scholarly journals Heterologous production of active form of beta-lytic protease by Bacillus subtilis and improvement of staphylolytic activity by protein engineering

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Takahiro Hioki ◽  
Daichi Yamashita ◽  
Masatoshi Tohata ◽  
Keiji Endo ◽  
Akihito Kawahara ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Bacillus Subtilis ◽  
Protein Engineering ◽  
Expression System ◽  
Active Form ◽  
Saturation Mutagenesis ◽  
Heterologous Production ◽  
Heterologous Proteins ◽  
Extracellular Proteases ◽  
Secretory Production

Abstract Background Most of the proteases classified into the M23 family in the MEROPS database exhibit staphylolytic activity and have potential as antibacterial agents. The M23 family is further classified into two subfamilies, M23A and M23B. Proteases of the M23A subfamily are thought to lack the capacity for self-maturation by auto-processing of a propeptide, which has been a challenge in heterologous production and application research. In this study, we investigated the heterologous expression, in Bacillus subtilis, of the Lysobacter enzymogenes beta-lytic protease (BLP), a member of the M23A subfamily. Results We found that B. subtilis can produce BLP in its active form. Two points were shown to be important for the production of BLP in B. subtilis. The first was that the extracellular proteases produced by the B. subtilis host are essential for BLP maturation. When the host strain was deficient in nine extracellular proteases, pro-BLP accumulated in the supernatant. This observation suggested that BLP lacks the capacity for self-maturation and that some protease from B. subtilis contributes to the cleavage of the propeptide of BLP. The second point was that the thiol-disulfide oxidoreductases BdbDC of the B. subtilis host are required for efficient secretory production of BLP. We infer that intramolecular disulfide bonds play an important role in the formation of the correct BLP conformation during secretion. We also achieved efficient protein engineering of BLP by utilizing the secretory expression system in B. subtilis. Saturation mutagenesis of Gln116 resulted in a Q116H mutant with enhanced staphylolytic activity. The minimum bactericidal concentration (MBC) of the wild-type BLP and the Q116H mutant against Staphylococcus aureus NCTC8325 was 0.75 μg/mL and 0.375 μg/mL, respectively, and the MBC against Staphylococcus aureus ATCC43300 was 6 μg/mL and 3 μg/mL, respectively. Conclusions In this study, we succeeded in the secretory production of BLP in B. subtilis. To our knowledge, this work is the first report of the successful heterologous production of BLP in its active form, which opens up the possibility of industrial use of BLP. In addition, this study proposes a new strategy of using the extracellular proteases of B. subtilis for the maturation of heterologous proteins.

scholarly journals Novel Expression Vector for Secretion of Cecropin AD in Bacillus subtilis with Enhanced Antimicrobial Activity

2009 ◽  
Vol 53 (9) ◽  
pp. 3683-3689 ◽  
Author(s):  
Xiang Chen ◽  
Faming Zhu ◽  
Yunhe Cao ◽  
Shiyan Qiao
Keyword(s):  
Staphylococcus Aureus ◽  
Antimicrobial Activity ◽  
Bacillus Subtilis ◽  
Signal Peptide ◽  
Expression System ◽  
Proteinase K ◽  
Farm Animals ◽  
Commercial Application ◽  
Protease Gene ◽  
Recombinant Product

ABSTRACT Cecropin AD, a chimeric antimicrobial peptide obtained from cecropins, is effective at killing specific microorganisms. However, a highly efficient expression system is still needed to allow for commercial application of cecropin AD. For the exogenous expression of cecropin AD, we fused the cecropin AD gene with a small ubiquitin-like modifier (SUMO) gene and a signal peptide of SacB, while a Bacillus subtilis expression system was constructed based on Bacillus subtilis cells genetically modified by the introduction of an operon including an isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible Spac promoter, a signal peptide of amyQ, and the SUMO protease gene. The recombinant cecropin AD was expressed, and 30.6 mg of pure recombinant protein was purified from 1 liter of culture supernatant. The purified cecropin AD displayed antimicrobial activity against some pathogens, such as Staphylococcus aureus and Escherichia coli, and was especially effective toward Staphylococcus aureus, with MICs of <0.05 μM (0.2 μg/ml). Stability analysis results showed that the activity of cecropin AD was not influenced by temperatures as high as 55°C for 20 min; however, temperatures above 85°C (for 20 min) decreased the antimicrobial activity of cecropin AD. Varying the pH from 4.0 to 9.0 did not appear to affect the activity of cecropin AD, but some loss of potency was observed at pH values lower than pH 4.0. Under the challenge of several proteases (proteinase K, trypsin, and pepsin), cecropin AD maintained functional activity. The results indicated that the recombinant product expressed by the designed Bacillus subtilis expression system was a potent antimicrobial agent and could be applied to control infectious diseases of farm animals or even humans.

