Generation of a broad esterolytic subtilisin using combined molecular evolution and periplasmic expression

Grazyna E. Sroga and Jonathan S. Dordick,1

Department of Chemical Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180-3590, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Concomitant activity improvement of an evolved enzyme toward two very different ester substrates was achieved when a unique combination of functional periplasmic enzyme expression in Escherichia coli, random mutagenesis, DNA shuffling and cell-based kinetic screenings was applied. Specifically, we focused on the conversion of subtilisin E into an enzyme with broader esterase activity as opposed to its native amidase activity. Cell-based microtiter assays were performed on N-acetyl-D,L-phenylalanine p-nitrophenyl ester (Phe-NPE) and sucrose 1'-adipate (S1'A), as well as on the tetrapeptide amide substrate N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide. After a single modified cycle of directed molecular evolution, we isolated a number of clones exhibiting increased activity toward Phe-NPE. In the following rounds of screenings, mutants with improved activity on Phe-NPE were also tested on S1'A. Three mutants were identified with increased esterolytic activity on Phe-NPE and S1'A, while having similar amidase activity to that of the parental enzymes. Because the two ester substrates are structurally distinct, we have evolved a more general esterolytic subtilisin and this may have important applications in synthesis.

Keywords: cell-based kinetic assays/esterolytic activity/molecular evolution/periplasmic expression/subtilisin


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Altering the substrate specificity of an enzyme is an important yet challenging task. Enzymes are fine-tuned by nature to exhibit a reaction specificity that often does not coincide with desired properties and activities useful for biotechnological applications. Although recombinant DNA technology and molecular biological techniques allow the creation of a protein with nearly any amino acid sequence, our present understanding of enzymes is insufficient for the de novo design of enzyme function. More recently, combinatorial protein engineering approaches that mimic evolutionary processes have become increasingly popular (Orencia et al., 2000Go). Combinatorial molecular evolution of available proteins is most commonly performed by creating libraries of random mutants, which are generated by mutagenic (error-prone) PCR, cassette mutagenesis, DNA shuffling (Stemmer, 1994Go), domain swapping (Hopfner et al., 1998Go; Nixon et al., 1998Go) or generation of interspecies enzyme chimeras (Campbell et al., 1997Go). In the present work, we extend molecular evolution approaches to the transformation of enzyme reactivity from one reaction class to another. Specifically, we endeavored to transform a protease into a broadly selective esterase.

Esterases and lipases catalyze a diverse array of esterolytic transformations on a large number of natural and unnatural ester substrates (Kazlauskas, 1994Go; Andersch et al., 1997Go; Schmid and Verger, 1998Go; Jaeger et al., 1999Go). These enzymes often exhibit high enantio- and regioselectivities (Reetz et al., 1997Go; Henke and Bornscheuer, 1999Go; Liebeton et al., 2000Go); however, there remains a need for more diverse esterolytic and ester synthesizing enzymes, particularly those with activities on a broad range of structurally complex molecules. Proteases (including amidases and peptidases) also catalyze ester hydrolysis (and synthesis in nonaqueous media) (Barbas et al., 1988Go; Kawashiro et al., 1996; Park et al., 2000Go; Kilbanov, 2001Go) and are well known to possess broad substrate specificities, particularly on complex compounds (Kraut, 1977Go). Nevertheless, proteases are more reactive on amides than on esters.

Subtilisin E (from Bacillus subtilis) was chosen as our model enzyme due to its natural amidase activity together with its relatively weak esterolytic activity. Many properties of subtilisins have been altered by classical site-directed mutagenesis (Carter and Wells, 1987Go; Wells and Powers, 1987Go; Wells et al., 1987aGo,bGo). Recently, directed molecular evolution of subtilisins was performed based on the expression of mutant libraries in the native host, Bacillus subtilis (You and Arnold, 1994Go; Ness et al., 1999Go; Zhao and Arnold, 1999Go) or by the unconventional product capture phage display applicable for a very specific catalytic event (Atwell and Wells, 1999Go). However, none of those studies addressed the esterolytic activity of the enzyme.

