Experiments on methods of applying end truncation, evolution, and re-elongation techniques to improve enzyme stability
Experiments on methods of applying end truncation, evolution, and re-elongation techniques to improve enzyme stability
Improving enzyme stability is an ideal design step for modifying enzymes to exercise their activity under extreme conditions, such as high temperatures, as well as reducing the enzyme's sensitivity to protease shear. Due to these advantages, researchers have invented many different methods and techniques to modify proteins, but so far these methods have had limited results. The source of this experiment is "A Guide to Modern Protein Engineering Experiments" [German] K.M. Arndt, K.M. Miller, eds.
Operation method
Application of end truncation, evolution, and re-elongation techniques to improve enzyme stability
Materials and Instruments
Plasmids Move This chapter focuses on conventional methods for improving the thermal stability of proteins without screening for enzyme activity at elevated temperatures. After the introduction of the β-lactamase model system (see 16.3.1 ), we describe the design schemes for end-truncations (see 16.3.2 and 16.3.3 ), and their effect on enzyme activity (see 16.3.4 ). Mutation libraries are generated by directed evolution of the enzyme and error-directed PCR (see 16.3.5) and then screened in vivo by applying different screening pressures (see 16.3.6). In the last part of this section, the re-elongation of truncation mutants (see 16.3.7), expression strategies and purification protocols, enzymatic methods (see 16.3.9), and stability assays (see 16.3.10) are discussed. For more product details, please visit Aladdin Scientific website.
DNase buffer EDTA solution TBE buffer Butanol Conversion salt reservoir Ammonia reservoir Suspension buffer Boric acid buffer
Water baths Spectrophotometers Spectrofluorometers
3.1 β-lactamase model systems
To illustrate that truncation-optimization-re-extension can be used to improve the thermal stability of proteins, lactamase was chosen as a model system (Fig. 16.1A ). As a clinically pathogenetically relevant research factor and important antibody, the family to which it belongs has been extensively studied and multiple crystal structures have been resolved. Many mutations to the enzyme have been used to better study and interpret structure-function correlations as well as to rationally design truncated segments. In addition, stabilized β-lactamases have been used to study the activity of potential drugs for cancer therapy in order to obtain potential drug precursor compounds [27, 28].
The A family of β-lactamases (EC 3.5.2.6 ) are bacterial cytoplasmic enzymes with diverse amino acid sequences and varying stability, but with very similar three-dimensional structures. Although the peptide backbones of the A family of β-lactamases overlap well in the core region, they do not overlap well at the two termini. Differences in terminal structure are also compatible with amino acid length and amino acid composition, making this class of enzymes suitable for studying sequence homology and the related importance of function.
3.2 Designing end truncations
The correct design of deletion mutants is important: if too many terminal truncations are made, the resulting truncation mutants will be non-functional due to insufficient structural stability and misfolding; similarly, if not enough truncations are made, there is a high probability of not obtaining the desired phenotypes, and the selection pressure will not be able to select for a stable structure.
If the structure of the target protein has already been resolved, it is possible to use the Protein Data Bank (PDB; http : //www.pdb.org/), SWISSPROT ( http : //www. expasy. org/sprot/ and http : //www.ebi. ac.uk/swissprot), and SCOP ( http : //scop. mrc-lmb. cam . ac . uk/ scop/ ) databases and structural analysis tools such as the molecular modeling program WHATIF (http : //swift cmbi.
kuru nl/WIWWWI) and CE algorithms (http : //cl. sdsc . edu /ce . edu /ce . html) for structural comparison and analysis. In addition, contact maps can be used to identify possible non-significant and significant interactions, which are also important in the design process. If high resolution structures are not available, other databases (e.g. HSSP) and the SWISS MODEL homology model server (http : //swissmodel. expasy. org) can be used, or one or both of the ammonia
or two amino acids cut by cut to determine the maximum cutoff number.
