Characterization and thermal denaturation kinetic analysis of

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Characterization and thermal denaturation kinetic analysis of recombinant L-amino acid ester hydrolase from Stenotrophomonas maltophilia Md Saddam Hossain, Takahiro Tanaka, Junji Hayashi, Kazuyoshi Takagi, Yoichi Takeda, and Mamoru Wakayama J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04573 • Publication Date (Web): 02 Oct 2018 Downloaded from http://pubs.acs.org on October 3, 2018

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Journal of Agricultural and Food Chemistry

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Characterization and thermal denaturation kinetic analysis of recombinant

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L

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Md Saddam Hossaina, Takahiro Tanakaa, Junji Hayashia, Kazuyoshi Takagib, Yoichi Takedaa,

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Mamoru Wakayamaa,*

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a

-amino acid ester hydrolase from Stenotrophomonas maltophilia

Dept of Biotechnology, College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga

525-8577, Japan b

Dept of Applied Chemistry, College of Life Sciences, Ritsumeikan University, Kusatsu,

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Shiga 525-8577, Japan

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*Corresponding author: E-mail: [email protected]

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TEL: 077-561-276

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ACS Paragon Plus Environment


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Abstract

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Stenotrophomonas maltophilia HS1 exhibits L-amino acid ester hydrolase (SmAEH) activity,

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which can synthesize dipeptides such as Ile–Trp, Val–Gly, and Trp–His from the

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corresponding amino acid methyl esters and amino acids. The gene encoding SmAEH was

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cloned and expressed in Escherichia coli, and was purified and characterized. SmAEH shared

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77% sequence identity with a known amino acid ester hydrolase (AEH) from Xanthomonas

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citri, which belongs to a class of β-lactam antibiotic acylases. The thermal stability of

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SmAEH was evaluated using various mathematical models to assess its industrial potential.

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First-order kinetics provided the best description for the inactivation of the enzyme over a

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temperature range of 35°C to 50°C. Decimal reduction time ranged from 212.76 to 3.44 min,

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with a z value of 8.06°C, and the deactivation energy was 204.1 kJmol−1.

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Keywords: Stenotrophomonas maltophilia HS1; L-amino acid ester hydrolase; Cloning;

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Purification; Inactivation kinetics

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Introduction

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In recent years, the use of peptides has led to major progress in medicine and the chemical

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industry, and they have gradually replaced classical drug approaches for regulating many

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physiological processes1. Dipeptides and tripeptides, in particular, have attracted considerable

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attention because of the possibility of their oral administration and the ease of performing

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structural, molecular, and quantitative functional characterization2. The isolation and

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characterization of new biologically active peptides are an ongoing endeavor and will

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undoubtedly continue in the future3. For example, γ-Glu–Val–Gly is found in natural food

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products, and received increased attention as a novel food additive, because it has a strong

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taste of kokumi. Kokumi enhances the five basic tastes, particularly sweet, salty, and umami,

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and modifies the thickness and richness of food4. Several food-derived anti-oxidant peptides

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such as glutathione (γ-Glu–Cys–Gly), carnosine (β-Ala–His), and His–Pro have recently

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received increased attention as novel food additives1. Val–Tyr, Ile–Trp, Ile–Tyr, and Lys–Trp

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are strong anti-hypertensive peptides and are derived from hydrolysates of fish-meat,

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seaweeds, etc5. Because of this growing interest, it is equally important to develop methods

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for the synthesis of dipeptides on a commercial scale. Consequently, significant research

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efforts have been expended to synthesize peptides using chemical or enzymatic approaches6.

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Development of cost-effective methods for synthesizing di- and tripeptides will be crucial in

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food science, since such small peptides have the ability to be absorbed through the

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gastrointestinal tract and reach target organs via the circulatory system1.

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L-Amino

acid ester hydrolases (AEHs) are well known for their application to the

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biocatalytic synthesis of semisynthetic β-lactam antibiotics since 19727. Only α-amino acid

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derivatives act as substrates, and owing to its high preference for esters over amides, the

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enzyme has been formally named α-amino acid ester hydrolase (EC 3.1.1.43)8. AEHs were

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never used for peptide synthesis until 2001 because, in addition to having only a few reports



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on such enzymes to that date, no data was available on their activity with respect to peptide

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synthesis and hydrolysis9. However, several recent studies on AEHs that successively and

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effectively produced dipeptides have revealed that they can synthesize oligo-peptides as

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well10–11. Owing to their preference for esters, it is conceivable that greater product

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accumulation could be achieved in synthesis reactions using these enzymes rather than with

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L

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reports on natural microbial strains that produce this enzyme have been published10. By

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contrast, however, reports on recombinant AEHs are limited because the attempts made to

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clone an aeh gene have been unsuccessful12–13. To date, recombinant AEHs from

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Xanthomonas citri14, A. turbidans15, and X. campestris pv. campestris16 have been

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characterized, but there remains great interest in further expanding the number of

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characterized species isoforms in this class of enzymes to enable improvement in their

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properties by combinatorial or data-driven protein engineering techniques. The cloning of aeh

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genes and the overproduction of their products would be particularly worthwhile since their

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expression level in their natural hosts is low, varying from 0.5% to 3% of total cellular

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protein17–18.

-amino acid ligase (Lal)5. Since the initial study of AEH from Acetobacter turbidans8, many

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Previously, we screened microorganisms exhibiting AEH activity that can synthesize

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dipeptides such as Ile–Trp from Ile–OMe and Trp and isolated several bacteria with this

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property. Among the isolates, Stenotrophomonas maltophilia HS1 exhibited higher AEH

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activity than the other isolates. The fermentation conditions for AEH production were

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investigated18, but AEH production was insufficient to produce a material for further

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investigation, such as conducting detailed kinetic studies with highly purified protein or

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assessing its application to dipeptide synthesis. In this study, we thus aimed to clone the aeh

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gene from S. maltophilia HS1 and to characterize its recombinant AEH protein, which could

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potentially be useful for the synthesis of the dipeptide Ile–Trp, an important anti-hypertensive


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agent. A BLAST search revealed that the 16s RNA sequence of wild-type S. maltophilia HS1

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had 99% sequence identity with S. maltophilia R551-3. Furthermore, an analysis of the NCBI

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protein database revealed that the sequence of a putative X-pro dipeptidyl-peptidase domain

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protein (Smal_2367) of S. maltophilia R551-3 is 80% identical to a known AEH from X. citri.