Therefore, the goal of this work was to use molecular evolution strategies to convert a protease into a broadly selective esterase. Our particular interest is the extent to which one can change these two functions by manipulating subtilisin E at the molecular level. Thus, we focused our effort on evolving enzymes with altered activities toward different esters, including sugar esters and we have developed a set of general cell-based screening procedures. In the process, we also developed a strategy that combines periplasmic expression of subtilisin E in Escherichia coli with directed molecular evolution for generation of subtilisin E mutants with broader catalytic activity toward ester substrates.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Materials

N-acetyl-D,L-phenylalanine p-nitrophenyl ester (Phe-NPE), N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (AAPF-NA) and bromothymol blue (BTB) were from Sigma (St. Louis, MO). Sucrose1'-adipate (S1'A) synthesized according to our published procedure (Park et al., 2000Go). Plasmids pBE4M, pTG5-3HT and p12M were provided by Frances H.Arnold (California Institute of Technology, Pasadena, CA). These plasmids contained the wild-type (WT) gene of subtilisin E (sprE), the temperature resistant gene (TeR) and the dimethylformamide (DMF)-resistant gene.

Construction of plasmids

Unless otherwise stated, all molecular biology methods used in our study were performed according to the standard protocols described by Sambrook et al. (Sambrook et al., 1989Go). WT-, TeR- and DMF-sprE genes were transferred into the pBAD/gIII prokaryotic expression vector (Invitrogen, Carlsbad, CA) giving pGES201 (WT-sprE), pGES202 (DMF-sprE) and pGES203 (TeR-sprE) plasmids, respectively. PCR was used to create 5'- and 3'-sprE DNA protruding ends in frame with the gene III secretion signal and the C-terminal myc-epitope tag followed by the 6xHis tag. Escherichia coli TOP10 was used as the bacterial host in all experiments. After identification of the respective clones, the cloning of the genes in the correct frame was confirmed by sequencing of both strands.

Restriction of DNA for mutagenesis

Plasmids pGES201, pGES202 and pGES203 were cut with NheI and XbaI (New England Biolabs, Beverly, MA) according to the manufacturer's protocol. The resulting linear DNA was electrophoresed in a 0.8% agarose gel and the bands corresponding to 1.2 kb were excised and the respective DNA was purified using the Gel Purification Kit (Qiagen, Chatsworth, CA).

Generation of mutant libraries by mutagenic-PCR

Initially, gel-purified WT subtilisin E gene (1154 bp) was subjected to a single random mutagenesis by PCR (M-PCR) performed under different conditions. The sequences of the M-PCR primers used were as follows: (5'-GCAATTTAA-GTATGTTAACGC-3') and (5'-TTGTTCTAGAATTCCTTGTGC-3'). The standard PCR buffer (Sigma) was supplemented with 6.5 mM MgCl2, 0.20 mM dGATPs, 0.80 mM dTCTPs, 2.2 µg of single strand binding (SSB) protein (Promega, Madison, WI) (Sroga et al., 1996Go), 0.40 µM of forward and reverse PCR primers, 10 ng of WT-sprE DNA as the PCR template and 5.0 units of Taq DNA polymerase (Sigma). The concentration of MnCl2 varied and equaled 0.10 mM (M-PCR I), 0.15 mM (M-PCR II) or 0.20 mM (M-PCR III) in the tested PCR mixes. The reactions were run in a Mastercycler Gradient 5331 (Eppendorf Sci. Inc., Westbury, NY) according to the following cycle program: 40 cycles of: 1 min at 94°C, 1 min at 50°C and 2 min at 72°C. The standard PCR, in which the M-PCR primers were used, was done according to the Sigma protocol included with the Taq DNA polymerase.

Generation of mutant libraries using combined mutagenic-PCR and DNA shuffling by StEP

Each gel-purified subtilisin E gene (WT-, DMF-, TeR-sprE) of 1154 bp was subjected to random mutagenesis by M-PCR. The PCR primers were the same as of the ones used in a simple M-PCR. The respective PCR mixtures (100 µl) contained 10 mM Tris–HCl pH 8.30, 50 mM KCl, 6.5 mM MgCl2, 0.15 mM MnCl2, 0.20 mM dGATPs, 0.80 mM dTCTPs, 2.2 µg of SSB protein (Sroga et al., 1996Go), 0.50 µM of forward and reverse PCR primers, a total of 10 ng of DNA templates and 2.5 units of Taq DNA polymerase. The reactions were run in a Mastercycler Gradient 5331 (Eppendorf Sci. Inc.) according to the following cycle program: 13 cycles of: 1 min at 94°C, 1 min at 50°C and 1 min at 72°C, followed by a 10 min extension reaction at 72°C.

