extract signal sequences, and glycosylate proteins. Both eukaryotic and prokaryotic species maintain reducing conditions in the cytoplasm. Hence, the formation of disulfide bonds is inhibited. These bonds are formed in eukaryotic cells during the export cycle, under the oxidizing conditions of the endoplasmic reticulum. Bacteria do not have an advanced subcellular space, but in the oxidizing periplasm, they exhibit both export systems and enzymatic activities directed at the formation and quality of disulfide bonds. Here, we discuss current techniques used to target eukaryotic and prokaryotic species for the generation of correctly folded proteins with disulfide bonds.

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Aspergillus oryzae S2 AmyA amylase expression in Pichia pastoris: production, purification and novel properties

Cited by 11 publications

References 53 publications

AmyZ1: a novel α-amylase from marine bacterium Pontibacillus sp. ZY with high activity toward raw starches

AmyZ1: a novel α-amylase from marine bacterium Pontibacillus sp. ZY with high activity toward raw starches

Native to designed: microbial α-amylases for industrial applications

Strategies for Optimizing the Production of Proteins and Peptides with Multiple Disulfide Bonds

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