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Cited by 36Enzyme Production and CharacterizationMicrobial Metabolic Engineering and BioproductionFungal and yeast genetics research

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

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Ma, Yingfang, Shen, Wei, Chen, Xianzhong, Liu, Long, Zhou, Zhemin, Xu, Fei, Yang, Haiquan·2016·Journal of Biological Engineering
DOI 10.1186/s13036-016-0035-2PMC PMC5067897
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Abstract

Alkaline 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. The 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#. The 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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Fig. 1 Novel ARTP and HTS operational procedures for the mutation and screening of B. subtilis
Fig. 1 Novel ARTP and HTS operational procedures for the mutation and screening of B. subtilisCC BY · PubMed Central
Fig. 2 Comparison of B. subtilis mutants with the wild type strain for alkaline amylase production and strain growth. a alkaline amylase production. The inner is SDS-PAGE analysis. Arrow: alkaline amylase, 1: Wild type B. subtilis 168, 2: B. subtilis 168 mut-16#, M: standard protein marker. b specific production rate. c DCW. d specific growth rate
Fig. 2 Comparison of B. subtilis mutants with the wild type strain for alkaline amylase production and strain growth. a alkaline amylase production. The inner is SDS-PAGE analysis. Arrow: alkaline amylase, 1: Wild type B. subtilis 168, 2: B. subtilis 168 mut-16#, M: standard protein marker. b specific production rate. c DCW. d specific growth rateCC BY · PubMed Central
Fig. 3 Effect of agitation speed on alkaline amylase production and growth of B. subtilis. a alkaline amylase production. b specific production rate. c DCW. d specific growth rate
Fig. 3 Effect of agitation speed on alkaline amylase production and growth of B. subtilis. a alkaline amylase production. b specific production rate. c DCW. d specific growth rateCC BY · PubMed Central
Fig. 4 Effect of different soluble starch/soy peptone (SSSP) concentrations on alkaline amylase production and growth of B. subtilis. a alkaline amylase production. b specific production rate. c DCW. d specific production rate. Soluble starch and soy peptone ratios were consistent
Fig. 4 Effect of different soluble starch/soy peptone (SSSP) concentrations on alkaline amylase production and growth of B. subtilis. a alkaline amylase production. b specific production rate. c DCW. d specific production rate. Soluble starch and soy peptone ratios were consistentCC BY · PubMed Central
Fig. 5 Effect of different feeding compositions on alkaline amylase production and growth of B. subtilis. a Effect of feeding hydrolyzed starch on alkaline amylase production and growth of B. subtilis. The inner: pH change curve during fermentation. b Effect of feeding a combination of hydrolyzed starch and soy peptone on alkaline amylase production and growth of B. subtilis
Fig. 5 Effect of different feeding compositions on alkaline amylase production and growth of B. subtilis. a Effect of feeding hydrolyzed starch on alkaline amylase production and growth of B. subtilis. The inner: pH change curve during fermentation. b Effect of feeding a combination of hydrolyzed starch and soy peptone on alkaline amylase production and growth of B. subtilisCC BY · PubMed Central
Fig. 6 Effect of different feeding methods on alkaline amylase production and growth of B. subtilis. a Effect of fed-batch with single feeding on alkaline amylase production and growth of B. subtilis. b Effect of fed-batch with continuous feeding on alkaline amylase production and growth of B. subtilis
Fig. 6 Effect of different feeding methods on alkaline amylase production and growth of B. subtilis. a Effect of fed-batch with single feeding on alkaline amylase production and growth of B. subtilis. b Effect of fed-batch with continuous feeding on alkaline amylase production and growth of B. subtilisCC BY · PubMed Central
Fig. 7 Effect of different feeding times on alkaline amylase production and growth of B. subtilis. Effect of feeding at (a) 8 h and (b) 12 h on alkaline amylase production and growth of B. subtilis
Fig. 7 Effect of different feeding times on alkaline amylase production and growth of B. subtilis. Effect of feeding at (a) 8 h and (b) 12 h on alkaline amylase production and growth of B. subtilisCC BY · PubMed Central

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Research works printed but not linked: 32

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Cited by

Metadata

Authors
Ma, Y., Shen, W., Chen, X., Liu, L., Zhou, Z., Xu, F., & Yang, H.
Affiliations
Jiangnan University (CN)
Corresponding author
Yang, Haiquan
ORCID
Venue
Journal of Biological Engineering
Publisher
Springer Science and Business Media LLC
Published
2016-10-17
Volume
10
Issue
1
Pages
13-13
ISSN
1754-1611 (ISSN-L)
DOI
10.1186/s13036-016-0035-2
PMC
PMC5067897
OpenAlex
W2533170373
Cited by
36
References
1
Citations by year
2025: 3 · 2024: 1 · 2023: 1 · 2022: 6 · 2021: 4 · 2020: 6 · 2019: 7 · 2018: 5 · 2017: 3
FWCI
4.37
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • Journal of Biological Engineering (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
  • 863 Program (2014AA021304)
  • Natural Science Foundation of Jiangsu Province (BK20140152)
  • National Natural Science Foundation of China (CN) (21406089)
  • the Open Project Program of the Key Laboratory of Industrial Biotechnology, Ministry of Education, China (KLIB-KF201509)
  • 111 Project (111-2-06)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biotechnology › Enzyme Production and Characterization
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Fungal and yeast genetics research
Keywords
Bacillus subtilis, recombinant protein expression, fermentation optimization, soy peptone, stirring speed, enzyme production, microbial cell factories

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