The mutagenized DNA fragments were then subjected to DNA shuffling by the StEP recombination protocol (Zhao et al., 1998Go). Our cycling program differed and was 5 min at 95°C followed by 90 cycles of: 30 s at 94°C, 6 s at 50°C. After StEP recombination, 1 µl samples of the respective shuffled DNA mixtures were amplified according to the PCR protocol described in the work of Sroga et al. (Sroga et al., 1996Go) and 1 µl of this final PCR mix was subjected to a second cycle of mutagenesis and shuffling. The Qiagen PCR purification kit was used for isolation of the PCR-amplified DNA fragments from the final reaction mixtures. After cleavage with NotI and XbaI, the PCR-DNA and pGES201 were ligated according to the manufacturer's protocol included with the T4 DNA ligase. Ligation mixtures were transformed into E.coli TOP10 according to the Invitrogen protocol submitted with the strain.

Functional expression of subtilisin E

Expression of functional subtilisin E was performed at 20°C in the complete minimal (CM) medium [g/l: K2HPO4, 10.50; KH2PO4, 1.13; (NH4)2SO4, 1.00; sodium citratex2H2O, 0.50; MgSO4x7H2O, 0.25; CaCl2x2H2O, 0.015] supplemented with 20 amino acids (each 40 µg/ml) and vitamin B1 (5 mg/l). Glycerol was used at a concentration of 0.20% (v/v). Ampicillin was used at the concentration of 100 µg/ml. Each induction experiment performed in 96-well microtiter plates (MT-plates) was preceded by cultivation of bacterial cells to a steady state of balanced growth (Shehata and Marr, 1970Go; Koch and Higgins, 1982Go). Thus, single-cell clones carrying subtilisin E plasmids were grown in 200 µl of nutrient medium with ampicillin in 96-well MT-plates overnight at 37°C with shaking at 225 r.p.m. (orbital shaker model 420 S/N 18069-130, Forma Scientific, Inc., USA). The cultures were re-inoculated into the CM medium and grown for an additional 8–9 h. These cultures were used as the low-density inocula of the CM medium (200 µl) supplemented with ampicillin and were then grown overnight. The resulting `balanced-for-growth' (`pseudo-synchronized') cultures (100 µl) were transferred to a new MT-plate, centrifuged and suspended in 200 µl (OD590 = ~0.40) of the respective CM medium, grown for an additional 2 h and then induced by 0.20% (w/v) arabinose. After 6 h of induction at 20°C, the cells were harvested and ready for use.

Screening of libraries for amidase activity

Proteolytic (amidase) activity of subtilisin E was measured spectrophotometrically by the release of p-nitroaniline from AAPF-NA. Pellets from 100 µl of induced cells or 25 µl of the respective periplasmic fractions, isolated according to the Invitrogen protocol included with the pBAD/gIII vector, were suspended in 200 µl of osmotic buffer I [OSI; 20 mM Tris–HCl pH 8.00, 2.5 mM EDTA, 2 mM CaCl2, 20% (w/v) sucrose] and 300 µM AAPF-NA. All reactions were performed for 60–90 min at 24°C. The amount of released p-nitroaniline ({varepsilon}405 = 9.33x10-3/µM/cm) was determined by measuring the absorbance at 405 nm in an HTS 7000 Plus Bio Assay Reader (Perkin-Elmer, Norwalk, CT). Initial reaction rate data were collected and calculated using the HTS 2.0 software. The respective extinction coefficients were taken into account in calculation of the initial reaction velocities.

Screening of libraries for esterase activity

Esterolytic activity of subtilisin E was measured for two substrates, Phe-NPE and S1'A. Hydrolysis of Phe-NPE was measured spectrophotometrically by the release of p-nitrophenol ({varepsilon}405 = 1.57x10-2/µM/cm) in a similar way as described for AAPF-NA hydrolysis. Hydrolysis of S1'A was performed under different conditions. Bacterial pellets from 100 µl of induced clones were suspended in 200 µl of osmotic buffer III (OSIII; 5 mM Tris–HCl pH 7.80, 1 mM EDTA, 2 mM CaCl2) with 1 mM of S1'A and 0.5 mM BTB. The reactions were performed at 24°C for 3–4 h. Formation of the basic form of BTB ({varepsilon}405 = 7.51x10-3/µM/cm) was monitored at 405 nm. The data were collected and calculated using the HTS 2.0 software.