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All of the key catalytic residues are conserved, including the catalytic triad, the carboxylate

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cluster, and the oxyanion hole (Figure S1(a), Supporting information). On the basis of these

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observations, the gene corresponding to Smal_2367 was cloned from the genomic DNA of S.

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maltophilia HS1, and the encoded protein was expressed in Escherichia coli. Since this

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protein exhibited properties of an AEH, the gene encoding it was named aehS. We succeeded

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in producing active AEH from S. maltophilia HS1 (SmAEH) in E. coli and characterized it.

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AEH is useful for producing value-added products16–17; however, its proper utilization in

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industrial applications requires engineering to facilitate optimization, design simulation, and

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control of industrial projects. The low thermal stability of AEH is the main obstacle to its

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industrial application19. Therefore, it is important to understand its thermal denaturation

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behavior in a properly controlled reaction to achieve maximum production efficiency; this

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includes evaluating the mutant enzymes from the view point of thermal stability. In the

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present work, we aimed at elucidating the thermal denaturation kinetics of recombinant

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SmAEH in detail. Various mathematical models that can predict enzyme residual activity as a

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function of time were analyzed statistically, and the kinetic model that best explains the

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thermal inactivation of SmAEH was selected. This model will facilitate inferences on the

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enzyme’s behavior during heat treatment, thereby improving procedure precision and the

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quality of the final products.

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Materials and Methods

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Materials

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L-Isoleucine

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methyl ester (L-Trp–OMe), L-tryptophan (L-Trp), L-glycine (L-Gly), and L-histidine (L-His)

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were purchased from Wako Pure Chemicals (Tokyo, Japan). Isoleucyl–tryptophan (Ile–Trp),

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valine–glycine (Val–Gly), and tryptophan–histidine (Trp–His) were from Watanabe

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Chemical Industry (Hiroshima, Japan). Diethylaminoethyl (DEAE)-Cellufine and DEAE-

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Sepharose were from JNC (Tokyo, Japan). The oligonucleotides for cloning of the aehS and

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DNA sequences were obtained from FASMAC (Kanagawa, Japan). The chemicals used in

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the DNA manipulation procedures were purchased from TaKaRa (Kusatsu, Japan) and used

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as recommended by the manufacturer. The other reagents were of analytical grade.

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Bacterial strains and plasmids

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S. maltophilia HS1 was grown at 30°C as described previously18. Chromosomal DNA was

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obtained using phenol/chloroform extraction and used as the template for PCR. The pET21a

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plasmid (Novagen) was used as an expression vector, and E. coli XL10-gold (Agilent

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Technologies) and E. coli Rosetta-gami B(DE3) (Novagen) were used for cloning and

140

recombinant protein expression, respectively.

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Cloning and protein expression

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Primers were initially designed based on the gene information of Smal_2367 (Figure S1(b),

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Supporting information) in order to isolate the complete open reading frame of the aehS gene

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from the chromosomal DNA of S. maltophilia HS1. The oligonucleotide primers used were

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5 ′ -ATGCTTTCCTGCCGGGAGGGGTTGTC-3 ′

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TCAGTACACCGGCAGATCGATGTAGCTGG-3′ (reverse). To construct the expression

methyl ester (L-Ile–OMe), L-valine methyl ester (L-Val–OMe), L-tryptophan


(forward)

and

5 ′ -

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vector, PCR was used with the 5′-GGAATTCCATATGCTTTCCTGCCGGGAGG-3′ as

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the forward primer and 5′-CCCAAGCTTTCAGTACACCGGCAGATCGAT-3′ as the

149

reverse. These primers contained Nde I and Hind III restriction sites (underlined),

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respectively. After DNA denaturation, the amplifications were carried out in 30 cycles of 10 s

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at 98°C, 30 s at 55°C, and 1 min at 68°C using KOD-plus neo as DNA polymerase according

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to the supplier’s instruction (TOYOBO, Osaka, Japan). The amplified DNA fragment was

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digested with Nde I and Hind III and ligated into pET21a linearized with the same restriction

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enzymes. This plasmid, pETSM, was used as an expression vector. The newly constructed

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plasmids were amplified in E. coli XL10-gold and then used to transform in E. coli Rosetta-

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gami B(DE3) cells for expression. The transformants were cultured at 30°C in 0.5 L of Luria

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Bertani medium containing 100 μg/mL ampicillin while shaking at 100 rpm. When the

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optical density at 600 nm reached about 0.8, isopropyl-β-D-thiogalactopyranoside was added

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to the culture medium at a final concentration of 0.2 mM. The cultures were incubated further

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at 20°C for 12 h, and cells were harvested by centrifugation (10,000 g for 10 min at 4°C).

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Protein purification

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The harvested cells were suspended in 20 mM Tris-HCl buffer (pH 8.0; buffer I) and treated

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with 0.5 mg/mL lysozyme at 4°C for 30 min. The cells were disrupted by sonication, and cell

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debris was removed by centrifugation. The resulting supernatant was dialyzed against buffer I

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and then loaded on a DEAE-Cellufine column pre-equilibrated with buffer I. SmAEH was

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eluted in the nonbinding fraction from the column and was dialyzed against buffer I. The

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SmAEH was then applied to a DEAE-Sepharose fast flow column equilibrated with buffer I

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and eluted with linear gradient from 0 to 150 mM NaCl. The SmAEH-containing fractions

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were collected and dialyzed against buffer I, and the purified enzyme was analyzed using



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sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and 10.5% gel.