scholarly journals Improved System for Protein Engineering of the Hydroxylase Component of Soluble Methane Monooxygenase

2002 ◽  
Vol 68 (11) ◽  
pp. 5265-5273 ◽  
Author(s):  
Thomas J. Smith ◽  
Susan E. Slade ◽  
Nicolas P. Burton ◽  
J. Colin Murrell ◽  
Howard Dalton
Keyword(s):  
Protein Engineering ◽  
Active Site ◽  
Methane Monooxygenase ◽  
Expression System ◽  
Active Form ◽  
Wild Type ◽  
Active Site Residues ◽  
Α Subunit ◽  
Soluble Methane Monooxygenase ◽  
Type Activity

ABSTRACT Soluble methane monooxygenase (sMMO) of Methylosinus trichosporium OB3b is a three-component oxygenase that catalyses the O2- and NAD(P)H-dependent oxygenation of methane and numerous other substrates. Despite substantial interest in the use of genetic techniques to study the mechanism of sMMO and manipulate its substrate specificity, directed mutagenesis of active-site residues was previously impossible because no suitable heterologous expression system had been found for expression in a highly active form of the hydroxylase component, which is an (αβγ)2 complex containing the binuclear iron active site. A homologous expression system that enabled the expression of recombinant wild-type sMMO in a derivative of M. trichosporium OB3b from which the chromosomal copy of the sMMO-encoding operon had been partially deleted was previously reported. Here we report substantial development of this method to produce a system for the facile construction and expression of mutants of the hydroxylase component of sMMO. This new system has been used to investigate the functions of Cys 151 and Thr 213 of the α subunit, which are the only nonligating protonated side chains in the hydrophobic active site. Both residues were found to be critical for the stability and/or activity of sMMO, but neither was essential for oxygenation reactions. The T213S mutant was purified to >98% homogeneity. It had the same iron content as the wild type and had 72% wild-type activity toward toluene but only 17% wild-type activity toward propene; thus, its substrate profile was significantly altered. With these results, we have demonstrated proof of the principle for protein engineering of this uniquely versatile enzyme.

Cell-associated degradation affects the yield of secreted engineered and heterologous proteins in the Bacillus subtilis expression system

Microbiology ◽  
2000 ◽  
Vol 146 (10) ◽  
pp. 2583-2594 ◽  
Author(s):  
Christina L. Jensen ◽  
Keith Stephenson ◽  
Steen T. Jørgensen ◽  
Colin Harwood
Keyword(s):  
Bacillus Subtilis ◽  
Expression System ◽  
Heterologous Proteins ◽  
Bacillus Subtilis Expression

scholarly journals Functional Implementation of the Posttranslational SecB-SecA Protein-Targeting Pathway in Bacillus subtilis

2011 ◽  
Vol 78 (3) ◽  
pp. 651-659 ◽  
Author(s):  
Liuyang Diao ◽  
Qilei Dong ◽  
Zhaohui Xu ◽  
Sheng Yang ◽  
Jiahai Zhou ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Protein Targeting ◽  
Heterologous Proteins ◽  
Gram Positive ◽  
Content Type ◽  
E Coli ◽  
New Strategy ◽  
Secretory Production ◽  
Close Relatives

ABSTRACTBacillus subtilisand its close relatives are widely used in industry for the Sec-dependent secretory production of proteins. Like other Gram-positive bacteria,B. subtilisdoes not possess SecB, a dedicated targeting chaperone that posttranslationally delivers exported proteins to the SecA component of the translocase. In the present study, we have implemented a functional SecB-dependent protein-targeting pathway intoB. subtilisby coexpressing SecB fromEscherichia colitogether with a SecA hybrid protein in which the carboxyl-terminal 32 amino acids of theB. subtilisSecA were replaced by the corresponding part of SecA fromE. coli.In vitropulldown experiments showed that, in contrast toB. subtilisSecA, the hybrid SecA protein gained the ability to efficiently bind toE. coliSecB, suggesting that the structural details of the extreme C-terminal region of SecA constitute a crucial SecB binding specificity determinant. Using a poorly exported mutant maltose binding protein (MalE11) and alkaline phosphatase (PhoA) as model proteins, we could demonstrate that the secretion of both proteins byB. subtiliswas significantly enhanced in the presence of the artificial protein targeting pathway. Mutations in SecB that do not influence its chaperone activity but prevent its interaction with SecA abolished the secretion stimulation of both proteins, demonstrating that the implemented pathway in fact critically depends on the SecB targeting function. From a biotechnological view, our results open up a new strategy for the improvement of Gram-positive bacterial host systems for the secretory production of heterologous proteins.