Sequences of selected mutants

Selected clones were streaked out and isolated as single-cell colonies, grown and induced in 10 ml scale batch cultures and their activity was tested again as described above. To identify selected mutants, DNA was isolated from the respective clones using the Mini-prep DNA Isolation Kit (Qiagen) and sequenced. We designed a forward sequencing primer that anneals upstream of the PBAD promoter and the respective reverse primer that anneals downstream of the terminator sequence of the pBAD/gIII vector. The DNA was sequenced on both strands using the Perkin-Elmer/Applied Biosystems Taq DyeDeoxy terminator sequencing kit and the Applied Biosystems model 373A DNA sequencer. DNA and amino acid sequence analysis was done using the Prophet 6.0 program (National Computing Resource for Life Sciences, NIH).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Strategies for directed molecular evolution and functional expression

Based on the methodology of Reetz and Jaeger (Reetz and Jaeger, 1999Go) and Liebeton et al. (Liebeton et al., 2000Go), we calculated that approximately 50% of active clones is optimal for random mutagenesis of a gene designated for in vitro molecular evolution, when the first and second generation of the respective mutants are to be evolved in a single mutagenic step. Therefore, we used 50% of active clones as a target for our DNA mutagenesis experiments. Mutagenic efficiency of our M-PCRs was determined by DNA and amino acid sequence analysis of created mutants, as well as by evaluation of mutant enzyme activities using an amidase screening assay.

Initially, we designed and tested a few simple mutagenic PCR protocols (Table IGo) for generation of subtilisin mutant libraries. We determined that manganese concentration influenced both the efficiency of the DNA mutagenesis (Tables I and IIGoGo) and the DNA yield (Figure 1Go). The highest DNA yield was observed at 0.10 mM MnCl2 (M-PCR I; Figure 1Go, lane 1). However, this did not correspond to the desired 50% level of inactive clones. As shown in Table IGo, approximately 83% clones were active (>40% WT-subtilisin E amidase activity) when a library was generated by M-PCR I. Conversely, at 0.20 mM MnCl2 not only was the DNA yield low (M-PCR III; Figure 1Go, lane 3), but restriction analysis of DNA from 40 randomly chosen clones revealed that only 10% of them had the correct size DNA fragment (942 bp) corresponding to subtilisin E (data not shown). Furthermore, among this subset of clones, only 2% were active (at least 10% WT subtilisin amidase activity; Table IGo, M-PCR III). Sequence analysis confirmed that simple M-PCRs either introduced too few mutations per gene (e.g. 0–1) or did not generate the desired (approximately 50%) number of active and inactive clones. As expected, the vast majority of tested mutants had similar amidase activity to WT subtilisin E (approximately 75–85% active clones per library; Table IGo, M-PCR I, II and IV) when the MnCl2 concentration was less than 0.20 mM.


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Table I. Frequency of active clones generated by different PCR protocols
 

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Table II. Comparison of unique mutations introduced to subtilisin E genes using different mutagenesis protocolsa
 


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Fig. 1. Agarose gel (1%) showing the DNA yield obtained from the three different M-PCRs. The PCR mixtures differed in the MnCl2 concentration. The 942 bp DNA fragment of subtilisin amplified by M-PCR I (lane 1, 0.10 mM MnCl2); M-PCR II (lane 2, 0.15 mM MnCl2); M-PCR III (lane 3, 0.20 mM MnCl2). Lane 4: Molecular Weight Standard No. X (Roche Molecular Biochemicals, Indianapolis, IN).

 
Because none of the tested simple mutagenic PCR protocols satisfied our requirements for mutagenesis efficiency, we extended a simple strategy for random evolution of an enzyme, which involves creating and screening libraries of enzyme variants that differ from the parent by only one or two amino acids (Reetz and Jaeger, 1999Go; Liebeton et al., 2000Go). Thus, we introduced a combined approach involving random point mutagenesis (M-PCR IV) and in vitro random recombination of sequence blocks (Stemmer, 1994Go) from three subtilisin E parental genes. Each of the three subtilisin E genes, WT-sprE, DMF-sprE and TeR-sprE, was mutagenized separately by PCR (M-PCR IV, Table IGo). As expected, due to base misincorporation and mispairing, the DNA fragments generated by simple M-PCR IV protocol contained a suitable low level of point mutations (from 1 to 2).

To increase the genetic diversity within created gene populations, the respective populations of mutants were shuffled directly after mutagenic PCR using the modified StEP protocol (Zhao et al., 1998Go). This cycle of mutagenesis and shuffling was then repeated. The important difference between our protocol and the ones described in the literature is that we performed the second mutagenic PCR and shuffling using the mixture of already once mutated and shuffled sprE genes as the DNA template (Tables I and IIGoGo). Thus, we omitted one intermediate step of transformation and screening for amidase and esterase activities. Using this approach, a total of five libraries were created, each one containing 1.2–1.8x104 clones with the desired population of active and inactive mutants per library (approximately 50%; Table IIGo). Therefore, we did not pursue additional rounds of mutation and shuffling. Among the populations of created mutant genes, there was a prevalence of both true and silent codon mutations (Table IIGo); however, opal stop codons and frameshift mutations were also observed.

