Clostridium thermocellum Nitrilase Expression and Surface Display on Bacillus subtilis Spores

2015 ◽  
Vol 25 (6) ◽  
pp. 381-387 ◽  
Author(s):  
Huayou Chen ◽  
Tianxi Zhang ◽  
Tengyun Sun ◽  
Zhong Ni ◽  
Yilin Le ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Clostridium Thermocellum ◽  
Surface Display ◽  
Effective Means ◽  
Free Enzyme ◽  
Reaction Conditions ◽  
Sds Page ◽  
Adverse Environmental Conditions ◽  
Anchoring Motif ◽  
Spore Coat Protein

Nitrilases are an important class of industrial enzymes. They require mild reaction conditions and are highly efficient and environmentally friendly, so they are used to catalyze the synthesis of carboxylic acid from nitrile, a process considered superior to conventional chemical syntheses. Nitrilases should be immobilized to overcome difficulties in recovery after the reaction and to stabilize the free enzyme. The nitrilase from<i> Clostridium thermocellum</i> was expressed, identified and displayed on the surface of <i>Bacillus subtilis </i>spores by using the spore coat protein G of <i>B. subtilis </i>as an anchoring motif. In a free state, the recombinant nitrilase catalyzed the conversion of 3-cyanopyridine to niacin and displayed maximum catalytic activity (8.22 units/mg protein) at 40°C and pH 7.4. SDS-PAGE and Western blot were used to confirm nitrilase display. Compared with the free enzyme, the spore-immobilized nitrilase showed a higher tolerance for adverse environmental conditions. After the reaction, recombinant spores were recovered via centrifugation and reused 3 times to catalyze the conversion of 3-cyanopyridine with 75.3% nitrilase activity. This study demonstrates an effective means of nitrilase immobilization via spore surface display, which can be applied in biological processes or conversion.

scholarly journals Production of endoglucanase A of Clostridium thermocellum in Bacillus subtilis

2014 ◽  
Vol 17 (4) ◽  
pp. 74-82
Author(s):  
Thang Luong Pham ◽  
Trang Thi Phuong Phan ◽  
Thuoc Linh Tran ◽  
Hoang Duc Nguyen
Keyword(s):  
Bacillus Subtilis ◽  
Catalytic Activity ◽  
Clostridium Thermocellum ◽  
Anaerobic Bacterium ◽  
Slow Growth ◽  
Bacterial Strains ◽  
Industrial Enzymes ◽  
Glucanase Activity ◽  
C Terminus ◽  
Sds Page

Endoglucanase A (CelA) is a component of thermostable complex - cellulosome which is produced by anaerobic bacterium Clostridium thermocellum. In this complex, CelA has the highest catalytic activity and it can cleave randomly β-1,4 glucosidic linkage of cellulose to smaller oligosaccharides. Bacillus subtilis a Gram positive and aerobic fast growth bacterium, is commonly used to produce industrial enzymes. This bacterium can be used to replace the anaerobic slow growth bacterium, C. thermocellum, to produce CelA. To create B. subtilis strains which can secret recombinant CelA enzyme, celA gene was amplified using C. thermocellum DNA genome by PCR and inserted in the C-terminus of signal peptide of amyQ (samyQ) in plasmid pHT43. The plasmid pHT43-celA was then transformed into B. subtilis 1012 and WB800N, an extracellular protease deficiency strain. Next, CelA production was induced by IPTG at different concentrations. Cultural supernatant was collected from 2 to 24 hours after addition of IPTG. The expression levels were evaluated by SDS-PAGE and endo-β-1,4- glucanase activity. This report demonstrates that CelA can be expressed in B. subtilis and its potentials for development of bacterial strains which can produce CelA to hydrolyze cellulose in the future.

scholarly journals Localization of the Transglutaminase Cross-Linking Sites in the Bacillus subtilis Spore Coat Protein GerQ

2006 ◽  
Vol 188 (21) ◽  
pp. 7609-7616 ◽  
Author(s):  
Alicia Monroe ◽  
Peter Setlow
Keyword(s):  
Bacillus Subtilis ◽  
Coat Protein ◽  
Cross Linking ◽  
Spore Coat ◽  
Bacillus Subtilis Spore ◽  
Binding Partners ◽  
Lysine Residues ◽  
Cross Links ◽  
Hydrolysis Of ◽  
Spore Coat Protein