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Enzymes were concentrated by ultrafiltration and stored at 4°C.

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Enzyme assays and determination of kinetic constants

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The amino-group transferring activity of AEH was routinely assayed at 20°C in 100 mM

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borate buffer (pH 10.5), 50 mM L-Ile–OMe as the acyl donor, and 100 mM Trp (pH 10.5) as

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the acyl acceptor. The formation of the Ile–Trp product was monitored by high-performance

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liquid chromatography (HPLC) with a COSMOSIL 5C18-MS-II column (4.6 × 250 mm;

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Nacalai Tesque, Inc., Kyoto, Japan) using the method of Tanaka et al17. Before analysis, the

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samples (5 µL) were quenched and diluted 100-fold by the addition of a derivatizing-

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fluorescent compound in the form of 10 mM Boc-Cys-OH and 20 mM phthalaldehyde in 400

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mM borate buffer (pH 9.0). Five µL of this mixture was then analyzed by HPLC. One unit of

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enzyme activity was defined as the amount required to produce 1 µmol Ile–Trp per minute

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under standard assay conditions. To determine the kinetics parameters, the enzyme was

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incubated with various concentrations of acyl donors in the range of 0–60 mM L-Ile–OMe, L-

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Val–OMe, or L-Trp–OMe, and acyl acceptors at 0–120 mM Trp and 0–250 or 0–250 mM His.

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The rates of dipeptide synthesis were estimated by measuring product formation by HPLC.

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Data were fitted using nonlinear regression using a Michaelis–Menten kinetics model, and

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the calculated kinetic parameters are presented with their standard deviations.

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MS analysis of SmAEH reaction products

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The reaction mixture was diluted 50-fold with Milli-Q water for matrix-assisted laser

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desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis. One µL

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of the sample was mixed with 1 µL of matrix solution on a plate, and the plate was dried and

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loaded onto the MALDI-TOF MS (autoflex speed, Bruker Daltonics K.K., Kanagawa, Japan).


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The matrix (2,5-dihydroxybenzoic acid; DHB) was dissolved in 0.1% trifluoroacetic

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acid:acetonitrile = 1:2.

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Heat inactivation experiments

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Thermal inactivation tests were performed in sealed tubes containing 1 mL of SmAEH. The

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tubes were immersed in a thermostatically controlled water bath (Personal-11 SM; Taitec

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Corporation, Japan), and the enzyme solutions were incubated at 35°C, 40°C, 45°C, or 50°C.

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After incubation, the tubes were immediately immersed in an ice bath, and SmAEH activity

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was measured using a mixture of 100 mM borate buffer (pH 10.5), 50 mM L-Ile–OMe, and

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100 mM Trp (pH 10.5). The activity after 1 min of heating was considered to be the initial

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activity (A0) for the purpose of calculating the residual activities in order to eliminate the

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effects of warming and to ensure evaluation of an isothermal process20. Assays were

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performed in triplicate. Residual activity data with respect to time at different temperatures

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were fitted to several models (Table 1) using nonlinear regression (Statistica 13.0; StatSoft,

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Inc., Tulsa, UK).

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Kinetic models of enzyme inactivation

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Kinetic models commonly used to describe the behavior of an enzyme during heat treatment

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were employed in this study (Table 1). In the equations, A represents the SmAEH activity at

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time t (min), A0 is the initial activity, and k (min−1) is the inactivation rate constant at a given

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temperature. Generally, first-order kinetics (Eq. 1) have been reported to describe the thermal

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inactivation of enzymes21. The temperature-dependent parameters for the first-order model

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are given by the Arrhenius equation, which describes the relationship of the thermal

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inactivation rate (k) and temperature and can be expressed algebraically:

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ln ( k ) = ln ( A ) −

Ea , RT


(6)


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where A is the Arrhenius constant, Ea is the activation energy, R is the universal gas constant

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(8.31 Jmol−1K−1), and T is the absolute temperature. However, we also considered other

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kinetics models; for equations 2 and 3, it has been suggested that the residual activity of an

219

enzyme could be the sum of two exponential decays, signifying the presence of a mixture of

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enzymes with different heat sensitivities and/or catalytic properties. These models were tested

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to check whether the SmAEH solution was composed of labile and resistant heat fractions,

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and kL and kR are the first-order reaction rate constants for the labile and resistant heat

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fractions, respectively. Moreover, coefficient “a” represents the active fraction of the

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thermolabile fraction in terms of the total activity, and Ar implies the nonzero activity of an

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enzyme after prolonged heating and assumes the presence of an extremely heat-resistant

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fraction22. Descriptions of nth-order decay (Eq. 4) for thermal inactivation of enzymes are

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also not exceptional23. The Weibull distribution (Eq. 5) is based on the assumption that, under

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the conditions examined, the momentary rate of thermal sensitivity to heat is only a factor of

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the transient heating intensity and residual activity but not of the rate at which the residual

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activity has been reached24. The values n and b characterize this model and represent the

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shape of the distribution curve and its scaling, respectively. Its temperature dependence can

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be expressed by a log-logistic equation (Eq. 7)25:

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b(T) = loge{1+ exp[k′(T −Tc )]},

234

where T marks the temperature level of the inactivation’s onset and K ' is the slope of b(T ) . c

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Comparison of kinetic models

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In general, the statistical standards for the assessment of models are the coefficient of

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determination(r2), chi-square (χ2), and standard error mean (SEM)26. χ2 was used to compare

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models of enzyme thermal inactivation20,25 and was determined as follows:


(7)

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χ2 =

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∑ ( ameasured − a predicted ) m− p

2

.

(8)

The SEM was determined using 2  (a  ∑ measured − a predicted ) . SEM =    m  

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(9)

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Each model was evaluated by an optimization criterion based on their sum of squares and the

243

parameters, where m is the number of observations and p is the number of parameters.

