Recombinant Bacillus subtilis Cephalosporin C deacetylase (cah)

What is Bacillus subtilis Cephalosporin C deacetylase and what is its function?

Cephalosporin C deacetylase (CAH) is an esterase that catalyzes the removal of acetyl groups from various O-acetylated substrates. It belongs to the alpha-beta hydrolase fold family and is classified as a member of carbohydrate esterase family 7. CAH specifically cleaves ester bonds in acetylated xyloses, short xylooligosaccharides, and importantly, cephalosporin C, but is known not to cleave amide bonds . The enzyme plays a critical role in the semi-synthetic production of β-lactam antibiotics by hydrolyzing the acetyl group from cephalosporin C and related compounds. While its natural function in B. subtilis may involve deacetylation of acetylated oligosaccharides, its capability to deacetylate cephalosporin C has made it industrially significant for pharmaceutical applications .

What is the structural composition of Cephalosporin C deacetylase from B. subtilis?

Cephalosporin C deacetylase from B. subtilis exists as a homohexameric protein composed of identical subunits. Each monomer has a molecular weight of approximately 32-36 kDa, with the enzyme from B. subtilis CICC 20034 specifically reported to have a calculated mass of 35,607 Da . The gene encoding this enzyme contains an open reading frame of 957 base pairs that translates to 318 amino acids . Structurally, the enzyme features the classic alpha-beta hydrolase fold characteristic of esterases. The functional hexamer is arranged as two trimers, and interestingly, under conditions containing 1% sodium dodecyl sulfate (SDS), the enzyme can exist as a trimer while still maintaining activity . This ability to transition between hexameric and trimeric forms while retaining catalytic function is a distinctive characteristic of this enzyme. The active site contains a catalytic triad consisting of Ser181, His298, and Asp269, which is typical of serine hydrolases .

What is the catalytic mechanism of Cephalosporin C deacetylase?

The catalytic mechanism of Cephalosporin C deacetylase follows the classical serine protease mechanism involving a catalytic triad. The process occurs through the following steps:

• The serine residue (Ser181) is activated by the histidine (His298) of the Ser-His-Asp catalytic triad, with Asp269 stabilizing the histidine through electrostatic interactions .

• The activated serine acts as a nucleophile, attacking the carbonyl carbon of the acetyl group in the substrate.

• This nucleophilic attack results in the formation of a tetrahedral intermediate.

• The intermediate collapses, leading to the elimination of the acetate group and the formation of an acyl-enzyme intermediate.

• Water then attacks this intermediate, cleaving the enzyme-substrate bond to regenerate the active site.

• The deacetylated product is released, completing the catalytic cycle .

The oxyanion hole, formed by the main chain nitrogen atoms of Gln182 and Tyr91, stabilizes the negative charge that develops on the oxygen atom during the reaction, facilitating the catalytic process .

What substrates can be processed by Cephalosporin C deacetylase?

Cephalosporin C deacetylase demonstrates activity on a variety of substrates, with specificity for ester bonds but not amide bonds. The primary substrates include:

• Cephalosporin C (CPC) - a key substrate in pharmaceutical applications

• 7-aminocephalosporanic acid (7-ACA)

• Acetylated xylose

• Short xylooligosaccharides

• Other O-acetylated small molecules

The enzyme from B. subtilis CICC 20034 has shown exceptionally high activity toward CPC and 7-ACA, reported at 484 U/mg and 888 U/mg respectively, which is the highest among acetyl xylan esterases from carbohydrate esterase family 7 . This high activity toward pharmaceutical precursors makes CAH particularly valuable for selective deacetylation reactions in antibiotic synthesis.

How is the cah gene organized in B. subtilis?

The cah gene in B. subtilis contains an open reading frame of 957 base pairs encoding a protein of 318 amino acids . The gene sequence shares significant identity with acetyl xylan esterases from other Bacillus species, including Bacillus sp. 916, B. subtilis 168, and Bacillus pumilus Cect5072 . The gene encodes a signal peptide at the N-terminus that directs the protein for secretion. When expressing the recombinant enzyme, this native signal peptide can be replaced with other signal peptides to optimize secretion efficiency .

