molecules Article
Site-Specific, Covalent Immobilization of Dehalogenase ST2570 Catalyzed by Formylglycine-Generating Enzymes and Its Application in Batch and Semi-Continuous Flow Reactors Hui Jian, Yingwu Wang *, Yan Bai, Rong Li and Renjun Gao * Key Laboratory for Molecular Enzymology and Engineering, the Ministry of Education, School of Life Science, Jilin University, Changchun 130012, China;
[email protected] (H.J.);
[email protected] (Y.B.);
[email protected] (R.L.) * Correspondence:
[email protected] (Y.W.);
[email protected] (R.G.); Tel.: +86-138-4407-2416 (Y.W.); +86-431-8515-5212 (R.G.); Fax: +86-431-8515-5200 (R.G.) Academic Editor: Roberto Fernandez-Lafuente Received: 2 April 2016; Accepted: 5 July 2016; Published: 11 July 2016
Abstract: Formylglycine-generating enzymes can selectively recognize and oxidize cysteine residues within the sulfatase sub motif at the terminus of proteins to form aldehyde-bearing formylglycine (FGly) residues, and are normally used in protein labeling. In this study, an aldehyde tag was introduced to proteins using formylglycine-generating enzymes encoded by a reconstructed set of the pET28a plasmid system for enzyme immobilization. The haloacid dehalogenase ST2570 from Sulfolobus tokodaii was used as a model enzyme. The C-terminal aldehyde-tagged ST2570 (ST2570CQ) exhibited significant enzymological properties, such as new free aldehyde groups, a high level of protein expression and improved enzyme activity. SBA-15 has widely been used as an immobilization support for its large surface and excellent thermal and chemical stability. It was functionalized with amino groups by aminopropyltriethoxysilane. The C-terminal aldehyde-tagged ST2570 was immobilized to SBA-15 by covalent binding. The site-specific immobilization of ST2570 avoided the chemical denaturation that occurs in general covalent immobilization and resulted in better fastening compared to physical adsorption. The site-specific immobilized ST2570 showed 3-fold higher thermal stability, 1.2-fold higher catalytic ability and improved operational stability than free ST2570. The site-specific immobilized ST2570 retained 60% of its original activity after seven cycles of batch operation, and it was superior to the ST2570 immobilized to SBA-15 by physical adsorption, which loses 40% of its original activity when used for the second time. It is remarkable that the site-specific immobilized ST2570 still retained 100% of its original activity after 10 cycles of reuse in the semi-continuous flow reactor. Overall, these results provide support for the industrial-scale production and application of site-specific, covalently immobilized ST2570. Keywords: immobilization; dehalogenase; formylglycine-generating enzyme; Sulfolobus tokodaii
1. Introduction Enzymes are biocatalysts that catalyze reactions under mild conditions with high efficiency, and stereospecificity or enantioselectivity, and they also reduce or avoid adverse conditions. Enzymes are used in organic synthesis, brewing, food, medicine and other fields. However, enzymes exhibit limited mechanical and storage stability for some reactions and cannot be reused. With the emergence of protein modification and immobilization techniques, enzymes have become conducive to automatisation and continuous use for industrial production. Molecules 2016, 21, 895; doi:10.3390/molecules21070895
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Enzyme immobilization may alter enzyme property and performance in many different ways, which depends on the methods used, a proper immobilization may improve stability and, in certain cases, activity, while a bad selection of the immobilization strategy may drive to the enzyme inactivation and even decrease enzyme stability [1]. Physical methods including physical adsorption and entrapment etc., which are simple, however the immobilized enzymes with those methods maybe leaked out from the matrix during operation [2]. The immobilization of enzymes via multi-point chemical covalent binding sometimes may result in a certain loss of observed activity, however, the chemical stability will be significantly enhanced and the enzyme leakage could be minimized [3]. In addition, immobilization may provide other benefits such as creating compatible environment for enzyme, and avoiding multi-layer packing of enzymes etc., such that improved specific activity may be observed after immobilization in certain cases [4]. Enzyme orientation