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To discriminate between kinetic models, statistical and physical criteria should be considered.

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The model with the lowest χ2 and SEM and highest r2 for the residual enzyme activity is the

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best choice from a statistical perspective20. Estimation of a negative value for parameters is a

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criterion to reject the inactivation model27.

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Thermodynamic analysis

249

Activation energy (Ea) can be estimated from the Arrhenius equation (Eq. 6). Using this value

250

of Ea, the inactivation enthalpy (∆H#) for each temperature was calculated by

251

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∆H # = Ea − RT .

(10)

The free energy of inactivation (∆G#) can be determined using the following expression:

 kh  ∆G # = − RT ln  ,  kBT 

(11)

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where h (6.6262 × 10−34 Js) is the Planck’s constant, KB (1.3806 × 10−23 JK−1) is the

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Boltzmann’s constant, and k (s−1) is the inactivation rate constant for each temperature.

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From equations 10 and 11, it is possible to calculate the inactivation entropy (∆S#) as follows:

∆S # =

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∆H # − ∆G # . T

(12)

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Nucleotide sequence accession number

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The nucleotide sequence for SmAEH has been submitted to the DNA Database of Japan and

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has been assigned the accession no. LC319754.

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Results and discussion

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Since 1974, amino acid sequence information on only two AEHs that have been

265

experimentally characterized has been available in the NCBI database, although they have

266

been repeatedly assessed for use in biocatalysts. To clone the gene encoding the AEH of S.

267

maltophilia (SmAEH), we designed oligonucleotide primers based on the first six amino

268

acids of the putative X-pro dipeptidyl-peptidase domain protein (NCBI GenBank ID

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ACF52070.1) and used them with the chromosomal DNA of S. maltophilia to produce a PCR

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product of 2.0-kb. Sequencing indicated that this product shared approximately 94%

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sequence identity with a putative X-pro dipeptidyl-peptidase domain protein of S. maltophilia

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R551-3 encoded with a polypeptide of 645 amino acids with a calculated molecular mass of

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72,019 Da. Database searches for homologous proteins using position-specific iterated PSI-

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BLAST28 found that the deduced amino acid sequence of SmAEH was homologous with

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several protein sequences, the majority of which are from genome-sequencing projects and

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have an unidentified function; however, protein information retrieved from the UniProt

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database suggested that most of the homologous proteins were either AEHs or glutaryl-7-

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ACA acylases (Table S1, Supporting information). Moreover, these homologous proteins

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encoded the consensus sequence GXSYXG, where X is a nonconserved amino acid,


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suggesting that the proteins are serine hydrolases. Surprisingly, this motif was also conserved

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in SmAEH, suggesting that it functions as a serine hydrolase; this was further supported by

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the observation that SmAEH is inactivated by PMSF (data not shown). Until a few years ago,

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it was believed that all lipases and carboxyl esterases contain the consensus sequence motif

284

GXSXG around the active site serine29. However, some exceptions to this have recently been

285

described.

286

To verify the identity of the ester hydrolase gene, the recombinant plasmid pETSM

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was introduced into E. coli RGB DE3 competent cells for heterologous expression of the

288

enzyme, and cell extracts were analyzed with an AEH activity assay. The extracts had a high

289

level of AEH activity compared with those produced from cells containing the pET21(a)

290

plasmid. The recombinant SmAEH was purified by chromatography on DEAE-Cellufine,

291

followed by DEAE-Sepharose resins. The specific activity of SmAEH increased at each step

292

to approximately 11.3 U/mg, representing a seven-fold purification (Table 2). A clear single

293

band corresponding to a protein with a molecular mass of around 72 kDa was observed by

294

SDS–PAGE (Figure 1). This is consistent with the masses of purified native AEHs from A.

295

turbidans and the Xanthomonas family, which are 74 and 72 kDa protein, respectively14,15.

296

The activity of SmAEH was measured at different temperatures and pH using

297

isoleucine methyl ester and tryptophan as substrates. The optimal pH for SmAEH activity

298

was 10.5, and the optimal temperature was 20°C (Figures S2a and S2b, Supporting

299

information). The activity of the enzyme was almost stable between pH 9.0 and 11.0, and it

300

did not exhibit any high requirements for any metals for activity (Figure S2c, Supporting

301

information). A slight inhibition was observed in the presence of Fe3+ and Co2+ ions, with a

302

decrease of around 30% of its original activity, and one of 20% in the presence of K+. A

303

similar study showed that AEH from Elizabethkingia sp. has no significant requirement for

304

metal ions, and its maximum activity occurred at pH 9.0 and a temperature of 25°C17. In



14 305

another study, AEH from Empedobacter brevis ATCC14234 exhibited the highest activity at

306

pH 8.5 and a temperature of 30°C30. However, Bacillus mycoides AEH has a maximum

307

activity at pH 8.0 and a temperature of 45°C9.

308

HPLC analyses of SmAEH reaction mixtures (Figure S3, Supporting information)

309

showed that products were formed in a time-dependent manner. In particular, Ile–Trp (peak

310

no. 3) was detected after 30 min and increased further after 1 h. Moreover, in comparisons

311

with standards and zero-min HPLC peaks, time-dependent reaction studied showed a new

312

peak with a retention time of 34 min, presumably reflecting presence of Ile-Trp; however, no

313

peaks corresponding to the formation of Ile–Ile–OMe were observed. For checking

314

hydrolysis of the product by the enzyme, enzymatic reaction using standard Ile-Trp as a

315

substrate instead of Ile-OMe was performed under the same condition as synthetic reaction.