The 5′ untranslated region (5′ UTR) plays a crucial role in translation initiation and efficiency. Research has shown that modifications in this region can dramatically impact expression levels. For instance, an unexpected insertion of a 73-bp sequence in the 5′ UTR resulted in an 82-fold increase in extracellular activity, likely due to an increase in the translation initiation rate (TIR) .

What strategies can be employed for high-level secretory expression of Cephalosporin C deacetylase in B. subtilis?

Achieving high-level secretory expression of Cephalosporin C deacetylase in B. subtilis involves several strategic approaches:

• Signal Peptide Optimization: Screening multiple signal peptides is crucial for identifying the most efficient secretion leader. Research has demonstrated that the YncM signal peptide achieved the highest extracellular CAH activity (13.5 U/ml) when compared to other signal peptides like Bpr, AmyE, Vpr, LipA, Epr, YwbN, YvgO, and OppA . Some signal peptides (AnsZ, WapA, and YclQ) failed to result in detectable extracellular CAH activity, highlighting the importance of proper signal peptide selection .

• Promoter Enhancement: Replacing the native or initial promoter with stronger ones can significantly improve expression. For example, substituting the HpaII promoter with the stronger P43 promoter increased CAH activity from 13.5 U/ml to 18.9 U/ml .

• 5′ UTR Modification: Alterations in the 5′ untranslated region can dramatically impact expression levels. An unexpected insertion of a 73-bp sequence in the 5′ UTR resulted in a remarkable 82-fold increase in extracellular activity (reaching 1,548.1 U/ml), likely due to enhanced translation initiation .

• Media and Culture Condition Optimization: Using rich media like TB (Terrific Broth) supplemented with appropriate antibiotics and optimizing growth conditions (37°C, 220 rpm for 48-72 hours) has been reported as effective . Monitoring growth curves helps determine optimal harvest time. Research shows that while biomass peaks at around 14 hours (OD600 of 23.3), enzyme activity continues to increase, reaching maximum levels at around 60 hours (2,042.8 U/ml) .

• Host Strain Selection: Using B. subtilis strains with reduced protease activity, such as WB600, minimizes degradation of the secreted enzyme .

Implementing these strategies collectively can lead to significant improvements in the secretory production of CAH in B. subtilis, with reported activity levels exceeding 2,000 U/ml in optimized systems.

How can directed evolution be applied to enhance Cephalosporin C deacetylase catalytic efficiency?

Directed evolution has proven to be a powerful approach for improving CAH properties through the following methodological framework:

• Site-Directed Saturation Mutagenesis: This technique targets residues in the substrate-binding pocket based on molecular docking studies. The R157 position has been identified as a key catalytic site for substrate deacetylation . A library of variants can be created by replacing the target residue with all 20 amino acids to explore the full range of possible substitutions.

• Screening Strategy: The mutant library is expressed in a suitable host (B. subtilis or E. coli), and a high-throughput assay is developed to measure deacetylase activity, allowing for the selection of mutants with improved catalytic parameters.

• Characterization of Improved Variants: Promising mutants are purified and their kinetic parameters (Km, Vmax, kcat) are determined. For example, the R157T mutant demonstrated significantly improved catalytic efficiency compared to the wild type:

  • Specific activity increased from 2,047.3 U/mg to 3,112.2 U/mg

  • Km decreased from 7.04 mM to 5.19 mM

  • Vmax increased from 5.11 μM s-1 to 7.56 μM s-1

• Structural Analysis: Molecular modeling helps understand the structural basis for improved activity. For CAH, the R157 residue is located at the center of the enzyme, and there are hydrogen bond interactions between this residue and the substrate . The R157G mutant completely lost catalytic activity, confirming the critical nature of this position.

• Iterative Improvement: The best-performing mutant can serve as the template for subsequent rounds of mutagenesis, targeting additional residues or combining beneficial mutations for further enhancement.