during immobilization may play a critical role in the recovered activity, for example, when the substrate is large and the active center is oriented towards the surface of the support this may raise some steric hindrances, or when the interaction of a specific area of the enzyme with the enzyme may drive to their inactivation [5]. Modifying enzymes by adding a tag can maintain enzyme activity and avoid enzyme inactivation caused by chemical reactions. Various techniques have been proposed for the chemical modification of proteins [6]. Introduction of short peptides to the target protein is a commonly useful method to achieve selective and site-specific modification of a proteins. With this method, target proteins can be recognized selectively and modified by a co-expressed enzyme in vivo or in vitro [7–13]. The reaction between the protein and the supports is therefore more controlled. Table 1 lists the enzyme-mediated systems used for the modification of target proteins. Prokaryotic formylglycine-generating enzyme (FGE) is used for protein labeling and was first identified in 2003 [14]. Several researchers reported that FGE can recognize a six-residue sulfatase submotif (LCTPSR) and oxidize the cysteine residue selectively within the motif to form an aldehyde-bearing formylglycine (FGly) residue [15,16]. The target protein can be fused with a LCTPSR submotif for modification by a co-expressed FGE. Thus, the free aldehyde group on the surface of the target protein can be used for site-specific covalent binding to amino-functionalised fluoresceins or immobilization supports through a Schiff base reaction [10]. Haloacid dehalogenases exhibit a potential for pharmaceutical and chemical industrial use and for the remediation of contaminated land [17]. In previous work, haloacid dehalogenase from the thermophile archaeon Sulfolobus tokodaii str.7 (ST2570) was cloned and characterized [18]. This enzyme belongs to the L-2-halogen acid (L-2-HAD) superfamily and is vital for industrial applications because of its efficient catalytic activity, stereoselectivity, thermal stability and tolerance to a wide pH range. As a monomer globin, ST2570 was a suitable model enzyme for testing site-specific covalent immobilization [19]. Many scientists have successfully fused a sulfatase sub-motif (also called an “aldehyde tag”) to target proteins by PCR reaction using a specific primer design before the construction of an expression plasmid. In this study, the pET28a plasmid was reconstructed, as shown in Scheme 1A. The gene encoding the peptide LCTPSR was introduced at different positions in pET28a, and a new set of vectors were obtained: pET28aDH, pET28aCQ, pET28aNQ and pET28aDQ. Thus, the sulfatase submotif can be easily fused to any target protein at different positions without designing specific primers. Two expression plasmids containing the ST2570 gene and FGE gene would be transformed into bacteria for co-expression. The host that retains both plasmids will co-express ST2570 and FGE in the presence of L-pectinose and isopropyl β-D-1-thiogalactopyranoside (IPTG). In this study, four proteins that are either wildtype enzymes or modified enzymes with aldehyde groups in different positions will be obtained (Scheme 1B).
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Table 1. Enzymes-mediated systems used in protein modification. Enzymes
Recognition Motif
Mechanism of Enzyme-Mediated System
Ref(s)
sortase A
LPXTG
A cysteine residue in sortase catalyzes the cleavage of the peptide bond between the threonine and glycine residues within the motif. Sortase will accept the N-terminus of an oligoglycine nucleophile.
[7]
microbial transglutaminase (mTG)
WALQRPH
TG2 mediates the incorporation of functionalized amines into a high-affinity Gln-substrate peptide tag.
[8]
farnesyltransferase (PFTase)
CAAX
PFTase catalyzes the transfer of a farnesyl isoprenoid group from farnesyl pyrophosphate to a sulfur atom present in the cysteine residue of the CAAX box.
[9]
formylglycine generating enzyme (FGE)
LCXPXR
FGE converts cysteine to formylglycine (FGly) within the LCTPSR motif. Proteins bearing a unique aldehyde group were chemically modified by selective reaction with hydrazide- or aminooxy-functionalized reagents.
[10]
biotin ligase (BirA)
GLNDIFEAQKIEWHE
BirA catalyzes the sequence-specific ligation of the biotin or the ketone isostere of biotin to the lysine side-chain of a 15-amino-acid acceptor peptide (AP).
