Production of a Thermostable Pullulanase in <i>Bacillus subtilis</i> by Optimization of the Expression Elements

Pang, Bo; Zhou, Li; Cui, Wenjing; Liu, Zhongmei; Zhou, Zhemin

doi:10.1002/star.202000018

Cited by 5 publications

(3 citation statements)

References 46 publications

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“…The ribosome binding site (RBS) and spacer between the RBS and the start codon were also components that allowed gene expression to be regulated at the translational level [ 17 , 18 , 19 , 20 ]. Pang et al, for example, used a mix of promoter and RBS engineering methods to greatly improve pullulanase yield [ 21 ]. Previously, a type I L-asparaginase from Bacillus licheniformis Z-1 (BlAase) with good enzymatic characteristics was successfully produced and secreted in B. subtilis [ 22 ].…”

Section: Introductionmentioning

confidence: 99%

Cis-Element Engineering Promotes the Expression of Bacillus subtilis Type I L-Asparaginase and Its Application in Food

Niu

Yan

Shen

et al. 2022

IJMS

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Type I L-asparaginase from Bacillus licheniformis Z-1 (BlAase) was efficiently produced and secreted in Bacillus subtilis RIK 1285, but its low yield made it unsuitable for industrial use. Thus, a combined method was used in this study to boost BlAase synthesis in B. subtilis. First, fifteen single strong promoters were chosen to replace the original promoter P43, with PyvyD achieving the greatest BlAase activity (436.28 U/mL). Second, dual-promoter systems were built using four promoters (PyvyD, P43, PaprE, and PspoVG) with relatively high BlAase expression levels to boost BlAase output, with the engine of promoter PaprE-PyvyD reaching 502.11 U/mL. The activity of BlAase was also increased (568.59 U/mL) by modifying key portions of the PaprE-PyvyD promoter. Third, when the ribosome binding site (RBS) sequence of promoter PyvyD was replaced, BlAase activity reached 790.1 U/mL, which was 2.27 times greater than the original promoter P43 strain. After 36 h of cultivation, the BlAase expression level in a 10 L fermenter reached 2163.09 U/mL, which was 6.2 times greater than the initial strain using promoter P43. Moreover, the application potential of BlAase on acrylamide migration in potato chips was evaluated. Results showed that 89.50% of acrylamide in fried potato chips could be removed when combined with blanching and BlAase treatment. These findings revealed that combining transcription and translation techniques are effective strategies to boost recombinant protein output, and BlAase can be a great candidate for controlling acrylamide in food processing.

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Section: Introductionmentioning

confidence: 99%

Cis-Element Engineering Promotes the Expression of Bacillus subtilis Type I L-Asparaginase and Its Application in Food

Niu

Yan

Shen

et al. 2022

IJMS

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“…Simple RBS modification proved spectacularly successful in the case of the dual promoter P hpalI -P ylb -more than 130 times higher activity than the wild type in shake-flasks and another 1.7 times increase in the 5 L fermenter. Three vectors (pP43NMK, pMA0911, pSTOP1622), four singles (P ylb , P BH4 , P 43 , P hpaII ), seven dual promoters, and seven RBS modifications were investigated in that study [129]. Simple overexpression of 2,3-butanediol dehydrogenase (BDH, encoded by bdhA) under the control of the dual promoter P 43 -P laps in B. subtilis BS2, aided by optimization of the metabolic conditions, led to 36.4% more butanediol, 36.7% more acetoin, and 95.5% more TTMP (Tetramethylpyrazine) compared to P laps or P 43 alone [85].…”

Section: Fusion Promotersmentioning

confidence: 99%

Cloning Systems in Bacillus: Bioengineering of Metabolic Pathways for Valuable Recombinant Products

Arsov,

Armenova,

Gergov

et al. 2024

Fermentation

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Representatives of the genus Bacillus have been established as one of the most important industrial microorganisms in the last few decades. Genetically modified B. subtilis and, to a lesser extent, B. licheniformis, B. amyloliquefaciens, and B. megaterium have been used for the heterologous expression of numerous proteins (enzymes, vaccine components, growth factors), platform chemicals, and other organic compounds of industrial importance. Vectors designed to work in Bacillus spp. have dramatically increased in number and complexity. Today, they provide opportunities for genetic manipulation on every level, from point mutations to systems biology, that were impossible even ten years ago. The present review aims to describe concisely the latest developments in the shuttle, integrative, and CRISPR-Cas9 vectors in Bacillus spp. as well as their application for large-scale bioengineering with the prospect of producing valuable compounds on an industrial scale. Genetic manipulations of promoters and vectors, together with their impact on secretory and metabolic pathways, are discussed in detail.

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“…[3] These enzymes are mainly derived from plants, microorganisms, and animals and are widely used in different industries such as food, starch-based applications, bakery, textile, paper, detergent, and pharmaceutical. [4,5] Several strategies for improving enzymatic stability and activity achieve economic, industrial processes, such as enzyme immobilization, using compatible solutes, and enzyme cocktail development. [6][7][8] Enzyme combination increases the enzymatic activity and significantly improves bread quality due to the better conversion of polysaccharides to smaller sugars due to enzymes synergistic effect.…”

Section: Introductionmentioning

confidence: 99%

Synergistic Effect of Metagenome‐Derived Starch‐Degrading Enzymes on Quality of Functional Bread with Antioxidant Activity

et al. 2021

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Starch-degrading enzymes have gained particular importance in recent decades due to their crucial role in numerous industrial applications especially, in the bakery industry. In this study, a novel thermostable pullulanase (PersiPul1) is screened from the metagenomic data for improving the quality of functional bread. The novel PersiPul1 is active over a wide range of temperature (30-80 °C) and pH (4-9) and exhibits high thermal stability, retaining 58.70% of activity at 80 °C. A cocktail of thermostable pullulanase and 𝜶-amylase (PersiPul1and PersiAmy2) is developed to be used in bread fortified with quinoa protein (Chenopodium quinoa Willd). The bread fortified with 7% quinoa protein isolate possess the highest reducing power and ABTS (73.64%) and DPPH (72.27%) radical scavenging activities. The addition of the enzyme mixture decreases the hardness and chewiness of the bread. The porosity, specific volume, and browning index of bread with pullulanase and 𝜶-amylase are higher than control. Also, sensory analysis of the functional bread reveals higher scores in the presence of enzymes. Based on the results, the novel starch-degrading enzyme cocktail is a promising alternative for improving the physical and sensory characteristics of the antioxidant bread enriched with quinoa protein isolate to be used in the bakery industry as a potential bio-additive.

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Production of a Thermostable Pullulanase in Bacillus subtilis by Optimization of the Expression Elements

Cited by 5 publications

References 46 publications

Cis-Element Engineering Promotes the Expression of Bacillus subtilis Type I L-Asparaginase and Its Application in Food

Cis-Element Engineering Promotes the Expression of Bacillus subtilis Type I L-Asparaginase and Its Application in Food

Cloning Systems in Bacillus: Bioengineering of Metabolic Pathways for Valuable Recombinant Products

Synergistic Effect of Metagenome‐Derived Starch‐Degrading Enzymes on Quality of Functional Bread with Antioxidant Activity

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