Separation, purification and properties of β-lactamase I and β-lactamase II from <i>Bacillus cereus</i> 569/H/9

Camelid-derived single domain VHH antibodies are highly heat resistant, and the mechanism of heatinduced VHH denaturation predominantly relies on the chemical modification of amino acids. Although chemical modification of disulfide bonds has been recognized as a cause for heat-induced denaturation of many proteins, there have been no mutagenesis studies, in which the number of disulfide bonds was controlled. In this article, we examined a series of mutants of two different VHHs with single, double or no disulfide bonds, and scrutinized the effects of these disulfide bond modifications on VHH denaturation. With the exception of one mutant, the heat resistance of VHHs decreased when the number of disulfide bonds increased. The effect of disulfide bonds on heat denaturation was more striking if the VHH had a second disulfide bond, suggesting that the contribution of disulfide shuffling is significant in proteins with multiple disulfide bonds. Furthermore, our results directly indicate that removal of a disulfide bond can indeed increase the heat resistance of a protein, irrespective of the negative impact on equilibrium thermodynamic stability.Keywords: antibody engineering/disulfide bonds/protein chemical modification/protein engineering/protein stability.Abbreviations: bLA, Bacillus cereus b lactamase; CD, circular dichroism; hCG, human chorionic gonadotropin; SPR, surface plasmon resonance; t 1/2 90 C , halflife of VHH at 90 C; T m , mid-point temperature of thermal unfolding; VHH, variable domain of camelid heavy chain antibody.The variable domain of the camelid heavy chain antibody (VHH) is known to be highly heat resistant.VHHs remain active even after incubation at high temperatures (>80 C) because of their ability to refold to their native structure from a heat-induced unfolded state (1 6). However, even for VHHs, a long incubation finally leads to denaturation: e.g. the half-life of VHH at 90 C (t 1/2 90 C ) was 3 h at neutral pH (7). Residual activity of VHH depends only on the incubation time at 90 C, and not on the number of heating (90 C)-cooling (20 C) cycles, indicating that folding and unfolding intermediates are irrelevant to the loss of activity. Heat denaturation of VHHs is independent of protein concentration; therefore, VHHs lose their activity in a unimolecular manner, not by aggregation. This strongly suggests that chemical modifications play an important role during VHH denaturation. Indeed, replacing the Asn residues, which are prone to chemical modification, increase the heat resistance of VHHs (7).VHHs typically have only one intramolecular disulfide bond. The introduction of a second non-canonical artificial disulfide bond connecting the amino acids at positions 49 and 69, with numbering according to Kabat et al. (8), results in a significantly higher midpoint temperature of equilibrium thermal unfolding (T m ) in comparison to wild-type protein (9 13). In spite of its superior equilibrium thermodynamic stability, the mutant with a second disulfide bond exhibited lower residual activity ...

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“…Purchased bLA was further purified by HiTrap SP HP (GE Healthcare) equilibrated by 10 mM Tris HCl (pH 7) and 1 mM ZnCl 2 (24).…”

Section: Methodsmentioning

confidence: 99%

The role of intra-domain disulfide bonds in heat-induced irreversible denaturation of camelid single domain VHH antibodies

2015

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“…Based on its kinetic parameters, ␤-lactamase BCL-1 belongs to group 2a of ␤-lactamases according to the BushMedeiros-Jacoby classification (7). IEF analysis showed that cultures of B. clausii NR gave multiple pIs ranging from 5.5 to 6, probably due to ragged amino termini, as shown for the ␤-lactamases produced by B. licheniformis 749/C (BL-␤L) (38) and B. cereus 569/H (␤-lactamase I) (13). IEF analysis of culture extracts of E. coli BL21(DE3)(pBCL-1) performed after 2 h of IPTG induction gave a major pI of 5.5.…”

Section: Resultsmentioning

confidence: 95%

“…Indeed, Colombo et al reported that YbxI of B. subtilis had a hydrolytic activity intermediate between those of penicillin-binding proteins and ␤-lactamases (12). Genomic analysis of B. clausii KSM-K16 showed that it does not seem to contain additional chromosomally encoded metallo-␤-lactamase in contrast to B. anthracis, which contains a gene encoding Bla2 (class B) in addition to a gene for Bla1 (class A) (9), or B. cereus, which contains a gene for BcII (class B) in addition to the BcI and BcIII (class A) genes (9,13).…”

Section: Resultsmentioning

confidence: 99%

Molecular and Biochemical Characterization of the Chromosome-Encoded Class A β-Lactamase BCL-1 from Bacillus clausii

