Purification and characterisation of thermostable α-amylases from microbial sources

Lim, Si Jie; Oslan, Siti Nurbaya

doi:10.15376/biores.15.1.2005-2029

Cited by 24 publications

(26 citation statements)

References 52 publications

(80 reference statements)

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“…Besides optimal temperature, the enzyme’s thermal stability is considered an important feature making its use possible in numerous biotechnological processes that require a wide range of temperatures [ 39 ]. The time course of BCA thermal inactivation was followed at temperatures ranging from 30 to 70 °C ( Figure 3 b).…”

Section: Resultsmentioning

confidence: 99%

A Novel Digestive α-Amylase from Blue Crab (Portunus segnis) Viscera: Purification, Biochemical Characterization and Application for the Improvement of Antioxidant Potential of Oat Flour

Maâlej

Maalej

Affes

et al. 2021

IJMS

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This study reports on the purification and characterization of a digestive α-amylase from blue crab (Portunussegnis) viscera designated Blue Crab Amylase (BCA). The enzyme was purified to homogeneity by ultrafiltration, Sephadex G-100 gel filtration and Sepharose mono Q anion exchange chromatography, with the final purification fold of 424.02, specific activity of 1390.8 U mg−1 and 27.8% recovery. BCA, showing a molecular weight of approximately 45 kDa, possesses desirable biotechnological features, such as optimal temperature of 50 °C, interesting thermal stability which is enhanced in the presence of starch, high stability towards surfactants (Tween 20, Tween 80 and Triton X-100), high specific activity, quite high storage and broad pH range stability. The enzyme displayed Km and Vmax values, of 7.5 ± 0.25 mg mL−1 and 2000 ± 23 μmol min−1 mg−1 for potato starch, respectively. It hydrolyzed various carbohydrates and produced maltose, maltotriose and maltotetraose as the major end products of starch hydrolysis. In addition, the purified enzyme was successfully utilized for the improvement of the antioxidant potential of oat flour, which could be extended to other cereals. Interestingly, besides its suitability for application in different industrial sectors, especially food industries, the biochemical properties of BCA from the blue crab viscera provide novel features with other marine-derived enzymes and better understanding of the biodegradability of carbohydrates in marine environments, particularly in invasive alien crustaceans.

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Section: Resultsmentioning

confidence: 99%

A Novel Digestive α-Amylase from Blue Crab (Portunus segnis) Viscera: Purification, Biochemical Characterization and Application for the Improvement of Antioxidant Potential of Oat Flour

Maâlej

Maalej

Affes

et al. 2021

IJMS

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“…AH1. The inhibition effect of EDTA on activity suggests that α-amylases from S. cerevisiae are metalloenzymes, as demonstrated by Lim et al [38]. These enzymes require divalent metal cations such as Ca 2+ in their active site to be fully active, like most of amylases which are Ca 2+dependent.…”

Section: Effect Of Chemical Agents On α-Amylase Activitymentioning

confidence: 85%

“…Similar results were obtained with a variety of fungal species such as A. niger [37], A. oryzae [35], Talaromyces pinophilus 1-95 [6] and A. flavus NSH9 [2], which exhibited optimal amylolytic activity at pH 5.0-6.0. Therefore, AMY1 and AMY2 are acidic amylases like most fungal amylases [38]. The pH stability in 0.1 M sodium acetate buffer showed a maximal stability at pH values ranging from 4.6 to 5.6 for these two isoenzymes (Fig.…”

Section: Optimum Ph and Stability Of α-Amylasementioning

confidence: 88%

Production, Partial Purification and Characterization of Two α-Amylase Isoforms from Saccharomyces cerevisiae strain YOP 1/2-2 Isolated from Tchapalo (Côte d’Ivoire)

Ouattara

Thierry

Evrard

et al. 2020

JABB

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Amylases play an important role in biotechnology and find applications in several industrial fields such as pharmaceutical, food, paper, cosmetics and detergents. Thus, it appears necessary to identify new sources of amylase, especially from microbial origin, due to the low energy consumption, cost-effective, high metabolic diversity, rapid cell growth, non-toxic and eco-friendly characteristics. In the present report, we carried out the production and partial purification of α-amylase by Saccharomyces cerevisiae strains isolated from Tchapalo, a traditional alcoholic beverage of Côte d'Ivoire. Five fungal isolates were screened initially for α-amylase production by using method of wells on Yeast Extract Peptone Dextrose Agar medium, a complete medium for yeast growth. One step DEAE-Sepharose Fast Flow was achieved for partial purification of α-amylase obtained. Among yeasts, isolate S. cerevisiae YOP 1/2-2 was able to provoke starch hydrolysis halo of 15.067±0.12 mm on starch agar plate after 48 h of incubation at 30°C. The partial purification of resulting enzyme showed two protein peaks, designated α-amylase 1 (AMY1) and α-amylase 2 (AMY2) with amylolytic activity and specific activities of 1.57-1.58 U/mg protein. Both isoforms (AMY1 and AMY2) were thermostable with optimal activity at 50 and 55°C, respectively, and at pH ranged from 4.5 to 5.3 in 0.1 M sodium acetate buffer. EDTA and Cd2+ strongly inhibited α-amylase activity by 72-75% and 64-65%, respectively, whereas cations Ca2+ and Mn2+ showed 85-99% and 71% increased amylolytic activity, respectively. All these properties show the potential uses of α-amylases from S. cerevisiae in the industrial transformation of starch.

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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%

“…In addition, most industrial processes are performed at non-physiological conditions encompassing elevated temperature, extreme pH, high salinity, organic solvents, and surfactants, where some usages require specific substrates or products generated ( Nigam, 2013 ; Sudan et al, 2018 ). Microbial -amylases with desirable properties are preferable since they can be natively isolated from wild type host, heterologously expressed in the recombinant host or engineered for the desired traits ( Nigam, 2013 ; Lim, Oslan & Oslan, 2020 ).…”

Section: Introductionmentioning

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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Purification and characterisation of thermostable α-amylases from microbial sources

Cited by 24 publications

References 52 publications

A Novel Digestive α-Amylase from Blue Crab (Portunus segnis) Viscera: Purification, Biochemical Characterization and Application for the Improvement of Antioxidant Potential of Oat Flour

A Novel Digestive α-Amylase from Blue Crab (Portunus segnis) Viscera: Purification, Biochemical Characterization and Application for the Improvement of Antioxidant Potential of Oat Flour

Production, Partial Purification and Characterization of Two α-Amylase Isoforms from Saccharomyces cerevisiae strain YOP 1/2-2 Isolated from Tchapalo (Côte d’Ivoire)

Native to designed: microbial α-amylases for industrial applications

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