[11]
microbial lipoic acid ligase (LplA)
DEVLVEIETDKAVLEVPGGEEE
LplA catalyzes the specific attachment of octanoic acid, 6-thio-octanoic acid, selenolipoic acid and alkyl azide to an engineered LplA acceptor peptide (LAP). The alkyl azide was selectively derivatized with cyclooctyne conjugates to various probes.
[12]
N-myristoyl transferase (NMT)
GXXXS
NMT specifically attaches myristate or modified myristate to peptides with amino-terminal Gly residues. Peptides with Asn, Gln, Ser, Val or Leu penultimate to the amino terminal Gly are substrates.
[13]
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Scheme 1. Schematic of the co-expression system thethe aldehyde tagging of proteins in Scheme 1. Schematic of the E. E. colicoli co-expression systemused usedforfor aldehyde tagging of proteins vivo. (A) the reconstructed plasmids; (B) the expression of four kinds ST2570 that are either wildtype in vivo. (A) the reconstructed plasmids; (B) the expression of four kinds ST2570 that are either wildtype enzymes or modified enzymes with aldehyde groups in different positions; (C) the one-step specific enzymes or modified enzymes with aldehyde groups in different positions; (C) the one-step specific immobilization of ST2570; (D) the immobilized ST2570. immobilization of ST2570; (D) the immobilized ST2570.
It is well-known that both the method and the support used might impact the efficiency of immobilization. Inorganic supportsand can form manageable particles large the surface areas, of Itenzyme is well-known that both the method the support used mightwith impact efficiency are reactive towards derivatizing/functionalizing agents and present excellent thermal and microbial enzyme immobilization. Inorganic supports can form manageable particles with large surface areas, resistance, reasonable mechanical strength and chemical resistance under operational conditions. On are reactive towards derivatizing/functionalizing agents and present excellent thermal and microbial the other hand, mesoporous supports not only exhibit a very high specific surface (up to 500 m2/g), resistance, reasonable mechanical strength and chemical resistance under operational conditions. but can also host the enzyme molecules within pores and protect against physical and mechanical2 On thedamages other hand, mesoporous supports not only exhibit a very high specific surface (up to 500 m /g), [20]. Fortunately, SBA-15 has all the advantages above and was selected as the support. but can also host the enzyme molecules withinand pores and protect and mechanical Amino functionalized SBA-15 is also low-cost convenient for theagainst one-stepphysical specific immobilization damages [20]. (Scheme Fortunately, of ST2570 1C). SBA-15 has all the advantages above and was selected as the support. Amino functionalized SBA-15 is also low-cost and convenient for the one-step specific immobilization 2. Results and 1C). Discussion of ST2570 (Scheme 2.1. Expression and Purification 2. Results and Discussion The aldehyde tag (LCTPSR) was ligated into the C or N-terminal or both terminals of ST2570
2.1. Expression Purificationplasmid pET28a. However, results showed that the aldehyde tag variably using theand reconstructed affected the protein expression was levelligated and enzyme (Figure 1). Unlike proteins,ofall of The aldehyde tag (LCTPSR) into activity the C or N-terminal or wildtype both terminals ST2570 the aldehyde-tagged ST2570 reduced the level of expression, which can evidently affect the enzyme using the reconstructed plasmid pET28a. However, results showed that the aldehyde tag variably activity. The C-terminal tag improved the activity of enzyme, despite a reduced level of expression, affected the protein expression level and enzyme activity (Figure 1). Unlike wildtype proteins, all of but the enzyme with double-sided aldehyde tags lost almost all