We transferred the entire subtilisin E gene together with its pre-pro-region into the pBAD/gIII vector. This was crucial as the pre-pro-sequence not only directs the extracellular secretion of the enzyme, but also functions as the intramolecular chaperon critical for subtilisin E maturation (Ikemura et al., 1987Go; Zhu et al., 1989Go; Bott and Betzel, 1996Go). Depending on the expression level, subtilisin production could be toxic to E.coli cells (Bott and Betzel, 1996Go) and this could lead to spontaneous mutations outside our target region of subtilisin E. In order to distinguish clones generated by our mutagenesis process from those that emerged spontaneously, we designed our own sequencing primers, where the forward primer anneals upstream of the PBAD promoter and the reverse primer anneals downstream of the terminator. Thus, we monitored sequences of the entire subtilisin E flanks including the PBAD promoter, the pre-pro-region of subtilisin E mutants, the C-terminal myc epitope tag, the 6xHis tag and the terminator sequence, for any spontaneous mutations. It was not surprising to find a frameshift-knock out mutation in the pre-pro-region of subtilisin (one out of 45 sequenced clones; Table IIGo). However, none of the sequenced clones had spontaneous mutations within the C-terminal myc epitope tag, the 6xHis tag or the terminator sequence (Table IIGo). As described below, our protocol significantly improved the overall quality of the libraries created such that a relatively small number of screened clones was suitable to identify desired enzymatic traits.

Cell-based assays for the measurement of subtilisin esterolytic activity could not be developed based on enzyme expression in its native host, B.subtilis, due to the presence of a number of endogenous lipases and esterases that hydrolyzed our ester substrates and interfered with screening for desired activities. To overcome this problem, we chose E.coli as the bacterial host, where such interfering enzyme activities were not observed. As a result, we were able to develop cell-based kinetic assays for measurement of subtilisin esterolytic activity. Using a combination of low (20°C) expression temperature and a relatively high [0.10–0.15% (w/v)] concentration of the arabinose inducer, we increased the expression level of functional subtilisin approximately 8- to 9-fold as compared to the level of expression at 30°C. This was possible because the arabinose inducer is not toxic to E.coli, and thus, can be used at much higher concentrations than, for example, IPTG (IPTG alone is toxic to E.coli) used with the Ptac promoter (Takagi et al., 1988Go). The cells were cultivated to a steady state of balanced growth (Shehata and Marr, 1970Go; Koch and Higgins, 1982Go) before the induction of subtilisin E expression (see Materials and methods) and this resulted in uniform and further improved periplasmic production of recombinant subtilisin E libraries. All the aforementioned factors contributed to the development of three assays for measurement of subtilisin activity either in the periplasmic fractions or in the whole cells.

Two cell-based kinetic assays for screening of mutant libraries functioned directly in the OS I buffer which contained 20% (w/v) sucrose, and thus, bacterial cells remained intact. The assays were developed using Phe-NPE and AAPF-NA as the substrates, and were based on the generation of the characteristic yellow color attributed to the released p-nitrophenol or p-nitroaniline product. Both compounds were found to be outer membrane permeable (data not shown) and, therefore, accessible to active subtilisin E mutants expressed in the E.coli periplasm. The criterion of the developed screens was the ratio of initial reaction rates of amidase to esterase activity. Calculating ratios eliminated the influence of promoter-dependent and gene-dependent differential expression levels on enzyme activity for the different mutant enzymes generated. A third screening procedure was developed for S1'A hydrolysis that was based on the generation of protons during ester hydrolysis (Figure 2Go), which can be detected by BTB at 405 nm. Any other relevant pH indicator could be used instead of BTB as well.



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Fig. 2. Substrate and products of S1'A hydrolysis reaction catalyzed by subtilisin E and improved enzyme mutants. The screening is based on the release of vinyl adipate, which causes a pH decrease detected by BTB.