Influence of a Cell-Wall-Associated Protease on Production of α-Amylase by Bacillus subtilis

1998 ◽  
Vol 64 (8) ◽  
pp. 2875-2881 ◽  
Author(s):  
Keith Stephenson ◽  
Colin R. Harwood
Keyword(s):  
Cell Wall ◽  
Bacillus Subtilis ◽  
Bacillus Licheniformis ◽  
Potential Role ◽  
Heterologous Proteins ◽  
Extracellular Proteases ◽  
Serine Protease Activity ◽  
A Cell ◽  
Processed Products

ABSTRACT AmyL, an extracellular α-amylase from Bacillus licheniformis, is resistant to extracellular proteases secreted by Bacillus subtilis during growth. Nevertheless, when AmyL is produced and secreted by B. subtilis, it is subject to considerable cell-associated proteolysis. Cell-wall-bound proteins CWBP52 and CWBP23 are the processed products of the B. subtilis wprA gene. Although no activity has been ascribed to CWBP23, CWBP52 exhibits serine protease activity. Using a strain encoding an inducible wprA gene, we show that a product of wprA, most likely CWBP52, is involved in the posttranslocational stability of AmyL. A construct in whichwprA is not expressed exhibits an increased yield of α-amylase. The potential role of wprA in protein secretion is discussed, together with implications for the use ofB. subtilis and related bacteria as hosts for the secretion of heterologous proteins.

scholarly journals Hetero- and Autoprocessing of the Extracellular Metalloprotease (Mpr) in Bacillus subtilis

2004 ◽  
Vol 186 (19) ◽  
pp. 6457-6464 ◽  
Author(s):  
Chi Hye Park ◽  
Sang Jun Lee ◽  
Sung Gu Lee ◽  
Weon Sup Lee ◽  
Si Myung Byun
Keyword(s):  
Bacillus Subtilis ◽  
Glutamic Acid ◽  
Proteolytic Activity ◽  
Active Form ◽  
Single Amino Acid ◽  
Extracellular Proteases ◽  
Major Mechanism ◽  
Single Amino Acid Change

ABSTRACT Most proteases are synthesized as inactive precursors which are processed by proteolytic cleavage into a mature active form, allowing regulation of their proteolytic activity. The activation of the glutamic-acid-specific extracellular metalloprotease (Mpr) of Bacillus subtilis has been examined. Analysis of Mpr processing in defined protease-deficient mutants by activity assay and Western blotting revealed that the extracellular protease Bpr is required for Mpr processing. pro-Mpr remained a precursor form in bpr-deficient strains, and glutamic-acid-specific proteolytic activity conferred by Mpr was not activated in bpr-deficient strains. Further, purified pro-Mpr was processed to an active form by purified Bpr protease in vitro. We conclude that Mpr is activated by Bpr in vivo, and that heteroprocessing, rather than autoprocessing, is the major mechanism of Mpr processing in vivo. Exchange of glutamic acid for serine in the cleavage site of Mpr (S93E) allowed processing of Mpr into its mature form, regardless of the presence of other extracellular proteases, including Bpr. Thus, a single amino acid change is sufficient to convert the Mpr processing mechanism from heteroprocessing to autoprocessing.

The Study of Bacillus Subtils Antimicrobial Activity on Some of the Pathological Isolates

2019 ◽  
Vol 9 (02) ◽  
Author(s):  
Hussein A Kadhum ◽  
Thualfakar H Hasan2
Keyword(s):  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Antimicrobial Activity ◽  
Bacillus Subtilis ◽  
Bacterial Growth ◽  
Broad Spectrum ◽  
Inhibitory Activity ◽  
Bacterial Pathogens ◽  
Inhibitory Ability ◽  
Selection Of

The study involved the selection of two isolates from Bacillus subtilis to investigate their inhibitory activity against some bacterial pathogens. B sub-bacteria were found to have a broad spectrum against test bacteria such as Staphylococcus aureus and Pseudomonas aeruginosa. They were about 23-30 mm and less against Klebsiella sp. The sensitivity of some antibodies was tested on the test samples. The results showed that the inhibitory ability of bacterial growth in the test samples using B. subtilis extract was more effective than the antibiotics used.

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