In this study, four truncation mutants were designed based on a previous mutant study of β-lactamases [29], and structural homology sequences were aligned to the A family of β-lactamases, which were analyzed using the CE algorithm [30] and the SWIS& PDB viewer [31] (http: //www w. expasy. org/spdbv) (Figure 16.1B and Figure 16.1C). 16.1C) to analyze the structure. The truncated mutants attempt to introduce structural disturbances at physiological temperatures without rendering the protein completely nonfunctional. the first three amino acids at the N-terminus (His, Pro, and Glu) have high temperature factors (up to 23.8 A, average 13.1 A) and solvent accessibility (PDB no. 1btl ), and when the first three amino acids are removed, the resulting mutant NΔ3 has a relatively small effect on protein stability. The NΔ5 mutant removes the neighboring threonine and leucine, where the threonine is completely masked and forms a hydrogen bond with the Ser285 amino acid at the C-terminus. At the C-terminus, only one and three amino acids are removed (mutants C△1 and C△3 ), as previous studies have shown that the terminal tyrosine is the critical amino acid [32].
3. Design of vector plasmids
The vector plasmid was designed to be expressed as a fusion of wild-type β-lactamase or a truncated mutant with the N-terminal pelB signaling sequence used for mesenchymal localization. A short aspartate-glycine linkage was introduced to ensure equal processing efficiency of different mutants at the N-terminus. Finally, the C-terminal His-tag can be used to immobilize metal ion affinity columns for purification, even of non-wild-type β-lactamase variants (Fig. 16.2A). 
The procedure for vector construction is as follows:
( 1 ) The TEM-1 β-lactamase truncation mutants NA3, N△5 and C△1 and C△3 were amplified from the pUC-derived TEM-1 gene using its forward and reverse primers, respectively. The forward primer contains the 5' Sfi I cleavage site and also contains the C-terminal coding sequence of the pelB signaling peptide chain and the N-terminal modification (truncation) sequence of the TEM-1 truncation mutant. At the 3' end of the gene, the reverse sequence encodes the C-terminal sequence of the modified TEM-1, a short sequence (2 glycine residues), 5 His-tags, 2 stop codons, and the Hind lll cleavage site.
( 2 ) The PCR product was double digested by Sfi I/Hind III, purified, and cloned into the pKMENGRbla vector (provided by K.M.M.), which is a modification of the expression vector pKA400 [ 26 ] and contains a chloramphenicol resistance gene.
The resulting plasmids (pKJE_Bla-NA/CA series) expressed various TEM-1 mutant genes under lac manipulators. Although there was constitutive expression of lacI on the plasmid, the strong T7g10 Shine-Dalgarno sequence [ 35 ] had high expression levels, so all selection steps were performed under extreme conditions and did not overexpress.
3.4 Effect of terminal truncation on enzyme activity
To test the effect of end truncation on enzyme activity, the growth rate was measured in ampicillin-resistant incremental solid and liquid media. Typically, screening on solid plates is more demanding than in the liquid state. Differences in the systems were observed by background expression and IPTG-induced expression as well as induction at different temperatures.
( 1 ) If necessary, transform BL21 cells coated on LB/Cm plates using standard methods.
( 2 ) Pick single clones from glycerol or transformed overnight (16 h) cultured LB/Cm plates [ step (1) ].
( 3 ) Dilute the overnight cultured bacteria to an OD600 of 0.1 in new medium and incubate them to an OD600 of 1 (see Note 1).
( 4 ) Apply the pre-cultured bacterial solution [ Step (3) ] to ampicillin-resistant plates with and without IPTG at different concentrations. The amount of bacteria coated can be estimated as 2. 5X108 cells/ml, 1 OD600.
( 5 ) Add the bacterial solution from step (3) to liquid LB/Cm medium and incubate in different concentrations of ampicillin medium with and without IPTG.
( 6 ) The growth rate [ steps (4) and (5) ] can be repeated at different temperatures.
When incubated on solid plates at 37°C without induction, the number of clones of all truncation mutants decreased dramatically when the ampicillin resistance concentration was increased from 0 μg/ml to 50 μg/ml (Fig. 16.2B). Mutants N△5 and C△3 did not grow clones after 40 h of continuous culture, indicating the importance of terminal amino acids. When induced at 25°C, N△5 was able to grow in solid medium with up to 50 μg/ml of ampicillin, and it was still able to grow in liquid medium at 37°C with proper oxygenation. This suggests that both truncation mutants are able to interfere with the protein structure, but at the same time can fold into an active structure.The N∆5 mutant is less tolerant of antibiotics than C∆3, and is therefore more suitable for the next step of optimization.