activity. These results indicated that the aldehyde-tagged ST2570 reduced the level of expression, which can evidently affect the enzyme the fused polypeptide tag affects protein folding and the tertiary structure. activity. The C-terminal tag improved the activity of enzyme, despite a reduced level of expression, but the2.2. enzyme double-sided aldehyde tags lostProbe almost all activity. These results indicated that Labelingwith of Recombinant Proteins with Fluorescence the fused polypeptide tag affects protein folding and the tertiary structure. Although many protein modification methods were developed, these methods usually resulted in the selective labeling of a given functional group rather than the site-specific labeling of one residue in a given protein [8]. Thus, the fused aldehydes were used for site-specific protein modification and identification with an aminooxy functionalised probe.developed, A panel ofthese aldehyde-tagged proteins was in Although many protein modification methods were methods usually resulted incubated with Alexa Fluor 647 C5-aminooxyacetamide and the reaction was analyzed by SDS-PAGE the selective labeling of a given functional group rather than the site-specific labeling of one residue
2.2. Labeling of Recombinant Proteins with Fluorescence Probe
in a given protein [8]. Thus, the fused aldehydes were used for site-specific protein modification and identification with an aminooxy functionalised probe. A panel of aldehyde-tagged proteins was incubated with Alexa Fluor 647 C5-aminooxyacetamide and the reaction was analyzed by SDS-PAGE
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Molecules 2016, 21, 895 5 of 13 and Odyssey Near Infrared Laser Imaging system (Figure 2). In contrast, the wildtype protein and Odyssey Near Infrared Laser Imaging system (Figure 2). In contrast, the wildtype protein without without an aldehyde tag demonstrated no signals, the C-terminal modified proteins showed strong and Odyssey tag Near Infrared Laser (Figure 2). In contrast, the wildtype an aldehyde demonstrated noImaging signals,system the C-terminal modified proteins showedprotein strong without signals. signals. Additionally, the N-terminal and double-terminal modified proteins exhibited weaker signals. an aldehyde tag nodouble-terminal signals, the C-terminal modified signals. Additionally, thedemonstrated N-terminal and modified proteinsproteins exhibitedshowed weakerstrong signals. Some SomeAdditionally, LCTPSR peptide tags in these proteins may modified be covered during the process ofsignals. protein folding, the N-terminal and double-terminal proteins exhibited weaker Some LCTPSR peptide tags in these proteins may be covered during the process of protein folding, and LCTPSR tags in proteins may be covered during the process of protein folding, and and could not be modified bythese theco-expressed co-expressed FGE. selected ST2570CQ for immobilization because could notpeptide be modified by the FGE. WeWe selected ST2570CQ for immobilization because could not be modified by the co-expressed FGE. We selected ST2570CQ for immobilization because of its of excellent activity andand thethe availability groups. its excellent activity availabilityof offree free aldehyde aldehyde groups.
of its excellent activity and the availability of free aldehyde groups.
Figure 1. Effect of the fused aldehydetag tag on on the the expression expression level activity of the recombinant Figure 1. Effect of the fused aldehyde leveland and activity of the recombinant Figure 1. Effect of the fused aldehyde tag on the expression level and activity of the recombinant protein ST2570. protein ST2570. protein ST2570.
Figure 2. Selective labelling of aldehyde tagged proteins with Alexa Fluor 647 C5-aminooxyacetamide. Figure 2. Selective labelling of aldehyde tagged proteins with Alexa Fluor 647 C5-aminooxyacetamide.