Girlich

Leclercq

Naas

et al. 2007

Antimicrob Agents Chemother

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A chromosomal ␤-lactamase gene from Bacillus clausii NR, which is used as a probiotic, was cloned and expressed in Escherichia coli. It encodes a clavulanic acid-susceptible Ambler class A ␤-lactamase, BCL-1, with a pI of 5.5 and a molecular mass of ca. 32 kDa. It shares 91% and 62% amino acid identity with the chromosomally encoded PenP penicillinases from B. clausii KSM-K16 and Bacillus licheniformis, respectively. The hydrolytic profile of this ␤-lactamase includes penicillins, narrow-spectrum cephalosporins, and cefpirome. This chromosome-encoded enzyme was inducible in B. clausii, and its gene is likely related to upstreamlocated regulatory genes that share significant identity with those reported to be upstream of the penicillinase gene of B. licheniformis. The bla BCL-1 gene was located next to the known chromosomal aadD2 gene and the erm34 gene, which encode resistance to aminoglycosides and macrolides, respectively. Similar genes were found in a collection of B. clausii reference strains.Bacillus species are used as probiotics in both humans and animals for the prevention of infectious bacterial diarrhea (8,10,18,28) or preneoplastic lesions (29). Enterogermina (SanofiAventis, Milan, Italy) is a pharmaceutical preparation containing an aqueous suspension of a mix of viable antibiotic-resistant alkaliphilic Bacillus clausii strains (NR, OC, SIN, and T) (10), initially identified as Bacillus subtilis (37). B. clausii spores are able to germinate after exposure to acidic pH in the stomach and to grow in the presence of bile and with a limited amount of oxygen, consistent with the beneficial health effect of probiotics (8). In their vegetative form, B. clausii strains are able to induce an immune response (39), as recently described for "Bacillus polyfermenticus" (22). Bacillus spores are used as probiotics, although they may carry antibiotic resistance determinants and express toxins (14,19). Resistance to erythromycin is one of the reported features of B. clausii (3, 10), due to the presence of an erm-related gene (5). B. clausii strains are also resistant to penicillin G, cephalothin, and cefotaxime (4).Therefore, it was interesting to determine the resistance determinants responsible for resistance phenotype in B. clausii. The study was initiated with one of the strains, B. clausii NR, present in the Enterogermina mixture and with the B. clausii reference strain ATCC 21537. A ␤-lactamase with an uncommon hydrolytic profile that included cefpirome was characterized. MATERIALS AND METHODSBacterial strains. B. clausii NR, SIN, T, OC, and DSM 8716 were from Sanofi-Aventis. B. clausii ATCC 21537 was used as a reference strain. Most of the work reported below was performed in parallel with B. clausii NR and ATCC 21537 strains. Escherichia coli DH10B and E. coli BL21(DE3) (Invitrogen, Life Technologies, Cergy-Pontoise, France) were used for cloning experiments and ␤-lactamase overexpression, respectively (17).Antimicrobial agents and MIC determinations. The antimicrobial agents and their sources are described ...

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“…The production of two inducible P-lactamases is unusual, although it has previously been reported for strains of Aeromonas spp. (13), Bacillus cereus (10), and Yersinia enterocolitica (8). Some Aeromonas spp.…”

Section: Discussionmentioning

confidence: 99%

Biochemical properties of inducible beta-lactamases produced from Xanthomonas maltophilia

Paton

Miles

Amyes

1994

Antimicrob Agents Chemother

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Four different P-lactamases have been found in several strains of Xanthomonas maltophilia isolated from blood cultures during 1984 to 1991 at the Edinburgh Royal Infirmary. One was a metallo-o-lactamase with predominantly penicillinase activity and an isoelectric point of 6.8. Its molecular size as determined by gel filtration was 96 kDa but was only 26 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), suggesting a tetramer of four equal subunits. The enzyme hydrolyzed all classes of P-lactams except the monobactam aztreonam. This enzyme was not inhibited by potassium clavulanate or BRL 42715 but was inhibited byp-chloromercuribenzoate, mercuric chloride, and EDTA. The P-lactamase was unstable in 50 mM sodium phosphate buffer (pH 8.0) but stable in 50 mM Tris HCI (pH 8.0). The other j8-lactamases focused as a series of different isoelectric points, ranging from pl 5.2 to 6.6. Together, these enzymes exhibited a broad spectrum of activity, hydrolyzing most classes of j-lactams but not imipenem or aztreonam. Their molecular size was 48 kDa by Sephadex gel filtration and 24 kDa by SDS-PAGE, indicating that they were enzymes consisting of two equal subunits. They were inhibited by p-chloromercuribenzoate, mercuric chloride, potassium clavulanate, and BRL 42715 but not EDTA. This study demonstrated that X. maltophilia produces more than just the Li and L2 P-lactamases.Xanthomonas maltophilia is the only member of the genus Xanthomonas that is pathogenic to humans. This species rests uneasily within this genus, however, and a recent report has proposed that it be placed in a new genus, which includes a single species, Stenotrophomonas maltophilia (24). They are glucose-nonfermenting, gram-negative bacilli and are increasingly recognized as an important opportunistic pathogen, often affecting patients with lowered defense mechanisms (12,21,22,29). The species is commonly resistant to a wide range of ,-lactams as well as other classes of antimicrobial agents (11,15,22). This resistance has been attributed to the interplay between outer membrane impermeability (20) and the production of two potent ,B-lactamases. Li, a metallo-o-lactamase (31), and L2, described as an unusual cephalosporinase (30), hydrolyze virtually the entire spectrum of ,B-lactams. It has been assumed that all strains produced the Li and L2 enzymes.

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Separation, purification and properties of β-lactamase I and β-lactamase II from Bacillus cereus 569/H/9

Cited by 112 publications

References 32 publications

The role of intra-domain disulfide bonds in heat-induced irreversible denaturation of camelid single domain VHH antibodies

The role of intra-domain disulfide bonds in heat-induced irreversible denaturation of camelid single domain VHH antibodies

Molecular and Biochemical Characterization of the Chromosome-Encoded Class A β-Lactamase BCL-1 from Bacillus clausii

Biochemical properties of inducible beta-lactamases produced from Xanthomonas maltophilia

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