Efficient secretory production of large‐size heterologous enzymes in Bacillus subtilis : A secretory partner and directed evolution

2020 ◽  
Vol 117 (10) ◽  
pp. 2957-2968
Author(s):  
Shan Liu ◽  
Juan Wang ◽  
Zhiguang Zhu ◽  
Ting Shi ◽  
Yi‐Heng P. Job Zhang
Keyword(s):  
Bacillus Subtilis ◽  
Directed Evolution ◽  
Large Size ◽  
Secretory Production

scholarly journals Directed evolution of lipase A from Bacillus subtilis for the preparation of enantiocomplementary sec-alcohols

2021 ◽  
Author(s):  
Danyang Li ◽  
Xiaoyang Chen ◽  
Zhichun Chen ◽  
Xianfu Lin ◽  
Jian Xu ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Directed Evolution ◽  
Lipase A

Improved catalytic efficiency of Endo-β-1,4-glucanase from Bacillus subtilis BME-15 by directed evolution

2009 ◽  
Vol 82 (4) ◽  
pp. 671-679 ◽  
Author(s):  
Ling Lin ◽  
Xin Meng ◽  
Pengfu Liu ◽  
Yuzhi Hong ◽  
Gaobing Wu ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Directed Evolution ◽  
Catalytic Efficiency

Directed Evolution of Bacillus subtilis Lipase A by Use of Enantiomeric Phosphonate Inhibitors: Crystal Structures and Phage Display Selection

ChemBioChem ◽  
2005 ◽  
Vol 7 (1) ◽  
pp. 149-157 ◽  
Author(s):  
Melloney J. Dröge ◽  
Ykelien L. Boersma ◽  
Gertie van Pouderoyen ◽  
Titia E. Vrenken ◽  
Carsten J. Rüggeberg ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Phage Display ◽  
Directed Evolution ◽  
Crystal Structures ◽  
Lipase A

scholarly journals Directed Evolution of an Enantioselective Bacillus subtilis Lipase

2003 ◽  
Vol 21 (2) ◽  
pp. 1-1
Author(s):  
Susanne Aileen Funke ◽  
Andreas Eipper
Keyword(s):  
Bacillus Subtilis ◽  
Directed Evolution

scholarly journals Enhanced Secretory Production of a Single-Chain Antibody Fragment from Bacillus subtilis by Coproduction of Molecular Chaperones

1998 ◽  
Vol 180 (11) ◽  
pp. 2830-2835 ◽  
Author(s):  
Sau-Ching Wu ◽  
Ruiqiong Ye ◽  
Xu-Chu Wu ◽  
Shi-Chung Ng ◽  
Sui-Lam Wong
Keyword(s):  
Bacillus Subtilis ◽  
Molecular Chaperones ◽  
Total Yield ◽  
Antibody Fragment ◽  
Biologically Active ◽  
Production Yield ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Enhanced Production ◽  
Secretory Production

ABSTRACT Formation of inclusion bodies is a major limiting factor for secretory production of an antidigoxin single-chain antibody (SCA) fragment from Bacillus subtilis. To address this problem, three new strains with enhanced production of molecular chaperones were constructed. WB600BHM constitutively produces the major intracellular molecular chaperones in an appropriate ratio without any heat shock treatment. This strain reduced the formation of insoluble SCA by 45% and increased the secretory production yield by 60%. The second strain, WB600B[pEPP], overproduces an extracytoplasmic molecular chaperone, PrsA. An increase in the total yield of SCA was observed. The third strain, WB600BHM[pEPP], coproduces both intracellular and extracytoplasmic molecular chaperones. This led to a further reduction in inclusion body formation and a 2.5-fold increase in the secretory production yield. SCA fragments secreted by this strain were biologically active and showed affinity to digoxin comparable to the affinity of those secreted by strains without overproduction of molecular chaperones. Interestingly, accumulation of a pool of periplasmic SCA was observed in the PrsA-overproducing strains. This pool is suggested to represent the secreted folding intermediates in the process of achieving their final configuration.

scholarly journals Secretory Expression Fine-Tuning and Directed Evolution of Diacetylchitobiose Deacetylase by Bacillus subtilis

2019 ◽  
Vol 85 (17) ◽  
Author(s):  
Zhu Jiang ◽  
Tengfei Niu ◽  
Xueqin Lv ◽  
Yanfeng Liu ◽  
Jianghua Li ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Catalytic Efficiency ◽  
Molecular Engineering ◽  
Fine Tuning ◽  
Wild Type ◽  
Content Type ◽  
Chitosan Oligosaccharides ◽  
In The Wild ◽  
Secretory Production ◽  
Extracellular Activity

ABSTRACT Diacetylchitobiose deacetylase has great application potential in the production of chitosan oligosaccharides and monosaccharides. This work aimed to achieve high-level secretory production of diacetylchitobiose deacetylase by Bacillus subtilis and perform molecular engineering to improve catalytic performance. First, we screened 12 signal peptides for diacetylchitobiose deacetylase secretion in B. subtilis, and the signal peptide YncM achieved the highest extracellular diacetylchitobiose deacetylase activity of 13.5 U/ml. Second, by replacing the HpaII promoter with a strong promoter, the P43 promoter, the activity was increased to 18.9 U/ml. An unexpected mutation occurred at the 5′ untranslated region of plasmid, and the extracellular activity reached 1,548.1 U/ml, which is 82 times higher than that of the original strain. Finally, site-directed saturation mutagenesis was performed for the molecular engineering of diacetylchitobiose deacetylase to further improve the catalytic efficiency. The extracellular activity of mutant diacetylchitobiose deacetylase R157T reached 2,042.8 U/ml in shake flasks. Mutant R157T exhibited much higher specific activity (3,112.2 U/mg) than the wild type (2,047.3 U/mg). The Km decreased from 7.04 mM in the wild type to 5.19 mM in the mutant R157T, and the Vmax increased from 5.11 μM s−1 in the wild type to 7.56 μM s−1 in the mutant R157T. IMPORTANCE We successfully achieved efficient secretory production and improved the catalytic efficiency of diacetylchitobiose deacetylase in Bacillus subtilis, and this provides a good foundation for the application of diacetylchitobiose deacetylase in the production of chitosan oligosaccharides and monosaccharides.

Iterative multitarget evolution dramatically enhances the enantioselectivity and catalytic efficiency of Bacillus subtilis esterase towards bulky benzoate esters of dl-menthol

2016 ◽  
Vol 6 (7) ◽  
pp. 2370-2376 ◽  
Author(s):  
Yi Gong ◽  
Guo-Chao Xu ◽  
Qi Chen ◽  
Jin-Gang Yin ◽  
Chun-Xiu Li ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Directed Evolution ◽  
Catalytic Efficiency

Structure-based directed evolution has been successfully applied to Bacillus subtilis esterase to produce a mutant with higher enantioselectivity and elevated efficiency.

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