316

No breakdown of di-peptide, Ile-Trp was observed after 2-h reaction time. The reaction

317

mixture was also analyzed by MALDI-TOF MS, and a peak was detected at m/z 318.41,

318

which corresponds to [Ile–Trp+H]+, indicating the formation of Ile–Trp under these reaction

319

condition (Figure S4, Supporting information). Furthermore, other peaks such as those at m/z

320

132.18, 205.23, 227.23, 243.33, 281.36, 396.64, and 544.77, which correspond to [Ile+H]+,

321

[Trp+H]+, [Trp+Na]+, [Trp+K]+, [Ile–Ile–OMe+Na]+, [Ile–Ile–Ile+K]+, and [Ile–Ile–Ile–

322

Trp+H]+, were also detected, suggesting the formation of peptides other than Ile–Trp as well

323

as simple hydrolysis of the donor substrate. HPLC analysis estimated that the Ile–Trp

324

concentration reached approximately 5 mM from 50 mM L-Ile–OMe and 100 mM Trp and

325

that of dipeptide Val–Gly and Trp–His reached 8 and 4 mM, each of 50 mM acyl donor and

326

200 mM acyl acceptor, respectively. Yokozeki and Hara found that AEH from E. brevis

327

ATCC produced 12 and 4 mM Val–Gln and Ile–Gln, each of 100 mM acyl donor and 200

328

mM acyl acceptor, respectively10, and in another study, AEH from Sphingobacterium

329

siyangensis synthesized 10 and 2 mM Val–Gln and Ile–Gln30, where each of 100 mM acyl


Page 14 of 41

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donor and 200 mM acyl acceptor, respectively. To study the substrate specificity of

331

recombinant SmAEH, steady-state kinetic parameters were measured for a variety of

332

substrates, and Michaelis–Menten kinetics were analyzed. The Km values for the acyl donors

333

L-Ile–OMe, L-Val–OMe,

334

respectively, and those for the acyl acceptors L-Trp, L-Gly, and L-His were 35, 65, and 55

335

mM (Table 3), respectively. However, AEHs from X. citri and A. turbidans have Km of 90

336

and 7 mM for D-phenylglycine methyl ester and 1.8 and 0.34 mM for cephalexin,

337

respectively14–15. In our study, SmAEH exhibited variability in substrate concentration

338

requirements for different acyl donors and acceptors, which supports a previous finding that

339

the nature of the acyl group has a large influence on AEH enzyme kinetics15. Furthermore,

340

the hydrolysis capability of SmAEH for various acyl donors was investigated using a

341

previously described colorimetric method17. The hydrolytic activity assay for SmAEH

342

showed that it had a relatively strong substrate preference for hydrophobic amino acid methyl

343

esters (Figure S5, Supporting information). Unfortunately, owing to the lack of standard

344

dipeptides in our laboratory, the preferences of SmAEH for acceptor substrates could not be

345

evaluated in this study. Examination of the acceptor preference of SmAEH will be performed

346

in the future, since it will provide useful information for the synthesis of other valyl

347

derivatives.

and L-Trp–OMe were determined to be 10.4, 22, and 27 mM,

348

The most closely related proteins whose activities have been described are the AEHs

349

from X. citri (XcAEH) and A. turbidans (AtAEH); the activity of these enzymes against α-

350

amino acid esters has been reported, and their crystal structures have been determined14,31. In

351

our study, the deduced amino acid sequence of SmAEH shared 77% and 58% sequence

352

identity with XcAEH (PDB ID 1MPX) and AtAEH (PDB ID 2V9B). Homology modeling of

353

SmAEH was performed based on XcAEH as a template and using the Swiss model server32,

354

and the model was validated using an Internal Coordinate Mechanics (ICM)-based computer



16 355

program via a method adapted from Hossain et al33. The model structure resembled the

356

experimental structure (Figure S6, Supporting information), and the catalytic triad residues of

357

SmAEH were Ser-180, Asp-261, and His-294, which correspond to residues Ser-174, Asp-

358

255, and His-288 of XcAEH. A hypothesis on the AEH enzyme family by Barends et al.14

359

suggests that a carboxylate group cluster near the catalytic triad is responsible for AEH

360

specificity toward Nα-free amino acid esters. We aligned the sequences of experimentally

361

characterized AEHs with SmAEH and one unreported AEH from Elizabethkingia sp.

362

identified in our lab and found that this also supports the hypothesis of the presence of a

363

carboxylate cluster group (Figure 2a). We judged that the catalytic site of SmAEH is

364

structurally related to XcAEH and AtAEH, and hence, we superimposed the catalytic triad

365

and carboxylate cluster residues and found that the root-mean-square deviation values of

366

SmAEH were 0.07 Å with XcAEH and 0.20 Å with AtAEH (Figure 2b and 2c), suggesting

367

that the AEH catalytic sites are structurally unique.

368

From both scientific and technological perspectives, inactivation kinetics are essential

369

to enzyme characterization. AEHs catalyze the synthesis and hydrolysis of α-amino β-lactam

370

antibiotics and are used as alternatives to the more commonly used penicillin G acylase14–16.

371

Semi-synthetic β-lactam antibiotics make up approximately 65% of the total world market for

372

antibiotics of $15 billion34. Moreover, AEHs are of particular interest for use in the one-pot

373

synthesis of semi-synthetic antibiotics such as ampicillin directly from Penicillin G, thus

374

eliminating intermediate states16; however, inactivation behavior studies of AEH would be

375

essential for its incorporation into such industrial processes. This would lead to lower costs

376

and more environmentally friendly production of semi-synthetic antibiotics and peptides

377

compared to traditional chemical synthesis and existing enzymatic synthesis. In 2010, Blum

378

and Bommarius16 tried to determine the inactivation pattern of AEH from X. campestris pv.