 
Functional improvement of subtilisin E toward esterase activity

A total of 1139 randomly chosen clones were used for the development of the cell-based kinetic screening assays (Figure 3Go). Out of this initial pool, 639 clones were transferred to wells in seven MT-plates containing 200 µl of nutrient medium supplemented with ampicillin. These clones became our stock-clone cultures and were used in various experiments leading to the development of the two esterase assays. Each MT plate was divided into two sections. Thus, the same batch culture of the respective clone was tested simultaneously for both amidase and esterase activities (critical for accurate comparison of the amidase/esterase rate ratios). Three types of controls were always used, the buffers, the E.coli host carrying pBAD/gIII vector and E.coli expressing WT-subtilisin E (GES201). One additional reason for the regular use of the OSIII buffer (the pH/BTB assay) as the control was the assumption that environmental CO2 could interfere with the pH/BTB assay. However, the interference from environmental CO2, if any within the relatively short time of the assay, was neither observed nor detected spectrophotometrically. A very small drop of pH, most likely caused by the natural E.coli's pH of approximately 7.2–7.4, was observed in the microtiter wells where E.coli carrying the pBAD/gIII vector was used as the control.



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Fig. 3. Strategy of cell-based kinetic screenings leading to identification of GES206, GES207 and GES208 mutants.

 
After the first round of screens with Phe-NPE, 200 clones showing various levels of increased activity towards Phe-NPE were chosen and then re-screened resulting in the selection of 96 clones that consistently showed increased activity toward Phe-NPE. Three clones showed the highest activity on Phe-NPE and were designated GES206, GES207 and GES208. These three mutants were then streaked out, isolated as single-cell colonies, grown in 10 ml batch cultures and evaluated for their respective amidase and esterase activities. Periplasmic fractions were also isolated and analyzed. Notably, our preliminary data demonstrated that subtilisin E mutants could be captured via the 6xHis tag on Ni-NTA magnetic agarose beads (Qiagen) added to the respective periplasmic fractions (data not shown).

Native subtilisin E (GES201) hydrolyzes Phe-NPE approximately 15-fold more slowly than AAPF-NA (Table IIIGo). The other two parents, DMF-subtilisin E (GES202) and TeR-subtilisin E (GES203), were also substantially more reactive toward the amidase substrate. GES206 and GES207 had comparable activities on both AAPF-NA and Phe-NPE, while GES208 had nearly 5-fold higher activity on the ester substrate than on the amidase substrate (Table IVGo). For GES208, this represented an approximately 70-fold swing in the ratio of esterase/amidase activity when compared with WT subtilisin E. Closer inspection of the data in Table IIIGo reveals that the increased esterase activity was mostly responsible for the decreased amidase/esterase ratio. Specifically, the increase in esterase activity for GES208 versus the three parent enzymes was approximately 25-fold, whereas the amidase activity decreased 2.5-fold for GES208 as compared to the average of the three parents. Thus, we were able to improve the desired esterolytic activity of subtilisin E without crippling the overall enzyme activity; a critical requirement for synthetic utility.


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Table III. Amidase (v0[AAPF-NA]) and esterase(v0[Phe-NPE] and v0[S1'A]) initial reaction rates and the respective rates ratios for WT and selected subtilisin E mutants
 

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Table IV. Initial reaction rates for proteolytic (v0[AAPF-NA]) and esterolytic (v0[Phe-NPE]) activity of subtilisins measured in the periplasmic fractions of E.coli cells producing WT-, DMF- and TeR-subtilisin E as well as the respective rate ratios
 
We also screened the 96 mutants for hydrolysis of the S1'A. Once again, GES206, GES207 and GES208 emerged as the most active enzymes for sucrose ester hydrolysis. As detailed in Table IIIGo, GES206, GES207 and GES208 were approximately 4.8-, 7.4- and 3.8-fold more active, respectively, on S1'A than the parent enzymes. This represented approximately 8-, 9- and 9.6-fold swings in the ratio of esterase/amidase activity for the respective mutants when compared with WT subtilisin E. Due to the vastly different structures of Phe-NPE and S1'A, these results suggest that the three mutant enzymes possessed general esterase activity.

As shown in Table IVGo, the overall initial reaction rates were approximately 1.4–1.7-fold lower, when measured in the periplasmic fractions (25 µl/Periind), as compared to the values determined for the induced whole cells (100 µl/cellind). However, the respective initial reaction rate ratios for the three parental enzymes determined in the periplasmic fractions were the same as for the whole cells, indicating that the enzyme in the whole cells is expressed in the mature state in the periplasm.

Structural characteristics of mutant subtilisins

To investigate the correlation between structure and observed activity, the respective DNA samples of GES206, GES207 and GES208 were sequenced. The DNA makeup of the parents and the three clones is shown in Figure 4Go. Sequence analysis revealed that the GES206 mutant is mainly the descendant of WT-sprE and TeR-sprE parents with an insert of approximately 120–123 bp from the DMF-sprE. The GES207 mutant derives from the DMF-sprE and WT-sprE parents. Finally, the GES208 mutant inherited the most of its genetic material from the WT-sprE parent with short insertions from the DMF-sprE (approximately 40 bp) and TeR-subtilisin E (approximately 30 bp) parents (Figure 4Go). Thus, in two cases (GES206, GES208) all three parents contributed to the genetic makeup of the evolved mutants and in one case (GES207) the genetic material originated from two parents instead of three.



