Heterologous expression, purification, immobilization and characterization of recombinant α-amylase AmyLa from Laceyella sp. DS3

El-Sayed, Ahmed K. A.; Abou‐Dobara, M. I.; El‐Fallal, Amira A.; Omar, Noha F.

doi:10.1016/j.ijbiomac.2019.04.010

Cited by 24 publications

(13 citation statements)

References 48 publications

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“…The loss in enzyme activity may be because of different factors like the denaturation of protein due to its repeated use at high temperature or as a result of the inhibition of enzyme activity by end product of the reaction. 63,64 The insignificant loss of enzyme activity was because of the leakage of very small amount of enzyme during its consecutive usage. The laccase enzyme immobilized onto magnetic β-cyclodextrin-modified chitosan maintained 70% of its initial activity at the temperature 55 °C.…”

Section: Discussionmentioning

confidence: 99%

Effective utilization of magnetic nano-coupled cloned β-xylanase in saccharification process

et al. 2022

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Section: Discussionmentioning

confidence: 99%

Effective utilization of magnetic nano-coupled cloned β-xylanase in saccharification process

et al. 2022

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“…Sephadex G-100 has a molecular weight fractionation range of 1-100 kDa, thus, the AmyLa from Laceyella sp. DS3 was shown to have 51.5 kDa from SDS-PAGE was small enough to enter the pores of the resin and was within the intermediate period of elution (El-Sayed et al 2019).…”

Section: Size-exclusion Chromatographymentioning

confidence: 98%

“…Referring to Table 6, the most frequently equipped SEC matrix is Sephadex G-100 (Chen et al 2015;Baltas et al 2016;Allala et al 2019;El-Sayed et al 2019). This matrix shown promising yields while purifying α-amylases produced by Anoxybacillus thermarum A4 strain (74.6%), Bacillus subtilis WB800 (41.7%), Escherichia coli BL21 (76.53%), and Tepidimonas fonticaldi strain HB23 (41%).…”

Section: Size-exclusion Chromatographymentioning

confidence: 99%

“…In recent research, Sephadex G-100 was loaded with enzyme solution before eluting with 0.1 M phosphate buffer (pH 7.5) while purifying recombinant α-amylase AmyLa from Laceyella sp. DS3 expressed in E. coli BL21 (El-Sayed et al 2019). Sephadex G-100 has a molecular weight fractionation range of 1-100 kDa, thus, the AmyLa from Laceyella sp.…”

Section: Size-exclusion Chromatographymentioning

confidence: 99%

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Purification and characterisation of thermostable α-amylases from microbial sources

Lim

Oslan

2019

BioRes

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α-Amylases (E.C 3.2.1.1) hydrolyse starch into smaller moieties such as maltose and glucose by breaking α-1,4-glycosidic linkages. The application of α-amylases in various industries has made the large-scale productions of these enzymes crucial. Thermostable α-amylase that catalyses starch degradation at the temperatures higher than 50 °C is favourable in harsh industrial applications. Due to ease in genetic manipulation and bulk production, this enzyme is most preferably produced by microorganisms. Bacillus sp. and Escherichia coli are commonly used microbial expression hosts for α-amylases (30 to 205 kDa in molecular weight). These amylases can be purified using ultrafiltration, salt precipitation, dialysis, and column chromatography. Recently, affinity column chromatography has shown the most promising result where the recovery rate was 38 to 60% and purification up to 13.2-fold. Microbial thermostable α-amylases have the optimum temperature and pH ranging from 50 °C to 100 °C and 5.0 to 10.5, respectively. These enzymes have high specificity towards potato starch, wheat starch, amylose, and amylopectin. EDTA (1 mM) gave the highest inhibitory effect (79%), but Ca2+ (5 mM) was the most effective co-factor with 155%. This review provides insight regarding thermostable α-amylases obtained from microbial sources for industrial applications.

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“…The intermediate temperature stable (ITS) Laceyella sp. DS3 α-amylases (AmyLa; wild type and recombinant) were said to be desirable in the baking industries as an antistaling agent while exhibiting the optimum temperature at 50 • C and 55 • C, respectively (El-Sayed et al, 2019). Nevertheless, α-amylase supplementation was also expected to improve the texture, flavour and shelf-life of the bread.…”

Section: Enzymes Characterizations For Industrial Applicationsmentioning

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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Heterologous expression, purification, immobilization and characterization of recombinant α-amylase AmyLa from Laceyella sp. DS3

Cited by 24 publications

References 48 publications

Effective utilization of magnetic nano-coupled cloned β-xylanase in saccharification process

Effective utilization of magnetic nano-coupled cloned β-xylanase in saccharification process

Purification and characterisation of thermostable α-amylases from microbial sources

Native to designed: microbial α-amylases for industrial applications

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