3.5 DNA mashups and random mutations
The key optimization step to reverse structural disturbances caused by end-truncation or deletion mutations is to create highly variable mutation libraries at the genetic level. The combination of random mutagenesis and DNA recombination meets the need for such mutations.
DNA hybridization [ 15, 16] is a widely used method for DNA recombination in vitro. The mixing of genes of different origins by DNA mashups is a key step in directed evolution, and its main features are briefly described as follows: after random point mutations are performed on whole genes, the mutation libraries obtained mainly by error-prone PCR (see 16.3.7) can be used for DNA fragmentation, resulting in small, slightly different, interchangeable DNA fragments of 50-200 base pairs in length. DNA fragments. One or more nucleotide-different homologous sequences are overlapped and re-amplified into long fragments by PCR.
Initially, a low-temperature annealing is used to ensure that even the smallest fragments can be subjected to PCR, and as the number of cycles increases, the annealing temperature is gradually increased to select the longer, interspersed fragments. Finally, the full-length gene is synthesized using primers on both sides of the gene, and the appropriate cleavage site is introduced for the next step of cloning.
The following section describes the use of error-prone PCR and DNA mixing to create diverse mutant libraries of sufficient complexity. It is important to note that the steps presented here are specific to our system and will need to be altered as the length and composition of the target genes change. Consideration needs to be given to the coverage of the reaction prior to the introduction of the mutation by error-prone PCR; the first random mutation, the second combination, cannot be used to determine the error rate during the mixing process (see Note 2 ).
( 1 ) To obtain a sufficient amount (5-10 μg) of the target gene, PCR can be performed with homologous sequences at both ends of the target gene or with restriction endonuclease gene-end shearing. If PCR is used, Taq DNA polymerase should be used to synthesize the product. In our example, we amplified the truncated β-lactamase with primers Pr_sfi_pelB_DG_ shuffl and Pr_GG_his5 _ hind_shuffl using pKJE_Bla_NΔ5 as template.
( 2 ) 4~5 μg of the target gene was digested with 0.2 U of DNase l in 50 μl of DNaSe I for 12 min at room temperature (25°C).
( 3 ) Terminate the endonuclease reaction by adding 4 μl of EDTA solution and place on ice.
( 4 ) Analyze the DNA fragments obtained on 2% agar gel and recover DNA fragments of 50~150 bp in length.
( 5 ) Recover the DNA fragments with the Qiaex II Gum Recovery Kit (see Note 3).
( 6 ) To 50 μl of the system, add approximately 100-300 μg of purified fragments for primer extension PCR (see Note 4). The reaction mixture consists of 2.5 U DNA polymerase, 0.4 mmol/L dNTP 2 mmol/L MgCl2. In a PCR instrument (Eppendorf), the PCR reaction is carried out with the following program: 94°C for 3 min; 94°C for 1 min (denaturation), (45 + 0.3)°C for 1 min per cycle (annealing), 72°C for 1 min (extension) (10 cycles). extension) (10 cycles); 5 min at 72°C after the last cycle; 1 min at 94°C, (50 + 0.4)°C 1 min per cycle, 72°C 1 min (15 cycles); 5 min at 72°C after the last cycle; 1 min at 94°C, (56 + 0.5)°C 1 min per cycle, 72°C 1 min (10 cycles) (10 cycles); last cycle
cycle after 72°C 5 min; 94°C 1 min, ( 61 + 0.5)°C 2 min per cycle, 72°C 2 min (10 cycles); last cycle after 72°C 5 min. Alternatively, a simple program such as 94°C 3 min, 35 cycles: 92°C 30s, (30 + 1)°C 1 min per cycle, 72°C 1 min + 4s/cycle, 5 min at 72°C after the last cycle.
