Improvement of the acid stability of Bacillus licheniformis alpha amylase by site-directed mutagenesis

Liu, Yihan; Huang, Lin; Jia, Leibo; Gui, Shuang; Fu, Yu; Dong, Zheng; Guo, Wanshou; Lu, Fuping

doi:10.1016/j.procbio.2017.04.040

Cited by 31 publications

(14 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Similar strategy to reduce the pKa of nucleophile and hydrogen donor in catalytic residues was performed through the SDM of H293R/H316R/H327R in BLA, resulting in the triple mutants ability to maintain 31% of residual activity at pH 4.5 and 70C for 40 min ( Liu et al, 2017 ). Interestingly, loop deletion is not only capable of increasing the enzyme thermostability, but a study concerning the deletion of the flexible loop (R179-G180) in B. stearothermophilus -amylase (AmyS) also showed its ability to improve the acid-resistance of AmyS to pH 4.56.0 with decreased optimal pH at pH 5.5 ( Gai et al, 2018 ).…”

Section: Survey Methodologymentioning

confidence: 99%

Native to designed: microbial α-amylases for industrial applications

Lim

Oslan

2021

PeerJ

View full text Add to dashboard Cite

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.

show abstract

Section: Survey Methodologymentioning

confidence: 99%

Native to designed: microbial α-amylases for industrial applications

Lim

Oslan

2021

PeerJ

View full text Add to dashboard Cite

show abstract

“…Liu et al, 2017) lipase A (BsLA) contains 12 mutations, four of which involves the introduction of carboxyl amino acids to improve solvent interaction and formation of hydrogen bonds and salt bridges (Singh, Bulusu, & Mitra, 2015). Liu et al, 2017) lipase A (BsLA) contains 12 mutations, four of which involves the introduction of carboxyl amino acids to improve solvent interaction and formation of hydrogen bonds and salt bridges (Singh, Bulusu, & Mitra, 2015).…”

Section: Surface Charge Modification Of Enzymes By Genetic Engineeringmentioning

confidence: 99%

“…α-amylase (Y. Liu et al, 2017) lipase A (BsLA) contains 12 mutations, four of which involves the introduction of carboxyl amino acids to improve solvent interaction and formation of hydrogen bonds and salt bridges (Singh, Bulusu, & Mitra, 2015). The well-studied charge mutations in BsLA were also merged without considering other type of mutations, and the selected charge mutations displays additive effects on activity, thermostability as well as IL tolerance of mutants.…”

Section: Surface Charge Modification Of Enzymes By Genetic Engineeringmentioning

confidence: 99%

“…In the case of α-amylase from Bacillus licheniformis (BLA) that lost the activity at low pHs, five histidine (H) residues at positions 281, 289, 293, 316, and 327 in BLA were selected to be substituted by arginine residues and aspartates based on the analysis of 3D structure of BLA (Y. Liu et al, 2017). At pH 4.5 and 6.5, H281D, H289D, and H293D showed low activity and H327D exhibited no activity.…”

Section: Genetic Engineering Of α-Amylasementioning

confidence: 99%

See 1 more Smart Citation

Genetic and chemical approaches for surface charge engineering of enzymes and their applicability in biocatalysis: A review

Pedersen

Zhou

Guo

et al. 2019

Biotech & Bioengineering

View full text Add to dashboard Cite

Surface charge engineering has received considerable interest from the scientific and industrial community in the last few decades. Although it was previously hypothesized that the surface charge-charge interactions were not a fundamental force to determine protein folding and stability, many studies today show that surface charge plays a key role determining protein structure and activity. This review aims to (a) highlight the value of surface charged engineering of proteins to improve enzyme stability and activity in aqueous media and in the presence of ionic liquids (ILs) and organic solvents, (b) describe the existing approaches (genetic engineering or chemical modifications) for surface charged engineering, and (c) demonstrate the applicability of these surface charged enzymes in biocatalysis. The review provides a new foundation for the scientific and research community to exploit the surface engineering of protein concept for the development of new enzymes that are more active and stable in the presence of ILs and organic solvents, thereby offering new opportunities for industrial biocatalysis. Furthermore, this review is a useful tool for researchers to decide the best available technology to improve their enzyme system/ process.

show abstract

“…Protein engineering, including directed evolution and rational design, is an effective technology for improving enzyme properties [9]. Directed evolution is the construction of a mutant library by random mutagenesis, and the mutants are then screened according to the target performance.…”

Section: Introductionmentioning

confidence: 99%

Rational Design of Alginate Lyase from Microbulbifer sp. Q7 to Improve Thermal Stability

Yang

Liu

et al. 2019

Marine Drugs

View full text Add to dashboard Cite

Alginate lyase degrades alginate by the β-elimination mechanism to produce oligosaccharides with special bioactivities. The low thermal stability of alginate lyase limits its industrial application. In this study, introducing the disulfide bonds while using the rational design methodology enhanced the thermal stability of alginate lyase cAlyM from Microbulbifer sp. Q7. Enzyme catalytic sites, secondary structure, spatial configuration, and molecular dynamic simulation were comprehensively analyzed. When compared with cAlyM, the mutants D102C-A300C and G103C-T113C showed an increase by 2.25 and 1.16 h, respectively, in half-life time at 45 °C, in addition to increases by 1.7 °C and 0.4 °C in the melting temperature, respectively. The enzyme-specific activity and kcat/Km values of D102C-A300C were 1.8- and 1.5-times higher than those of cAlyM, respectively. The rational design strategy that was used in this study provides a valuable method for improving the thermal stability of the alginate lyase.

show abstract

Improvement of the acid stability of Bacillus licheniformis alpha amylase by site-directed mutagenesis

Cited by 31 publications

References 30 publications

Native to designed: microbial α-amylases for industrial applications

Native to designed: microbial α-amylases for industrial applications

Genetic and chemical approaches for surface charge engineering of enzymes and their applicability in biocatalysis: A review

Rational Design of Alginate Lyase from Microbulbifer sp. Q7 to Improve Thermal Stability

Contact Info

Product

Resources

About