379

campestris ATCC33913, but their data did not follow first-order kinetics, and this issue


Page 16 of 41

Page 17 of 41


17 380

remains unresolved. Therefore, from an industrial perspective on the AEH enzyme, it is

381

essential to assess the effects of heat treatment on residual activity without performing

382

numerous trial runs; with this in mind, several mathematical equations have been proposed to

383

explain the behavior of SmAEH during thermal degradation. A graphical representation of

384

the residual activities during heat inactivation fitted to a first-order equation is presented in

385

Figure 3, showing a satisfactory fit of the experimental data to the predicted curves. Table 4

386

presents the results from five inactivation kinetic models that were tested to fit the

387

experimental data for heat treatments of SmAEH. Among these, fractional conversion (Eq. 3)

388

produced negative parameters, which are physical criteria to reject the models, since the

389

residual activity for SmAEH must be positive. And, in the analysis of two fractions (Eq. 2),

390

the data showed equal parameters and were rejected as well. The nth order model (Eq. 4)

391

produced low r2 and high SEM and χ2 values compared with the other models, so it was also

392

rejected. In general, enzyme inactivation is characterized by a first-order model. However, in

393

recent years, the appropriateness and utility of the Weibull distribution have been widely

394

discussed35, and it has been proposed to explain the thermal inactivation of both enzymes and

395

microorganisms27. Thus, the Weibull distribution was tested for its suitability for fitting the

396

experimental data from SmAEH inactivation. Both first-order and Weibull distribution

397

models produced good fits for the data (Table 4) with very similar r2, χ2, and SEM values.

398

The r2 values for the Weibull distribution model ranged from 0.980 to 0.997, and those for

399

the first-order model ranged from 0.989 to 0.998. The χ2 values for the first-order model were

400

lower than those for the Weibull pattern, ranging from 0.022 to 0.00027 and from 0.0482 to

401

0.00137, respectively. The SEM ranged from 0.026 to 0.00077 for the first-order model and

402

from 0.027 to 0.00224 for the Weibull. The data obtained for the analyzed conditions were

403

very similar for both models, making it difficult to choose between them. Therefore, the

404

temperature-dependent parameters obtained for the first-order (k values) and Weibull



18 405

distribution (b values) models were fitted to the Arrhenius (Eq. 6) and log-logistic (Eq. 7)

406

equations, as described elsewhere20. The r2 for the Arrhenius equation gave a good fit to the

407

data, with a value of 0.984 (Figure 4). For the Weibull model parameters, the log-logistic

408

equation produced a comparatively low r2 value of 0.74 (data not shown). This comparatively

409

poor fit of the Weibull model to the experimental data supports the conclusion that the first-

410

order equation is the best model to explain the thermal inactivation of SmAEH.

411

Thermal inactivation concepts (D and z values) are generally used to represent a first-

412

order reaction. The decimal reduction time (D value) is the time needed for a 10-fold

413

reduction of the initial activity at a given temperature, and it is obtained by plotting activity

414

on a log scale against inactivation time. The z value is the temperature needed to reduce the D

415

value one log unit and is obtained by plotting D values on a log scale against the

416

corresponding temperatures20. The data obtained for the k, D, and z values for SmAEH for

417

heat treatments between 35°C and 50°C are shown in Table 5. In general, the rate constant

418

increased with higher heating temperatures while D values decreased, indicating a faster

419

inactivation at higher temperatures. D values ranged between 212.76 and 3.44 min at

420

temperatures between 35°C and 50°C, respectively. The effect of the temperature on the D

421

values is shown in Figure 5, and the calculated z value for the temperature range studied was

422

approximately 8.06°C.

423

In our study, data for the residual SmAEH activity best fit the first-order model and

424

yielded decent statistical criteria for the conditions evaluated. The values for r2, SEM, and χ2

425

were comparable to those calculated for model acceptance in other investigations20–21,27,35.

426

This is the first of such report on an AEH enzyme, although there are broad classes of

427

enzymes36,37, bio-active substances20, and peptides38 that obey first-order kinetics. Figure 3

428

signified data that SmAEH exhibits a single-step exponential behavior and provides

429

suggestions for a possible deactivation mechanism. Since alterations in pH do not strongly


Page 18 of 41

Page 19 of 41


19 430

interfere with the enzyme deactivation rate (data not shown), it is unlikely that inactivation is

431

due to reactions such as reshuffling or β-elimination of cysteine residues, which are

432

extremely pH dependent36, although SmAEH enzymes have four cysteine residues. The pH

433

range studied also ruled out inactivation from the hydrolysis of the peptide chain. In our study,

434

enzyme activity decreased as heating time increased. It is apparent in single step first-order

435

reaction that only one reason controlling overall inactivation rate leading to enzyme

436

denaturation37. In this study, the most likely cause of inactivation with increasing temperature

437

is the deamidation of the asparagine or glutamine residues. The sequence of SmAEH consists

438

of approximately 7% glutamine (4.5%) and asparagine (2.5%). This high percentage may be

439

one of the causes of enzyme inactivation at elevated temperatures. Furthermore, it has been

440

reported that a temperature increase of 15°C above the optimum resulted in a 75% reduction

441

in the half-life of an asparagine peptide39. These observations support the hypothesis that

442

deamidation is temperature sensitive and that proteins exposed to high temperatures are

443

especially prone to deamidation and subsequent enzyme inactivation40. Another study found

444

that, during an increase in temperature from 5°C to 65°C, peptide chain deamidation behaves

445

in accordance with the Arrhenius equation41. However, a direct biochemical analysis is

446

required to validate that deamidation actually occurs during the heat inactivation of SmAEH.

447

The temperature dependence of the k-values obtained for the first-order model was

448

fitted to the Arrhenius equation (Eq. 6), and the result is shown in Figure 4. The r2 value was

449

0.984, indicating that 98.4% of the total variation is explained by the linear regression. The

450

deactivation energy (Ea) can be considered to represent the energy that needs to be absorbed

451

or released for the molecules to be able to react42 and it can be estimated from the Arrhenius

452

equation. In general, the higher the Ea, the higher the energy barrier that must be crossed for

453

enzyme inactivation to occur; higher values therefore indicate increased stability. The

454

estimated Ea for SmAEH inactivation was 204.1 kJ mol−1, suggesting that energy needed to



20 455

be absorbed from the external medium to initiate inactivation at temperatures between 35°C

456

and 50°C; the data fitted well, which enabled the calculation of thermodynamic parameters

457

(Table 5) using transition state theory (Eqs. 10–12). ∆H# (enthalpy) increased, and ∆G# (free

458

energy) decreased with increasing temperature, suggesting that destabilization of SmAEH

459

occurred42. Positive ∆H# values are indicative of the endothermic character of the inactivation

460

process43. However, the ∆S# (entropy) values did not exhibit a continuous behavior,

461

suggesting that the increase of temperature destroyed ordered structures, leading to a fragile

462

state. ∆H# and ∆S# represent heat and entropy changes, respectively, and these two

463

parameters also provide a measure of the number of noncovalent bonds broken and the net

464

change in enzyme/solvent disorder associated with the formation of the transition state44.