This systematic approach to directed evolution has successfully improved the catalytic efficiency of CAH, providing a valuable tool for industrial applications in pharmaceutical synthesis.

What role does the 5′ untranslated region play in the expression of Cephalosporin C deacetylase?

The 5′ untranslated region (5′ UTR) plays a critical role in regulating gene expression at the translational level for CAH production:

The significant impact of 5′ UTR modifications on CAH expression highlights the importance of this region as a target for expression optimization, particularly when high-level protein production is desired for industrial applications.

What methods are optimal for purification of recombinant Cephalosporin C deacetylase?

Purification of recombinant Cephalosporin C deacetylase can be achieved through a systematic approach leveraging the enzyme's unique properties:

• Expression and Initial Processing: Harvest the culture supernatant from recombinant B. subtilis after 48-72 hours of cultivation and centrifuge to remove cells and debris .

• Heat Treatment: Exploit the enzyme's exceptional thermal stability by incubating the culture supernatant at 85°C for 30 minutes. This step effectively removes most thermally unstable proteins, as CAH remains stable at this temperature . Following heat treatment, centrifugation removes denatured proteins.

• Filtration: Filter the heat-treated supernatant through a 0.45-μm-pore-size membrane to remove any remaining particulates before chromatographic purification .

• Chromatographic Purification: Ion exchange chromatography using an anion-exchange column (such as HiTrap Q HP) is effective for high-resolution purification. The column is equilibrated with a suitable buffer (e.g., 50 mM sodium phosphate buffer, pH 7.0), the filtered enzyme solution is applied, and the enzyme is eluted using a gradient of increasing salt concentration (e.g., 0-500 mM NaCl in the same buffer) .

• Quality Assessment: Evaluate purification efficiency using SDS-PAGE to confirm the presence of a single band at approximately 32 kDa, which is the expected molecular weight for CAH . Measure specific activity to assess purification yield and enzyme quality.

This purification protocol takes advantage of CAH's inherent thermal stability, which provides a simple and effective initial purification step, followed by conventional chromatographic techniques to achieve high purity enzyme suitable for detailed characterization and application studies.

How can molecular docking be utilized to identify key catalytic sites in Cephalosporin C deacetylase?

Molecular docking is a powerful computational approach for understanding enzyme-substrate interactions and identifying critical catalytic residues in Cephalosporin C deacetylase:

• Structural Preparation: A high-resolution crystal structure of CAH (such as PDB entry 1l7a for the related enzyme from B. subtilis strain 168) serves as the starting point . The protein structure is prepared by adding hydrogen atoms, assigning protonation states, and minimizing energy to resolve any structural inconsistencies.

• Substrate Modeling: Accurate three-dimensional models of substrates such as cephalosporin C, 7-ACA, or acetylated oligosaccharides are generated, ensuring proper representation of substrate conformational flexibility.

• Docking Simulation: The binding site is defined based on known catalytic residues (Ser181, His298, Asp269) or using cavity detection algorithms . Docking simulations are performed using appropriate software packages to generate multiple poses, which are scored based on binding energy and geometric complementarity.

• Analysis of Docking Results: Residues that directly interact with the substrate are identified, particularly those forming hydrogen bonds or other non-covalent interactions. For CAH, docking studies revealed that R157 is located at the center of the enzyme and forms hydrogen bond interactions with the substrate . The critical nature of R157 was confirmed experimentally, as the R157G mutant completely lost catalytic activity.

• Validation through Mutagenesis: Site-directed mutagenesis experiments test the role of identified residues. Creating variants (such as R157T, R157W, R157H) explores the impact of different amino acid substitutions . Characterizing the resulting mutants correlates computational predictions with experimental outcomes.

The successful application of this approach in identifying R157 as a key catalytic site in CAH demonstrates the value of molecular docking for rational enzyme engineering. The R157T mutant exhibited significantly improved catalytic properties, validating the computational predictions and providing insights for further optimization.