Engineering, expression, purification, and production of recombinant thermolysin

Inouye, Kuniyo; Kusano, Masayuki; Hashida, Yasuhiko; Minoda, Masashi; Yasukawa, Kiyoshi

doi:10.1016/s1387-2656(07)13003-9

Cited by 35 publications

(27 citation statements)

References 64 publications

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“…The pre-proenzyme is processed into the proenzyme by the signal peptidase. The prosequence then acts as a molecular chaperone leading to an autocleavage of the peptide bond linking the pro and mature sequences (Inouye et al, 2007). The EOs acted at some point of this regulatory mechanism.…”

Section: Discussionmentioning

confidence: 99%

Inhibitory action of essential oils against proteases activity of Paenibacillus larvae, the etiological agent of American Foulbrood disease

Pellegrini

Zalazar

Fuselli

et al. 2018

Span. j. agric. res.

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American foulbrood (AFB) is a disease affecting the larva of Apis mellifera. The etiological agent is Paenibacillus larvae, which releases metalloproteases involved in the degradation of larval tissues. Through quorum sensing (QS) mechanism, bacteria are able to activate specific genes such as virulence factors. The exoproteases regulation of P. larvae could be associated with QS. A promising mechanism of AFB control is to block QS mechanism with essential oils (EO). The aim of this study was to investigate the potential presence of QS signals in the regulation of P. larvae proteases and the effect of seven EOs on the exoproteases activity of P. larvae. From growth curves and evaluation of the presence of proteases by milk agar plates assay, it was observed protease activity during the late exponential phase of growth. Early production of protease activity (15 hours earlier than control) was observed when a low density culture was incubated with late exponential spent medium (SM) suggesting the presence of factor(s) inducing this activity. SM was obtained by the ultrafiltration of P. larvae cultures on late growth phase and was free of proteases. Proteolytic activity was quantified on P. larvae cultures in presence of sublethal concentration of EO by azocasein method. The EOs, except S. chilensis EO, reduced significantly protease activity (more than 50%). We report for the first time evidence on the possible role of QS on P. larvae and the antiproteolytic activity of EOs (except for S. chilensis) on exoproteases, an interesting therapeutic strategy to control AFB.

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Section: Discussionmentioning

confidence: 99%

Inhibitory action of essential oils against proteases activity of Paenibacillus larvae, the etiological agent of American Foulbrood disease

Pellegrini

Zalazar

Fuselli

et al. 2018

Span. j. agric. res.

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“…Bacteria produce numerous proteases that disrupt normal human physiology and impair both innate and adaptive immunity (Häse and Finkelstein, 1993;Chung et al, 2006;Potempa and Pike, 2009;Cadot et al, 2010;Guillemet et al, 2010). Among these enzymes is thermolysin, a member of the M4 family of metallopeptidases that is a thermostable protein and requires one zinc ion for enzyme activity and four calcium ions for structural r Archives of Insect Biochemistry and Physiology, January 2015 stability (Inouye et al, 2007;Adekoya and Sylte, 2009). Members of this enzyme family are produced by bacteria and fungi that are pathogenic to animals (Häse and Finkelstein, 1993;Adekoya and Sylte, 2009;Vilcinskas, 2010).…”

Section: Introductionmentioning

confidence: 99%

Thermolysin Damages Animal Life Through Degradation of Plasma Proteins Enhanced by Rapid Cleavage of Serpins and Activation of Proteases

Kong

Lü

Guan

et al. 2014

Arch Insect Biochem Physiol

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Thermolysin, a metallopeptidase secreted by pathogenic microbes, is concluded as an important virulence factor due to cleaving purified host proteins in vitro. Using the silkworm Bombyx mori as a model system, we found that thermolysin injection into larvae induces the destruction of the coagulation response and the activation of hemolymph melanization, which results in larval death. Thermolysin triggers the rapid degradation of insect and mammalian plasma proteins at a level that is considerably greater than expected in vitro and/or in vivo. To more specifically explore the mechanism, thermolysin-induced changes to key proteins belonging to the insect melanization pathway were assessed as a window for observing plasma protein cleavage. The application of thermolysin induced the rapid cleavage of the melanization negative regulator serpin-3, but did not directly activate the melanization rate-limiting enzyme prophenoloxidase (PPO) or the terminal serine proteases responsible for PPO activation. Terminal serine proteases of melanization are activated indirectly after thermolysin exposure. We hypothesize that thermolysin induces the rapid degradation of serpins and the activation of proteases directly or indirectly, boosting uncontrolled plasma protein degradation in insects and mammalians.

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“…Most of the peptidases in the M4 family are bacterial extracellular metalloproteases [4]. The representative of the M4 family, thermolysin (EC 3.4.24.27) secreted by Bacillus thermoproteolyticus , has been studied in detail and extensively applied for the synthesis of the artificial sweetener aspartame [5]. Besides thermolysin, most of the M4 metalloproteases studied in detail are the virulence factors of some pathogens [6]–[8].…”

Section: Introductionmentioning

confidence: 99%

Structural and Functional Characterization of Mature Forms of Metalloprotease E495 from Arctic Sea-Ice Bacterium Pseudoalteromonas sp. SM495

Guo

Chen

et al. 2012

PLoS ONE

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E495 is the most abundant protease secreted by the Arctic sea-ice bacterium Pseudoalteromonas sp. SM495. As a thermolysin family metalloprotease, E495 was found to have multiple active forms in the culture of strain SM495. E495-M (containing only the catalytic domain) and E495-M-C1 (containing the catalytic domain and one PPC domain) were two stable mature forms, and E495-M-C1-C2 (containing the catalytic domain and two PPC domains) might be an intermediate. Compared to E495-M, E495-M-C1 had similar affinity and catalytic efficiency to oligopeptides, but higher affinity and catalytic efficiency to proteins. The PPC domains from E495 were expressed as GST-fused proteins. Both of the recombinant PPC domains were shown to have binding ability to proteins C-phycocyanin and casein, and domain PPC1 had higher affinity to C-phycocyanin than domain PPC2. These results indicated that the domain PPC1 in E495-M-C1 could be helpful in binding protein substrate, and therefore, improving the catalytic efficiency. Site-directed mutagenesis on the PPC domains showed that the conserved polar and aromatic residues, D26, D28, Y30, Y/W65, in the PPC domains played key roles in protein binding. Our study may shed light on the mechanism of organic nitrogen degradation in the Arctic sea ice.

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Engineering, expression, purification, and production of recombinant thermolysin

Cited by 35 publications

References 64 publications

Inhibitory action of essential oils against proteases activity of Paenibacillus larvae, the etiological agent of American Foulbrood disease

Inhibitory action of essential oils against proteases activity of Paenibacillus larvae, the etiological agent of American Foulbrood disease

Thermolysin Damages Animal Life Through Degradation of Plasma Proteins Enhanced by Rapid Cleavage of Serpins and Activation of Proteases

Structural and Functional Characterization of Mature Forms of Metalloprotease E495 from Arctic Sea-Ice Bacterium Pseudoalteromonas sp. SM495

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