ABSTRACT The Bacillus subtilis spore coat protein GerQ is necessary for the proper localization of CwlJ, an enzyme important in the hydrolysis of the peptidoglycan cortex during spore germination. GerQ is cross-linked into high-molecular-mass complexes in the spore coat late in sporulation, and this cross-linking is largely due to a transglutaminase. This enzyme forms an ε-(γ-glutamyl) lysine isopeptide bond between a lysine donor from one protein and a glutamine acceptor from another protein. In the current work, we have identified the residues in GerQ that are essential for transglutaminase-mediated cross-linking. We show that GerQ is a lysine donor and that any one of three lysine residues near the amino terminus of the protein (K2, K4, or K5) is necessary to form cross-links with binding partners in the spore coat. This leads to the conclusion that all Tgl-dependent GerQ cross-linking takes place via these three lysine residues. However, while the presence of any of these three lysine residues is essential for GerQ cross-linking, they are not essential for the function of GerQ in CwlJ localization.

Expression, Localization and Modification of YxeE Spore Coat Protein in Bacillus subtilis

2007 ◽  
Vol 142 (6) ◽  
pp. 681-689 ◽  
Author(s):  
R. Kuwana ◽  
H. Takamatsu ◽  
K. Watabe
Keyword(s):  
Bacillus Subtilis ◽  
Coat Protein ◽  
Spore Coat ◽  
Spore Coat Protein

scholarly journals Cloning and Over-expression of xynB Gene of Bacillus subtilis subsp. spizizenii W23 into Escherichia coli Origami Host Cells

2017 ◽  
Vol 3 (5) ◽  
pp. 139
Author(s):  
Mariana Wahjudi ◽  
Catherina . ◽  
Nita Marcelia Wangunhardjo ◽  
Ernest Suryadjaja ◽  
Xavier Daniel
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Recombinant Plasmid ◽  
Sodium Dodecyl ◽  
Xylanase Activity ◽  
Cellular Protein ◽  
Host Cells ◽  
Chromosomal Dna ◽  
Clear Zone ◽  
Sds Page

<p class="Els-Abstract-text">The <em>xyn</em>B gene of <em>Bacillus</em><em> subtilis</em> subsp. spizizenii W23 is predicted to encode a xylan 1,4-beta-xylosidase. Application of XynB enzymes in industries is wide. Production of this enzyme in its host cells is naturally restricted by repression process. It will give certain beneficial to over-expressed the enzymes in other host-cells under inducing promoter. This study aimed to clone the <em>xyn</em>B gene from <em>Bacillus</em><em> subtilis</em> subsp. spizizenii W23, to pMMB67EH plasmid, and to over-express the <em>xyn</em>B gene in <em>Escherichia coli </em>Origami as host cells. The <em>x</em><em>yn</em>B gene was successfully amplified by polymerase chain reaction (PCR) technique using a pair of primers flanking the gene sequence and chromosomal DNA of the W23 strain as a template. The <em>xyn</em>B gene inserted in recombinant plasmid was confirmed by PCR detection using primers pair’s specific for <em>xyn</em>B gene and for the vector, then continued by restriction analyses.  The result showed that transformants clone 9 and 10 bear the recombinant pMMB-<em>xyn</em>B plasmid. The xylanase activity of <em>xyn</em>B gene in <em>Escherichia coli</em> Origami clone 10 was detected by sodium-dodecyl-sulfate polyacrylamide gel analyses and with addition of isopropyl-β-D-thio-galactoside (IPTG) as an inducer. The protein seem to be over-expressed as intra- and extra-cellular protein detected on SDS-PAGE gel. Result from xylan degrading activity on Luria-Bertani-xylan-IPTG plate with addition of Congo Red, showed that the cells with pMMB-<em>xyn</em>B recombinant plasmid have clear zone around the colonies while the transformant bearing an empty plasmid showed no clear zone. It could be concluded that the <em>xyn</em>B gene of <em>Bacillus subtilis</em> subsp.spizizenii W23 has been successfully been cloned on pMMB67EH plasmid and over-expressed in the <em>Escherichia coli</em> Origami cells as intra- and extra-cellular protein, as observed on SDS-PAGE gel analysis. The protein has activity on xylan degradation.</p>

Development of a novel bacterial surface display system using truncated OmpT as an anchoring motif

2019 ◽  
Vol 41 (6-7) ◽  
pp. 763-777 ◽  
Author(s):  
Chang-ye Hui ◽  
Yan Guo ◽  
Lisa Liu ◽  
Hao-qu Zheng ◽  
Hong-min Wu ◽  
...  
Keyword(s):  
Surface Display ◽  
Display System ◽  
Bacterial Surface ◽  
Bacterial Surface Display ◽  
Anchoring Motif
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