( 7 ) Amplify 1/5 volume of overlapping PCR reactants (no purification required) to amplify the full-length gene and add the appropriate digest site for the next cloning step. This step can be performed under error-prone tendency conditions to amplify a diverse library of mutations. In addition to extension with Taq DNA polymerase, the mutation rate of the amplification reaction can be increased by the addition of 7 mmol/L MgCl2, 0.5 mmol/L MnCl2 ( see Note 5). In our case, we used the primer Pr_sfi_
pelB_DG_shuffl and Pr_GG_his5_hind_shuffl, each at a concentration of 0.5 μmol/L. The reaction mixture consisted of 7 mmol/L MgCl2, 0.5 mmol/L MnCl2, 0.4 mmol/L dNTP, and 2.5 U of Tag. The program used was 94°C for 3 min, 25 cycles: 94°C 1 min , 68°C 1 min, 72°C 1 min, and 7 min at 72°C after the last cycle.
( 8 ) The PCR product was purified using the GFX PCR DNA and cuttings recovery kit, digested with appropriate enzymes and cloned into an expression vector. The products were cloned into expression vectors and transformed as described in 16.3.6.
We performed 3 cycles of directed evolution (S1~S3) with DNA mixing and random mutagenesis combined with an in vivo screening process. The resulting clones were collected and used as templates for the next evolutionary step. In the last cycle of directed evolution, error-prone PCR after DNA mixing was replaced by a standard PCR procedure to prevent deleterious mutations in the recombinant clones.
16.3.6 Transformation and in vivo screening of mutant libraries
( 1 ) Take the plasmid mutant library and precipitate it with butanol. Use 10-20 μl of the ligation mixture and add double the amount of deionized water to bring the volume to 50 μl. Add 500 μl of butanol and centrifuge at 2000 g for 30 min at room temperature. remove the supernatant and allow it to dry at room temperature, adding 10-20 μl of water.
( 2 ) Add the precipitated DNA to 100 μl of electroreceptor E.coli XL-1 blue cells at 1.7 kV, 200 Ω, 25 μF. Immediately after transformation, add 900 μl of 2YT medium and 1/100 volume of Transformation Salt Reservoir.
( 3 ) Add the suspended cells into 10 ml glass tubes and incubate at 37°C for 60~70 min.
( 4 ) Determine the transformation efficiency by applying the transformation products onto LB/Cm plates with gradual dilution.
( 5 ) Determine the screening pressure by applying the transformed cells from another tube onto ampicillin-resistant LB/Cm plates at different concentrations (see Note 6). The mutant that grows at the highest ampicillin concentration is selected for the next step.
( 6 ) As a screen, the transformed cells were coated on ampicillin-resistant LB/Cm plates at different concentrations [ concentrations obtained from step (5 ) ] ( see Note 6 ). We used ampicillin at concentrations of 20 μg/ml, 100 μg/ml, and 200 μg/ml for the first to third screenings, respectively. the first screening was performed in liquid medium, and the last two screenings were on solid medium.
Mutant N∆5 was subjected to 3 cycles of directed evolution (including random mutation and DNA mixing) (see 16.3.5), and the impaired function was restored. In these 3 cycles 19X103, 147X103 and 260X103 clones were obtained, grown on 20 μg/ml, 100 μg/ml and 200 μg/ml ampicillin-resistant plates, respectively [ step (6 )], yielding 3600-7000 clones.
Table 16.1 summarizes the sequencing results of all the clones obtained in the directed evolution of the 3 mixing cycles (S1~S3 ). In addition, Table 16.1 includes the relative solvent accessibility and average side chain atomic temperature factor (by PDB 1btl ) for each wild-type residue [ 37] . Two of the five mutants after the second cycle had two mutations, M182T and T265M ( named after Ambler et al. These two mutations were already present after the first cycle. 
After the final optimization step, the mutant library was collected and the maximum ampicillin resistance concentration was determined without the use of IPTG induction, see 16.3.4 ( Figure 16.3). The resistance that can grow a significant number of clones is 700 μg/ml, and after 40 h of incubation, clones can grow on resistance containing 1000 μg/ml. In contrast, the wild type could only grow on 500 μg/ml of resistance. Twenty-six clones were picked from the plates containing 800 μg/ml and 1000 μg/ml of resistance and sequenced. 26 clones belonged to 15 different mutants (Table 16.1). All clones had two mutation sites in common, M 182T and A224V.
Previous studies have shown that the M182T mutant is able to complement folding defects [39] and stability deletions [29, 40]. Mutant A224V increases selection pressure in the third cycle. This mutant has been mentioned before, but there is no experimental data to confirm it [41].