465

Lals or proteases have also been reported as other enzymatic methods for synthesizing

466

dipeptides. Peptide synthesis using proteases has been extensively studied by protecting or

467

freeing either one or both N and C-terminal ends of acyl donors and acceptors45. Despite

468

numerous trials, proteases fail to meet industrial requirements in terms of productivity,

469

reaction rate, yield, proportion of acyl acceptors to acyl donors, and breadth of applicability

470

to a sufficiently wide range of peptides. In Lal-based methods, processes suffer from the low

471

accumulation and productivity of the target dipeptide5, reflecting technological nascence of

472

dipeptide formulations. By contrast, AEHs synthesize dipeptides efficiently and irreversibly

473

using Nα-free amino acid esters as acyl donors and a Nα- and Cα-free amino acids as an acyl

474

acceptors9,17. Although several reports on the chemical and enzymatic synthesis of dipeptides

475

are available, all of these processes have acknowledged limitations with respect to

476

incorporation of hydrophobic amino acids. Synthesis of highly hydrophobic dipeptides is a

477

difficult and challenging task because of their low solubility in both aqueous and organic

478

solvents. SmAEH in our study had optimum pH at 10.5, which makes it particularly

479

advantageous for dissolving hydrophobic amino acids, and the enzyme exhibited a wide


Page 20 of 41

Page 21 of 41


21 480

specificity toward hydrophobic substrates. This is because the alkaline buffer facilitated

481

dissolution of hydrophobic amino acids. Nothing has been published on the synthesis of the

482

dipeptide Ile–Trp, and this is the first report on the synthesis of this important anti-

483

hypertensive agent. Our study covered the characterization of SmAEH and the analysis of

484

inactivation kinetics, which should prove valuable for industrial applications of its bioproduct

485

synthesis capabilities. Our analysis will be useful for the future engineering of AEH enzymes.

486

Competing interests

487

The authors have declared that no competing interest exists.

488

Acknowledgments

489

We are grateful to Monbukagakusho, The Ministry of Education, Culture, Sports, Science

490

and Technology in Japan (MEXT), for financial support (MEXT scholarship).

491

Supporting information

492

Figure S1 Multiple sequence alignments and primer sequence information data

493

Table S1 Amino acid sequence similarities of the AEH of S. maltophilia HS1

494

Figure S2 Characterization of SmAEH

495

Figure S3 High-performance liquid chromatography (HPLC) profiles of SmAEH reactions

496

over time.

497

Figure S4 MALDI-TOF MS analysis of the SmAEH reaction

498

Figure S5 Substrate specificity of SmAEH for hydrolysis reaction

499

Figure S6 Homology model of SmAEH

500



22

501

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642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658


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659

Figure legends

660

Figure 1. SDS–PAGE of recombinant AEH protein from Stenotrophomonas maltophilia.

661

SDS–PAGE (10% separating and 3% stacking) gel stained with Coomassie brilliant blue.

662

Lane 1, pETSM-expressed protein lysate; lane 2, DEAE-Cellufine; and lane 3, DEAE-

663

Sepharose (2 µg protein). The band corresponding to SmAEH is indicated. Molecular mass

664

markers were loaded in the lane labeled M, and their masses are shown on the left in kDa.

665

Figure 2. (a) Partial alignment of SmAEH with experimentally characterized AEHs. The

666

sequences shown are AEHs from Stenotrophomonas maltophilia HS1 (SmAEH, in this

667

study), Elizabethkingia sp. (EsAEH, data from our lab), Acetobacter turbidans (AtAEH,

668

protein ID no. AAL60195), and Xanthomonas citri (XcAEH, protein ID no. AAO24758).

669

The alignment was performed using the online “MUSCLE” multiple sequence alignment

670

server46. Catalytic triad residues within sequences are shaded black, and carboxylate cluster

671

residues are shaded red. (b and c) Superposition of the catalytic triad and carboxyl cluster

672

residues of SmAEH with XcAEH (b) and AtAEH (c). SmAEH is shown as red sticks, and

673

XcAEH and AtAEH are in green. Residues are labeled black and are numbered. The figure

674

was prepared using Molscript47.

675

Figure 3. Thermal inactivation SmAEH. Data are averages of two independent experiments.

676

The standard deviations are always lower than 6%. Residual activity was determined after

677

incubation at 35°C (closed square), 40°C (closed rectangle), 45°C (closed angle), and 50°C

678

(closed circle). Data were fitted to the first-order model (Eq. 1).

679

Figure 4. Arrhenius plot for inactivation of SmAEH. The graph was obtained by plotting the

680

logarithm of the rate constant ( k ) versus the inverse temperature (1/T). The regression

681

equation was determined to be − ln ( k ) = 24552T −1 − 75.142 ( r 2 = 0.984 ) . Data are presented as

682

the mean ± standard errors of the mean (SEM) of three independent experiments.



Page 30 of 41

30 683

Figure 5. Correlation between log(D) and temperature for the thermal inactivation of SmAEH.

684

The graph was obtained by plotting the D values on a log scale against the corresponding

685

temperatures.

686

log ( D ) = −0.124T + 6.7357 r 2 = 0.987 . Data were presented as the mean ± standard errors of

687

the mean (SEM) of three independent experiments.