Figure 2. Selective labelling of aldehyde tagged proteins with Alexa Fluor 647 C5-aminooxyacetamide. 2.3. Modification of SBA15 2.3. Modification of SBA15 Silica-based mesoporous materials have been used as enzyme supports, biomarkers and 2.3. Modification of SBA15 Silica-based mesoporous materials have used asfor enzyme biomarkers and biosensors in medicine. However, its direct use been as a support enzymesupports, immobilization is limited Silica-based mesoporous materials have been used as enzyme supports, biomarkers biosensors in medicine. However, its direct use as a support for enzyme immobilization is limited because of its inert nature. Thus, chemically modifying the surface of these materials is necessary to and becausein ofan its inert nature. Thus, chemically modifying of these materials immobilization is necessary to biosensors medicine. However, its direct use asthea surface support for enzyme is introduce active group, and the most common modifier is aminopropyltriethoxysilane (APTES, introduce an active group, and the most common modifier is aminopropyltriethoxysilane (APTES, limited its inert nature.in Table Thus,2,chemically modifying the surface these and materials is C9Hbecause 23NO3Si) of [21–23]. As shown SBA-15 exhibited improved loading of capacity, the C 9H23NO3Si) [21–23]. As shown in Table 2, SBA-15 exhibited improved loading capacity, and the enzyme showed better immobilization but worse retention of activity (R) when necessary to introduce an active group, andefficiency the most (IE) common modifier is aminopropyltriethoxysilane enzyme showedwas better immobilization efficiency (IE) butdeficient worse retention of modification. activity (R) when immobilization performed support with 2, either or excessive (APTES, C9 H23 NO3 Si) [21–23]. Asusing shown in Table SBA-15 exhibited improved loading This capacity, immobilization was performed using support with either deficient or excessive modification. This outcome couldshowed be due better to the random combination and aggregation enzymes throughofphysical and the enzyme immobilization efficiency (IE) but of worse retention activity (R) outcome could becan dueenhance to the the random combination andand aggregation of formation enzymes of through physical which steric hindrance effect prevent the the monolayer. whenabsorption, immobilization was performed using support with either deficient or excessive modification. absorption, which can enhance the steric hindrance effect and prevent the formation of the monolayer. Moreover, over-modification with APTES will result in increased steric hindrance because of the high This outcome could be due to the random and aggregation of enzymes through physical Moreover, over-modification with APTEScombination will result in increased steric hindrance because of the high protein density. Therefore, SBA-15 modified with 2% APTES (v/v) was selected for the next experiment. absorption, which can enhance the steric hindrance effect and prevent the formation of the monolayer. protein density. Therefore, SBA-15 modified with 2% APTES (v/v) was selected for the next experiment.
Moreover, over-modification with APTES will with result in increased stericand hindrance Table 2. Modification of SBA-15 different ratios of APTES ethanol. because of the high Table 2. Modification of SBA-15 with different ratios of APTES and ethanol. protein density. Therefore, SBA-15 modified with 2% APTES (v/v) was selected for the next experiment. Specific Activity Specific Activity Immobilization Retention of Ratio of APTES Loading Capacity Specific Activity Specific Activity Immobilization Retention of Ratio of APTES carrier U/mg U/gcarrier Efficiency % Activity % and Ethanol % (v/v) Loading mg/gCapacity carrier U/g carrier Efficiency % Activity and Ethanol % (v/v)2. Modification mg/g 0 Table 4.31 103 443.93 90.79ethanol. 57.17 % of SBA-15U/mg with different ratios of APTES and 4.31 103 443.93 90.79 57.17 20 3.17 141.63 448.97 66.31 78.61 3.17 141.63 448.97 66.31 78.61 62 4.37 108.68 474.93 91.42 60.32 Ratio of APTES6 and Loading Capacity Specific Specific Activity Immobilization Retention of 4.37 108.68 474.93 91.42 60.32 10 4.23 109.56 478.78 88.49 60.81 Ethanol % (v/v) mg/g Activity U/mg U/g Efficiency % Activity % carrier carrier 10 4.23 109.56 478.78 88.49 60.81
0 2 6 10
4.31 3.17 4.37 4.23
103 141.63 108.68 109.56
443.93 448.97 474.93 478.78
90.79 66.31 91.42 88.49
57.17 78.61 60.32 60.81
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2.4. Immobilization of ST2570CQ Molecules 2016, 21, 895