The two mutants are 17A away from the truncated site, suggesting an independent complementation of structural disturbances caused by the truncation. Figure 16.4 shows some of the most commonly mutated residues to TEM- 1β-lactamase. Typically, in individually optimized mutants the vectorial changes are distributed throughout the structure rather than clustered locally. 
3.7 Construction of full-length mutants
The optimized N∆5-S3/6 was reextended to study the complementary effect of the mutation on the truncated unstable protein. According to Figure 16.2A, the truncated amino acids were re-added, resulting in a clone of the full-length gene - named FL-S3/6. This mutant localized to the intercellular matrix and was expressed in E.coli XL-1 Blue to assay in vivo function. Surprisingly, the number of clones grown on chloramphenicol- and 100 μg/ml ampicillin-containing plates at 37°C for 20 h was only 50% of the number grown on control plates without ampicillin. This is in contrast to the same number of clones obtained for NΔ5-S 3/6 on both plates. In order to study the protein
function and protein localization, an enzyme activation reaction was performed on crude extracts of FL-S 3/6 and N△5-S3/6 cells. No difference was seen in the hydrolysis reaction to the chromogenic substrate cefonithiophene, indicating that enzyme localization is important. Consistent with these data a reduction in signal peptide shear was also seen during purification of FL- S 3/6 (see 16.3.8). Consequently, FL- S3/6-cyt, which can be expressed in the cytoplasm, was cloned, which replaced the signal peptide as well as the Asp- Gly tag with methionine. in order to ensure a correct comparison under physiological conditions, a gene similar to the wild-type endocannabinoid enzyme (wt- done-cyt) was cloned.
3.8 Protein expression and purification
β-lactamase mutants (wild-type mesenchymal-localized, wild-type cytoplasmic-localized, N∆5, optimized N∆5-S3/6, N∆5- S5/S7, and FL-S3/6 cytoplasmic-localized mutants) were expressed in E.coli cells with His -tag fusions under the lac promoter (see 16.3.8.1). Purification is carried out by a two-step purification reaction, first by affinity chromatography of phenylborate columns with similar substrates (see 16.3.8.2 ) and then by IMAC ( see 16.3.8.3 ). This purification step is non
often applied to the purification of N∆5 (Fig. 16. 5 ) and N∆5- S3/7 mutants.
For some β-lactamase mutants (wild-type, N△5-S3/6, FL-S3/6), the purified proteins are highly contaminated and in an incomplete form (30%-65%; see Note 7), most likely due to overexpression or too fast folding. Because contamination can be introduced even for purification of intercellular extracts aimed at high yields, hydrophobic affinity chromatography (HIC ) was used to separate the natural forms of proteins from those of very hydrophobic unprocessed complete proteins (30%~65% ; see Note 7 ).
forms of proteins that are extremely hydrophobic and not fully processed (see 16.3.8.4).
Cytoplasmically expressed β-lactamase variants wt-clone-cyt and FL-S3/6-cyt are purified using phenylboronate affinity columns and IMAC. Further purification can be performed with ion exchange columns (see 16.3.8.5).
3.8.1 Protein Expression and Cell Fragmentation
( 1 ) Transform the plasmid into a suitable expression strain (e.g. BL21).
( 2 ) Pick bacteria from glycerol bacteria into 2YT/Cm culture medium and incubate at 28°C for 16~18 h ( see Note 8 ) .
( 3 ) Amplify four 1L-2YT/Cm cultures with overnight bacteria, starting with an OD600 of 0.15, and incubate the N∆5 clone at 24°C and the other clones at 25~30°C (see Note 9 ).
( 4 ) When the OD600 is 0.7, use 0.5 mmol/L IPTG to induce the cells. After 40 min of induction, 100 μg/ml ampicillin was added to screen for endoribonuclease-expressing cells.
( 5 ) After 4~5 h of induction ( OD600 of 4~5), the cells were harvested with GS-3 transporter and frozen at -80°C. The cells were then frozen at -80°C for 4 hours.
( 6 ) For purification, suspend the bacteria (1 L of expression broth) in suspension buffer (see Note 10) and add 200 U of benzonase.
