Secretion of recombinant Bacillus hydrolytic enzymes using Escherichia coli expression systems

Yamabhai, Montarop; Emrat, Suphap; Sukasem, Sirima; Pesatcha, Puntarika; Jaruseranee, Nanthnit; Buranabanyat, Bancha

doi:10.1016/j.jbiotec.2007.09.005

Cited by 85 publications

(40 citation statements)

References 27 publications

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“…Our results showed that the mature cutinase was partially located in the periplasm, while some was secreted into the culture medium (14). Secretion into the culture medium was not unexpected, since similar phenomena have been observed in the expression of other secretory proteins (15)(16)(17)(18)(19). After optimization of the cultivation conditions in shake flasks, the cutinase activity in the culture medium reached 149.2 U/ml (326 mg/liter) 52 h postinduction (14).…”

mentioning

confidence: 65%

Extracellular Location of Thermobifida fusca Cutinase Expressed in Escherichia coli BL21(DE3) without Mediation of a Signal Peptide

Woodard

Chen

et al. 2013

Appl Environ Microbiol

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Cutinase is a multifunctional esterase with potential industrial applications. In the present study, a truncated version of the extracellular Thermobifida fusca cutinase without a signal peptide (referred to as cutinase NS ) was heterologously expressed in Escherichia coli BL21(DE3). The results showed that the majority of the cutinase activity was located in the culture medium. In a 3-liter fermentor, the cutinase activity in the culture medium reached 1,063.5 U/ml (2,380.8 mg/liter), and the productivity was 40.9 U/ml/h. Biochemical characterization of the purified cutinase NS showed that it has enzymatic properties similar to those of the wild-type enzyme. In addition, E. coli cells producing inactive cutinase NS S130A were constructed, and it was found that the majority of the inactive enzyme was located in the cytoplasm. Furthermore, T. fusca cutinase was confirmed to have hydrolytic activity toward phospholipids, an important component of the cell membrane. Compared to the cells expressing the inactive cutinase NS S130A, the cells expressing cutinase NS showed increased membrane permeability and irregular morphology. Based on these results, a hypothesis of "cell leakage induced by the limited phospholipid hydrolysis of cutinase NS " was proposed to explain the underlying mechanism for the extracellular release of cutinase NS . Cutinase not only catalyzes the cleavage of the ester bonds of cutin, but is also capable of hydrolyzing soluble esters, insoluble triglycerides, and a variety of polyesters (1). In addition to its hydrolytic capability, cutinase is also used in ester synthesis (2, 3) and transesterification (4). Therefore, as a multifunctional enzyme, cutinase has potential uses in the food, chemical, textile, and other industries (5).Enzymes with cutinase activity have been found in both fungi and bacteria, such as Fusarium solani pisi and Thermobifida fusca, respectively (5-7). It has been demonstrated that cutinases from both groups belong to the ␣/␤-hydrolase superfamily, share similar spatial structures, and display similar catalytic properties, as well as substrate specificities. In addition, consistent with their ability to accommodate large substrates like cutin, they contain an open active site, and the lid structure normally seen in most lipases is absent in cutinase. Despite these similarities, there does not appear to be significant sequence homology between fungal and bacterial cutinases; thus, it has been suggested that they be classified into prokaryotic and eukaryotic cutinase subfamilies, respectively (6).To date, all the studied cutinases from microorganisms have been found to be secretory enzymes (6). Therefore, for heterologous expression, signal peptides are usually utilized to mediate the secretion of recombinant cutinase. Using this approach, F. solani cutinase has been expressed in a variety of host cells, such as Escherichia coli (8), Aspergillus awamori (9), Fusarium venenatum (10), Saccharomyces cerevisiae (11, 12), and Pichia pastoris (13). The highest yield of extracellu...