The

regression

(

equation

was

)


determined

to

be

Page 31 of 41


31

Table 1. Kinetic equations used to analyze inactivation of L-Amino acid ester hydrolase Equation no. Model 1

Equations

First-order20

A A0

= exp ( −kt )

2

Two-fraction48

A = aAL exp ( −kLt ) + (1 − a ) exp ( −kRt ) A0

3

Fractional conversion22

A = Ar + ( A0 − Ar ) exp ( −kt ) A0

4

nth order23

5

Weibull distribution24

1/ (1− n ) A = { A01−n + ( n − 1) kt} A0

A = exp ( −bt n ) A0



Page 32 of 41

32

Table 2. Purification of SmAEH from E. coli Rosetta-gami B (DE3) expressing pETSM plasmid

Steps

Total protein (mg)

Total activity (U)

Specific activity (U/mg)

Yield (%)

Purification fold

Cell free extract

150.0

250

1.7

100

1

DEAE-Cellufine

25.0

175

7.0

70.0

4.21

DEAE-Sepharose

4.5

51

11.3

20.4

6.80


Page 33 of 41


33

Table 3. Kinetics parameter of SmAEH

Substrates

Mean ± SE of:

Acyl donor Acyl Acceptor

Km (mM) acyl donor

Km (mM) acyl acceptor

kcat (s-1) acyl donor

kcat (s-1) acyl acceptor

kcat/Km acyl donor

kcat/Km acyl acceptor

L-Ile-OMe

L-Trp

10.4±0.8

35±1.5

17.26±0.8

17.5±1.0

1.65±0.15

0.50±0.05

L-Val-OMe

L -Gly

22.0±1.5

65±5.0

10.16±0.8

10.5±0.5

0.37±0.02

0.16±0.01

L-Trp-OMe

L -His

27.0±2.0

55±3.0

7.05±0.5

7.0±0.6

0.26±0.04

0.13±0.01



Page 34 of 41

34

Table 4. Statistical parameters of the kinetic models describing thermal inactivation of SmAEH

r2

χ2

SEM

Remarks

First-order (1)

0.989; 0.998

0.022; 0.00027

0.026;0.00077

Accepted: higher r2and low x ଶ and SEM

Two-fraction (2)

0.987; 0.998

0.0886; 0.0012

0.0511;.0026

Rejected: equal parameter estimates

Fractional conversion (3)

0.995; 0.998

0.0894; 0.02651

0.013;.0009

Rejected: negative parameter estimates

nth order (4)

0.945; 0.988

0.094; 0.026

0.0936;0.025

Rejected: low r2 and high x ଶ and SEM

Weibull distribution (5)

0.980; 0.997

0.0482; 0.00137

0.027; 0.00224

Rejected: low r2 and high x ଶ and SEM

Model (Eq.)


Page 35 of 41


35

Table 5. Kinetics and thermodynamics data for thermal inactivation of SmAEH

Temperature (°C)

k (min-1)

35

0.011

40

D (min)

∆H#

∆G#

∆S#

63.0

212.80

201.61

87.08

371.85

0.029

23.90

81.30

201.56

86.01

369.17

45

0.16

4.30

12.10

201.52

80.56

380.40

50

0.39

1.80

3.40

201.48

81.87

370.34

t 1/2 (min)



Page 36 of 41

36

(kDa)

M

1

2

3

204 114 84 72 kDa

62 46

27

14

Figure 1


Page 37 of 41


37

(a) SmAEH EsAEH AtAEH XcAEH

151 126 174 143

RVDHSTDAWDTIDWLVKNVPESNGKVGMLGSSYKGFTVVMALTDPHPALKVAAPQSPMVDGWMSDDWLNYGA-FRPGQLQ GIDESTDTFDTLEWLSKNLKNYNQKAGVYGISYPGFYSTTTLVNSHPTLKAVSPQAPVTNWYLGDDFHHKGAMFLNDAFM KTDETTDAWDTVDWLVHNVPESNGRVGMTGSSYEGFTVVMALLDPHPALKVAAPESPMVDGWMGDDWFHYGA-FRQGAFD EVDHATDAWDTIDWLVKNVSESNGKVGMIGSSYEGFTVVMALTNPHPALKVAVPESPMIDGWMGDDWFNYGA-FRQVNFD

SmAEH EsAEH AtAEH XcAEH

325 288 328 297

LKVPTMWLQGLWDQEDMWGANHAYQAMEGRDSGNNRNYLVMGPWRHSQ-VNYSGSELGALKFDGDTALQFRRDVLKPFFD PAV--MVVGGFFDAEDAYGTFETYKAIEKQNPKAN-NILVAGPWFHGGWVRGDGKQFGDIKFDHPTSIDYQQNLELPFFN PTVPMLWEQGLWDQEDMWGAIHAWQALKDADVKAP-NTLVMGPWRHSG-VNYNGSTLGPLEFEGDTAHQYRRDVFRPFFD LKVPTMWLQGLWDQEDMWGAIHSYAAMEPRDKRNTLNYLVMGPWRHSQ-VNYDGSALGALNFEGDTARQFRHDVLRPFFD

(b)

(c)

Figure 2



Page 38 of 41

38

1 0.9 0.8 0.7

A/A0

0.6 0.5 0.4 0.3 0.2 0.1 0 0

10

20

30

40

Time (min) Figure 3


50

60

70

80

Page 39 of 41


39

5 4.5 4

-ln(k)

3.5 3 2.5 2 1.5 1 0.5 0 0.00308 0.00311 0.00314 0.00317 0.0032 0.00323 0.00326 1/T (k-1)

Figure 4



Page 40 of 41

40

3 2.5

Log (D)

2 1.5 1 0.5 0 30

35

40 45 Temperature ( °C)

Figure 5


50

55

Page 41 of 41


41

TOC graphics (width, 3.30 inch; height 1.85 inch)


Characterization and thermal denaturation kinetic analysis of

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