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Fig. 4. Parental composition of the subtilisin E mutants based on DNA sequence analysis. Shuffled blocks are colored to parental origin: WT-subtilisin E (green), DMF-subtilisin E (blue), TeR-subtilisin E (red). The DNA base mutations shown are unique for each mutant. Boxes mark the inferred crossovers.

 
Next, the amino acid sequences of GES206, GES207 and GES208 were determined and compared with the parents (Table VGo). A total of 16 amino acid residues were changed by our molecular evolution strategy when compared to the three parents globally. Of these 16 residues, only three gave unique amino acids relative to the parent enzymes at specific positions; L16P for GES206, V139G for GES207 and V143H for all three mutants. Interestingly, the GES208 mutant, which gave the highest esterase activity on Phe-NPE, differed from the WT by only two amino acids—V143H and D181N (Table VGo), the latter clearly due to shuffling involving the short inserts from the DMF and TeR enzymes (Figure 4Go) as discussed above. Conversely, the GES207 mutant that differed from the WT subtilisin E by 11 amino acids gave the highest esterase activity on S1'A. Finally, the GES206 mutant differed from the WT subtilisin E by nine amino acids and gave comparable esterase activity on both Phe-NPE and S1'A.


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Table V. Amino acid (AA) mutations identified in the three evolved mutants in comparison with the parental sequences
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The choice of an appropriate expression system for our enzyme mutant libraries was based on two main factors, our screening requirements and the ester substrates to be used. Since our cell-based assays for the measurement of subtilisin esterolytic activity could neither be developed nor could be performed in B.subtilis, we used E.coli as the bacterial host. Additionally, we chose the E.coli periplasm as the site of library expression, which resulted in a significantly greater accessibility of the recombinant protein for in vivo assays and efficiently minimized the well known toxic effect of subtilisin expression on E.coli (Bott and Betzel, 1996Go). A secreted periplasmic expression system also offers some additional benefits, which may be useful for directed molecular evolution of enzymes. For example, periplasmic expression quite efficiently prevents recombinant proteins from proteolysis [only seven out of 25 known E.coli proteases are present in the periplasm (Swamy and Goldberg, 1982Go; Baynex and Georgiou, 1991)]. Periplasmic expression significantly minimizes the formation of inclusion bodies. For non-secreted proteins, the N-terminal amino acid residue is preserved identical to the natural gene product, as it is protected by the signal sequence. Purification of recombinant proteins is much simpler due to the overall lower number of proteins in the periplasm than in the cytoplasm. The periplasmic space also provides an oxidative environment, which is often required for maturation of various recombinant proteins (Wulfing and Pluckthun, 1994Go; Makrides, 1996Go). Bacterial libraries (currently super-transformation efficiency of bacteria can reach up to approximately 5x109; the Stratagene catalog) and microbial cell surface display technologies are very attractive for high-throughput screening strategies (Georgiou, 2000Go). Finally, whole bacterial cells can be used as biocatalysts, which is useful in screens, particularly for cofactor-dependent enzymes (Joo et al., 1999Go).

Active subtilisin has been expressed in the E.coli periplasm (Ikemura et al., 1987Go; Takagi et al., 1988Go); however, induction of subtilisin E in 100 µl suspensions of the respective E.coli cells according to the published protocol (Ikemura et al., 1987Go; Takagi et al., 1988Go) showed that the p-NA production, if any, was below the detection limit of our amidase assay. Therefore, we searched for a more efficient and versatile periplasmic system and settled on the pBAD/gIII vector. The pBAD/gIII vector enables the control of subtilisin E expression level that is critical for the adjustment of selection stringency at each screening step. As a result, significantly improved expression of functional subtilisin E was achieved and this was supported by the unique combination of growth conditions and the induction procedure specific for the aforementioned vector. Some additional benefits of using the pBAD/gII vector are introduction of the 6xHis tag to the C-terminus of mutant subtilisins that simplifies protein purification, as well as the introduction of the C-terminal myc epitope tag for easy immunological identification of the respective recombinant proteins.

