Integrating Terminal Truncation and Oligopeptide Fusion for a Novel Protein Engineering Strategy To Improve Specific Activity and Catalytic Efficiency: Alkaline α-Amylase as a Case Study

Yang, Haiquan; Liu, Long; Shin, Hyun‐Dong; Chen, Rachel R.; Li, Jianghua; Du, Guocheng; Chen, Jian

doi:10.1128/aem.02087-13

Cited by 19 publications

(9 citation statements)

References 28 publications

(42 reference statements)

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“…[ 1 – 3 ]. Many alkaline amylases have been heterologously expressed in recombinant hosts to improve their yield and optimize their properties [ 4 , 5 ]. Murakami et al heterologously expressed alkaline amylase from B. halodurans MS-2-5 in recombinant Escherichia coli , and under optimized cultivation conditions, the amylase yield increased 104-fold compared with yield from the wild-type strain [ 4 ].…”

Section: Introductionmentioning

confidence: 99%

Significantly enhancing recombinant alkaline amylase production in Bacillus subtilis by integration of a novel mutagenesis-screening strategy with systems-level fermentation optimization

Shen

Chen

et al. 2016

J Biol Eng

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BackgroundAlkaline amylase has significant potential for applications in the textile, paper and detergent industries, however, low yield of which cannot meet the requirement of industrial application. In this work, a novel ARTP mutagenesis-screening method and fermentation optimization strategies were used to significantly improve the expression level of recombinant alkaline amylase in B. subtilis 168.ResultsThe activity of alkaline amylase in mutant B. subtilis 168 mut-16# strain was 1.34-fold greater than that in the wild-type, and the highest specific production rate was improved from 1.31 U/(mg·h) in the wild-type strain to 1.57 U/(mg·h) in the mutant strain. Meanwhile, the growth of B. subtilis was significantly enhanced by ARTP mutagenesis. When the agitation speed was 550 rpm, the highest activity of recombinant alkaline amylase was 1.16- and 1.25-fold of the activities at 450 and 650 rpm, respectively. When the concentration of soluble starch and soy peptone in the initial fermentation medium was doubled, alkaline amylase activity was increased 1.29-fold. Feeding hydrolyzed starch and soy peptone mixture or glucose significantly improved cell growth, but inhibited the alkaline amylase production in B. subtilis 168 mut-16#. The highest alkaline amylase activity by feeding hydrolyzed starch reached 591.4 U/mL, which was 1.51-fold the activity by feeding hydrolyzed starch and soy peptone mixture. Single pulse feeding-based batch feeding at 10 h favored the production of alkaline amylase in B. subtilis 168 mut-16#.ConclusionThe results indicated that this novel ARTP mutagenesis-screening method could significantly improve the yield of recombinant proteins in B. subtilis. Meanwhile, fermentation optimization strategies efficiently promoted expression of recombinant alkaline amylase in B. subtilis 168 mut-16#. These findings have great potential for facilitating the industrial-scale production of alkaline amylase and other enzymes, using B. subtilis cultures as microbial cell factories.

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Section: Introductionmentioning

confidence: 99%

Significantly enhancing recombinant alkaline amylase production in Bacillus subtilis by integration of a novel mutagenesis-screening strategy with systems-level fermentation optimization

Shen

Chen

et al. 2016

J Biol Eng

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“…Based on the results of kinetic analysis, PersiAmy1 showed higher V max than detergent-stable alkaline α-amylase from Bacillus showing V max of 2.342 μmol.ml –1 .min –1 ( Priyadarshini and Ray, 2019 ). Moreover, another cold-adapted α-amylase from Monascus sanguineus and an alkaline α-amylase from Alkalimonas amylolytica showed V max of 22.07 μmol.ml –1 .min –1 and 37.8 μmol.ml –1 .min –1 , respectively, when starch was used as substrate ( Yang et al, 2013 ; Tallapragada et al, 2017 ).…”

Section: Discussionmentioning

confidence: 99%

Highly Efficient Computationally Derived Novel Metagenome α-Amylase With Robust Stability Under Extreme Denaturing Conditions

et al. 2021

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α-Amylases are among the very critical enzymes used for different industrial purposes. Most α-amylases cannot accomplish the requirement of industrial conditions and easily lose their activity in harsh environments. In this study, a novel α-amylase named PersiAmy1 has been identified through the multistage in silico screening pipeline from the rumen metagenomic data. The long-term storage of PersiAmy1 in low and high temperatures demonstrated 82.13 and 71.01% activities after 36 days of incubation at 4 and 50°C, respectively. The stable α-amylase retained 61.09% of its activity after 180 min of incubation at 90°C and was highly stable in a broad pH range, showing 60.48 and 86.05% activities at pH 4.0 and pH 9.0 after 180 min of incubation, respectively. Also, the enzyme could resist the high-salinity condition and demonstrated 88.81% activity in the presence of 5 M NaCl. PersiAmy1 showed more than 74% activity in the presence of various metal ions. The addition of the detergents, surfactants, and organic solvents did not affect the α-amylase activity considerably. Substrate spectrum analysis showed that PersiAmy1 could act on a wide array of substrates. PersiAmy1 showed high stability in inhibitors and superb activity in downstream conditions, thus useful in detergent and baking industries. Investigating the applicability in detergent formulation, PersiAmy1 showed more than 69% activity after incubation with commercial detergents at different temperatures (30–50°C) and retained more than 56% activity after incubation with commercial detergents for 3 h at 10°C. Furthermore, the results of the wash performance analysis exhibited a good stain removal at 10°C. The power of PersiAmy1 in the bread industry revealed soft, chewable crumbs with improved volume and porosity compared with control. This study highlights the intense power of robust novel PersiAmy1 as a functional bio-additive in many industrial applications.

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“…Therefore, C-terminal modifications have been explored as a novel protein engineering strategy (Yang et al, 2013). Studies have found that partially truncating the C-terminal sequence in keratinase can improve catalytic efficiency, thermophilicity, salt tolerance, and detergent tolerance (Fang et al, 2016a).…”

Section: C-terminal Engineeringmentioning

confidence: 99%

The tale of a versatile enzyme: Molecular insights into keratinase for its industrial dissemination

Gong

Qin

et al. 2020

Biotechnology Advances

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Integrating Terminal Truncation and Oligopeptide Fusion for a Novel Protein Engineering Strategy To Improve Specific Activity and Catalytic Efficiency: Alkaline α-Amylase as a Case Study

Cited by 19 publications

References 28 publications

Significantly enhancing recombinant alkaline amylase production in Bacillus subtilis by integration of a novel mutagenesis-screening strategy with systems-level fermentation optimization

Significantly enhancing recombinant alkaline amylase production in Bacillus subtilis by integration of a novel mutagenesis-screening strategy with systems-level fermentation optimization

Highly Efficient Computationally Derived Novel Metagenome α-Amylase With Robust Stability Under Extreme Denaturing Conditions

The tale of a versatile enzyme: Molecular insights into keratinase for its industrial dissemination

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