( 7 ) Pre-cooled cells are broken by 6 repeated cycles at 97 MPa (~14,000 psi), keeping the sample on ice at all times.
( 8 ) Centrifuge the supernatant with an SS-34 rotor at 41,000 g for 40 min. filter the supernatant through a 0.45 μm sulfopolyether injection filter.
3.8.2 Phenylboronic Acid Affinity Chromatography Columns
( 1 ) Suspend a 2 ml phenylborate column with suspension buffer, up-sample the supernatant obtained in step (8) of 16.3.8.1 into the affinity column, and rinse with suspension buffer until the absorption of 280 mn reaches baseline.
( 2 ) Elute the β-lactamase mutant with boric acid buffer. 
( 3 ) Protein purity was characterized by 12.5% polyacrylamide gel electrophoresis (SDS-PAGE), stained with Caulmers Brilliant Blue.
3.8.3 Metal affinity columns
( 1 ) Equilibrate a 4 ml Ni-NTA column with phosphate buffer containing 5 mmol/L imidazole or no imidazole and load the sample from step (2) of 16.3.8.2 onto the column.
( 2 ) Elute the sample with a gradient buffer containing imidazole at (5%, 10%, 16%, and 100%).
( 3 ) Protein purity is identified by 12.5% polyacrylamide gel electrophoresis (SDS-PAGE), stained with Caulmers Brilliant Blue.
( 4 ) For further purification, the purified proteins were dialyzed in 1 L of phosphate buffer containing 1 mmol/L EDTA (about 8 h) and repeated three times.
3.8.4 Hydrophobic interaction chromatography
( 1 ) Equilibrate a 1 ml phenylborate column with 1 mol/L ( NH4)2SO4.
( 2 ) Add ammonia sulfate to the sample obtained in step (2) of 16.3.8.3 or step (2) of 16.3.8.2 to a final concentration of 1 mol/L ( NH4)2SO4 (see Note 11), centrifuge (10 min; 27,000 g; 4°C; SSr-34 rotor), and filter (0.45 μm filters) to prepare a clarified sample.
( 3 ) The sample is loaded onto the HIC column and washed with 30 ml of linear gradient buffer containing 0-100% 0.5X phosphate. The more hydrophobic portion of the sample, the more tightly bound it is to the column.
( 4 ) If the HIC column is loaded directly onto the HIC column after phenylboronate affinity chromatography, the sample needs to be further purified by IMAC (see 16.3.8.3).
3.8.5 Ion Exchange Chromatography
( 1 ) Dialyze the sample obtained in step (2) of 16.3.8.3 3 in 1~1.5 L Tris-HCl buffer 3~4 times.
( 2 ) Equilibrate the Mono Q Ion Exchange Column with Tris-HCl buffer and apply the sample.
( 3 ) Elute with 30 ml of 0-100% Tris-HCl-NaCl linear gradient 0-100% buffer.
( 4 ) The eluted sample is dialyzed as described in Section 16.3.8.3 Step (4).
The amino acid composition of all mutants was analyzed by electrospray mass spectrometry to confirm that it was within the range of deviations from the calculated mass. The enzyme is dialyzed as described in Section 16.3.8.3 Step (4 ) and characterized within one week of purification. Prior to use, the enzyme solution is clarified by centrifugation (30 min; 15000 g; 10°C) and the protein concentration is determined by absorption spectroscopy at 280 nm (see Note 12).
3.9 Enzyme activity reactions
The β-lactamase mutant enzyme activity reaction parameters were determined from the spectrum of the chromogenic substrate cefnithiophene at 486 nm.
( 1 ) A dilution of 20 μl of the enzyme was added to 980 μl of cefdinitothiophene solution, mixed well, and immediately measured at 486 nm to determine the change in absorbance over approximately 1 min (see Note 13). We used a final concentration of the enzyme of 2.5 nmol/L for the start deletion mutation and 0.5 nmol/L for the other variants.
( 2 ) The above assay procedure was repeated at least twice and the standard deviation was calculated.
Kinetic constants were determined at 25°C in 50 mmol/L potassium phosphate buffer (pH 7.0) containing 0.5% dimethyl sulfoxide, using the β-lactam compound cefnithiophene as a substrate.