We modified a protocol for generation of low-level point-mutation libraries. A search of libraries containing low rates of random nucleotide substitutions has been dictated by both experimental limitations and theoretical considerations (Reetz and Jaeger, 1999Go; Liebeton et al., 2000Go). When a low rate of mutations is used, a large fraction of all the possible amino-acid substitutions (`sequence space') may be represented in a relatively small library. The theoretical number of different subtilisin E mutants with a single amino acid exchange per enzyme macromolecule is 5225 (calculated as described by Reetz and Jaeger, 1999Go). Also, a low-rate mutagenesis is considered necessary to maintain the fraction of deleterious mutations at a tolerable level (Kuchner and Arnold, 1997Go; Reetz and Jaeger, 1999Go). The iterative screening of relatively small libraries of mutants with a low frequency of nucleotide substitutions has proven to be very effective for the functional improvement of numerous proteins (Arnold, 2001Go).

Using the screening flexibility of the developed kinetic assays based on the pBAD/gIII periplasmic expression vector, and the power of directed evolution and gene shuffling, we quickly identified three mutant subtilisins with increased ratios of esterase to amidase activities. Importantly, the increased esterase activity was not obtained at the expense of overall subtilisin activity.

Figure 5Go depicts subtilisin's structure containing secondary structural elements (Siezen and Leunissen, 1997Go). Both V139G and V143H (the mutants in GES207) lie in an {alpha}-helix arm that protrudes away from the active site, while L16P (from GES206) lies in another {alpha}-helix near the amino terminus. All three mutations lie far from the active site. This result indicates that striking changes in substrate specificity can occur in regions of the enzyme distant from the active site. To date, we know that mutations away from the active site have pronounced effects in protein function, with very minor structural changes, especially to the active site cavity shape (Arkin and Wells, 1998Go; Oue et al., 1999Go). It is interesting that most of the proteins evolved by combinatorial molecular evolution that have been studied so far, have a very low number of mutations within the active site cavity itself. These global mutational changes are currently very difficult to predict a priori; thus, many lessons can be learned from the structural analysis of directed evolution derived enzymes.



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Fig. 5. Ribbon-plot representation of the secondary and tertiary structure of model subtilisin (PDB code: 2SNI; Siezen and Leunissen, 1997Go) used for superimposition of mutations (L16P, V139G, V143H) evolved in the subtilisin E mutants. {alpha}-Helices are shown as ribbons and ß-sheet strands as arrows. Side chains of the catalytic residues (S221, H64, D32) and oxyanion binding site (N155) are shown in ball-and-stick representation.

 
Another interesting finding is that the hydrophobic valine in the 143 position is conserved in all three parents, yet it is converted to histidine in all three mutants. The V143H mutation, therefore, appears to be pivotal in converting subtilisin E into a general esterase. An intriguing observation is that the 143 position lines the binding pocket for the peptide substrate. Thus, it is possible that the introduction of histidine in the binding pocket of the three mutants could facilitate improved binding (or positioning) of the respective ester substrates. It is important to note that the same V143H mutation evolved within the three different amino acid sequence contexts originating from the three different parents (Figure 4Go, Table VGo). Thus, the combination of the unique amino acid environment of each mutant with the V143H mutation could result in the observable catalytic differences among them.

While sequencing data alone cannot provide the information necessary to ascertain the order in which the mutations were introduced and through which of the applied mutagenic processes, they are sufficient for establishing the final parental makeup of each mutant (Figure 4Go, Table VGo). In contrast to classical breeding, more than two parents can contribute to each of the progeny depending on the molecular breeding format used. As shown here, all three parents contributed to the final genetic makeup of two mutants, GES206 and GES208, and only two of them contributed their genetic material to evolved GES207.

In conclusion, we developed a strategy for generating and identifying mutant subtilisins with esterase activity. Although the desired activity seldom emerges in a single step of directed evolution, rather it increases slowly during successive rounds of selection, we showed that some useful mutants could be isolated in one round of directed molecular evolution when high-quality libraries are used. Evolution of broad esterolytic activity from a proteolytic enzyme has not been previously reported. Enzymes with broad substrate specificity are in high demand for industrial applications for selective transformations over a wide array of structures (Schmid et al., 2001Go). By using the principle of micro-reversibility, the evolved mutants may be useful in regioselective ester synthesis in nonaqueous media. This is currently under evaluation for applications ranging from sugar-based polymers to synthetic nucleoside derivatives.


    Notes
 
1 To whom correspondence should be addressed. E-mail: dordick{at}rpi.edu Back


    Acknowledgments
 
This work was supported by Biotechnology Research and Development Corporation.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
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Received March 27, 2001; revised July 19, 2001; accepted August 7, 2001.





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