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confidence: 65%

Extracellular Location of Thermobifida fusca Cutinase Expressed in Escherichia coli BL21(DE3) without Mediation of a Signal Peptide

Woodard

Chen

et al. 2013

Appl Environ Microbiol

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“…It has been verified that both E. coli OmpA and native signal peptide could be used to direct the secretion of Bacillus extracellular enzymes in E. coli expression system [9,10]. It is the first report that the signal peptide of chitosanase from Microbacterium sp.…”

Section: Discussionmentioning

confidence: 95%

“…EF159153) [7,8]. It has been verified both the E. coli OmpA and native signal peptide could be used to direct the secretion of Bacillus extracellular enzymes in E. coli expression systems [9,10]. A signal peptide was predicted by online software (SignalP 4.1), in the deduced amino acid sequence of mschito gene at the position 1-25.…”

Section: Introductionmentioning

confidence: 96%

Heterologous Expression and Efficient Secretion of Chitosanase from Microbacterium sp. in Escherichia coli

2014

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A recombinant expression vector, pCT7-CHISP6H, was constructed for the secretory expression of mature peptide of chitosanase (mMschito) from Microbacterium sp. OU01. The vector contains several elements, including T7 promoter, signal peptide sequence of mschito, 6 9 His-tag sequence and PmaCI restriction enzyme cloning site. In pCT7-CHISP6H, mMschito was fused into signal peptide sequence of mschito gene to construct recombinant plasmid pCT7-CHISP6H-mMschito. The recombinant plasmid was transformed into Escherichia coli BL21(DE3) and then expressed. The recombinant protein was secreted into the Luria-Bertani broth and the chitosanase activity in supernatant of the culture could reach up to 67.56 U/mL. The rmMschito in the broth supernatant was purified using HisTrap TM FF Crude column and the purified rmMschito was shown to be apparent homogeneity by 12 % SDS-PAGE analysis. Detected by 4700 MALDI-TOF-TOF-MS, the molecular weight of the purified rmMschito was 26,758.1875 and it was consistent with the predicted molecular weight. Chitosan (degree of deacetylation of 99 %) was mostly hydrolyzed into chitopentaose, chitotriose, and chitobiose by the purified rmMschito.

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“…The modified agar diffusion method was used to visualize SCChi-1 chitinase activity (18,19). First, the colloidal chitin, glycol chitin, glycol chitosan, and soluble chitosan plates were prepared.…”

Section: Substrate Specificity Of Scchi-1mentioning

confidence: 99%

Overexpression and characterization of thermostable chitinase from Bacillus atrophaeus SC081 in Escherichia coli

Cho

Choi²,

Choi

2011

BMB Reports

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The chitinase-producing strain SC081 was isolated from Korean traditional soy sauce and identified as Bacillus atrophaeus based on a phylogenetic analysis of the 16S rDNA sequence and a phenotypic analysis. A gene encoding chitinase from B. atrophaeus SC081 was cloned in Escherichia coli and was named SCChi-1 (GQ360078). The SCChi-1 nucleotide sequences were composed of 1788 base pairs and 596 amino acids, which were 92.6, 89.6, 89.3, and 78.9% identical to those of Bacillus subtilis (ABG57262), Bacillus pumilus (ABI15082), Bacillus amyloliquefaciens (ABO15008), and Bacillus licheniformis (ACF40833), respectively. A recombinant SCChi-1 containing a hexahistidine tag at the amino-terminus was constructed, overexpressed, and purified in E. coli to characterize SCChi-1. H6SCChi-1 revealed a hydrolytic band on zymograms containing 0.1% glycol chitin and showed the highest lytic activity on colloidal chitin and acidic chitosan. The optimal temperature and pH for chitinolytic activity were 50 o C and pH 8.0, respectively. [BMB reports 2011; 44(3) : 193-198]

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Secretion of recombinant Bacillus hydrolytic enzymes using Escherichia coli expression systems

Cited by 85 publications

References 27 publications

Extracellular Location of Thermobifida fusca Cutinase Expressed in Escherichia coli BL21(DE3) without Mediation of a Signal Peptide

Extracellular Location of Thermobifida fusca Cutinase Expressed in Escherichia coli BL21(DE3) without Mediation of a Signal Peptide

Heterologous Expression and Efficient Secretion of Chitosanase from Microbacterium sp. in Escherichia coli

Overexpression and characterization of thermostable chitinase from Bacillus atrophaeus SC081 in Escherichia coli

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