3.9.1 Determination of the Mie constant and calculation of the conversion ratio
( 1 ) For the determination of the Mie constant ( KM ), the initial rate of the reaction was measured with 2.7-5.9 nmol/L NΔ5 or 0.5 nmol/L other variants at substrate concentrations of 10-500 μmol/L. The initial rate of the reaction was measured with 2.7-5.9 nmol/L NΔ5 or 0.5 nmol/L other variants.
( 2 ) The data were entered into the Mie equation as obtained by the MarquardtLevenberg algorithm provided by the program SigmaPlot (SPSS).
The KM value of N∆5 was unchanged from the wild type, but the conversion K was reduced by a factor of 6, indicating that the composition of the active site is sufficient for substrate binding but does not allow efficient catalytic reactions to occur (Table 16.2 ). 
3.9.2 Thermal activity description and half-life
In order to detect and compare the kinetic parameters for temperature-dependent activity and thermally induced inactivation, a thermal activity screen was performed. We determined the conversion of the reaction when the temperature was increased in the temperature range of 25 to 70°C (Fig. 16.6). 
( 1 ) To assay the thermal activity, a 292 nmol/L solution of the N∆5 mutant and a 25 nmol/L solution of the other mutants were prepared and partitioned into 3 equal parts.
( 2 ) The first aliquot was placed in a water bath at the given temperature (25-70°C) for 5 min. The remaining two aliquots were kept on ice.
( 3 ) Mix the sample lightly before testing.
( 4 ) Add 20 μl of sample to 980 μl of cefonithiophene solution pre-warmed at the reaction temperature, with an initial reaction rate of 5 to 25 s as measured by a heated spectrometer.
( 5 ) The second aliquot is preheated at the corresponding temperature for 30 s and the enzyme activity is measured as described in steps (3) and (4).
( 6 ) The third aliquot was placed on ice for 10 min and the enzyme activity was measured.
( 7 ) Repeat the above steps at different temperatures.
The thermal activity of the wild-type is largely dependent on the preheating treatment (Fig. 16.6A ). The optimal temperature for the reaction of the enzyme placed on ice or preheated for 30 s was 40°C. However, when the enzyme was preheated for 5 min, its optimal reaction temperature dropped to 35°C, indicating the emergence of temperature-dependent defolding.
The N∆5 mutant had the highest enzyme activity at 25°C (the lowest temperature detected) (Figure 16.6C). The enzyme activity decreases rapidly with increasing temperature and reaches zero when 40°C is reached. A detailed assay of enzyme activity at 40°C after a warm bath on ice showed that the enzyme was heat inactivated during the assay and that this occurred within only a few seconds of addition of the enzyme to the reaction solution (Fig. 16.7A ). The enzyme activity decreases with increasing temperature, and the exponential decay equation (see Note 14) gives a half-life of 7 s at 40°C (Fig. 16.7 B). The thermal activity of the NΔ5-S3/6 mutant at 0 s and 30 s is very similar to that of the wild type. However, the situation after 5 min preheating was quite different. The thermal activity of the N△5-S3/6 mutant was increased by approximately 8 °C compared to the wild type (Figure 16.6 A and F). 
The full-length optimized mutant FL-S3/6-cyt ( Fig. 16.6 E ) demonstrated better thermal stability than the optimized deletion mutant N△5-S3/6 and the corresponding wild-type wt-clone-cyt (Fig. 16.6 B and F). The activities of these enzymes were similar up to 40°C and were not strongly dependent on preheating. At temperatures above 50°C, FL-S 3/6-cy maintained a significantly higher enzyme activity, especially when the heat pressure was extended to 5 min preheating. The optimal catalytic activity of wild-type cytoplasmic lactamase placed on ice was at 45°C, whereas after 5 min of preheating it dropped to 35°C. In contrast, the optimal temperature (50°C) for FL-S 3/6-cyt remained unchanged. Only the reaction rate decreased after the warm bath. Interestingly, the conversion of the N-terminal amino acid (Met instead of AspGly) between wild-type and cytoplasmic wild-type endoribonuclease affects the stability of the enzyme.
Comparison of the thermal activity of all truncat