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One-step immobilization and purification of ST2570CQ were simultaneously performed through 2.4.base Immobilization ST2570CQ a Schiff reaction of between the amino groups in the support and the free aldehyde groups in the enzyme. This approach avoids further purification of proteins before the immobilization. The amount One-step immobilization and purification of ST2570CQ were simultaneously performed through of enzymes to thebetween carrier the andamino the activity of the thesupport immobilized depend on conditions a Schiff bound base reaction groups in and theenzyme free aldehyde groups in the enzyme. This approach avoids further purification of proteins before thetemperature, immobilization. The amount during immobilization, such as the availability of linking groups, pH, coupling time and of enzymes bound to the carrier and the activity of the immobilized enzyme depend on conditions initial enzyme amount. To achieve the optimal immobilization efficiency (IE), retention of activity (R) during immobilization, such as the of linking groups, pH, temperature, coupling time and and immobilized enzyme activity, theavailability conditions for immobilization were optimized. initial enzyme amount. To achieve the optimal immobilization efficiency (IE), retention of activity The ionization state of enzyme molecules was affected by the pH of the buffer used in the (R) and immobilized enzyme activity, the conditions for immobilization were optimized. immobilization process. Figure 3A shows the effect of pH on the activity of the enzymes during The ionization state of enzyme molecules was affected by the pH of the buffer used in the immobilization. The relative activity of immobilized SBA15-NH vary from 2 did notduring immobilization process. Figure 3A shows the effect ofST2570CQ pH on the on activity of the enzymes pH 6.0 to 10.0. The highest activity for of immobilized observed2atdid pH 6.5. The relative immobilization. The relative activity immobilizedenzymes, ST2570CQwas on SBA15-NH not vary from activity enzyme significantly increased when initial crude amounts pH of 6.0the to immobilized 10.0. The highest activity for immobilized enzymes, wasthe observed at pH ST2570CQ 6.5. The relative of the immobilizedwere enzyme significantly when the10initial crude amounts the used activity in the immobilization changed from increased 0 to 3.0 mg per mg of theST2570CQ carrier. However, used in the immobilization were changed from 0 to 3.0 mg per 10 mg of the carrier. However, the enzyme reached steady-state when its amounts was greater than 2.0 mg (Figure 3B). The operating ˝ enzyme reached steady-state when its amounts was greater than 2.0 mg (Figure 3B). The operating temperature of immobilization was 10–37 C, and the immobilization time ranged from 1 h to 10 h temperature of optimal immobilization was 10–37 °C, and the immobilization ranged from to 10optimal h (Figure 3C,D). The temperature for immobilization was 10 ˝ Ctime for more than 3 1h.hThe (Figure 3C,D). The optimal temperature for immobilization was 10 °C for more than 3 h. The optimal conditions for immobilization are 2 mg of crude enzyme per 10 mg of support in 50 mM PB buffer conditions for immobilization are 2 mg of crude enzyme per 10 mg of support in 50 mM PB buffer (pH 6.5) and incubation for 3 h at 10 ˝ C. Under the optimal conditions of immobilization, the enzyme (pH 6.5) and incubation for 3 h at 10 °C. Under the optimal conditions of immobilization, the enzyme loading capacity can can be as as 108 mg/g enzymeactivity activity can loading capacity bemuch as much as 108 mg/gsupport, support,and and the the immobilized immobilized enzyme can be be as muchasasmuch 450 U/g immobilized enzyme. as 450 U/g immobilized enzyme.
Figure 3. Conditions for the covalent immobilization of ST2570CQ on SBA-15. (A) The optimization of
Figure 3. Conditions for the covalent immobilization of ST2570CQ on SBA-15. (A) The optimization of pH for the immobilization system using either, 50 mM PB buffer (pH 6.0–9.0) or 50 mM Gly-NaOH pH for the immobilization system using either, 50 mM PB buffer (pH 6.0–9.0) or 50 mM Gly-NaOH buffer (pH 8.6–10.0); (B) The optimization of the initial amount of enzyme per 10 mg support for buffer (pH 8.6–10.0); (B) The of the initial amount of enzyme persystem; 10 mg(D) support immobilization system; (C) optimization The optimization of temperature for immobilization The for immobilization (C)the The optimizationThe of temperature immobilization (D) The optimizationsystem; of time for immobilization. immobilizationfor system consisted of 2system; mL 50 mM optimization time the immobilization. The system consisted of 2 activity mL 50 mM PB bufferof (pH 6.0)for containing 10 mg support in immobilization a 20 mL reactor. We set the best enzyme as PB buffer100%. (pH Three 6.0) containing 10 mg support in agroup. 20 mL reactor. We set the best enzyme activity as 100%. trials were performed for each Three trials were performed for each group.
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2.5. Characterization Characterization of of Immobilized Immobilized ST2570CQ ST2570CQ 2.5. 2.5.1. pH and Thermo-Stability of Immobilized ST2570CQ
pH and and thermo-stability thermo-stability of an an enzyme enzyme are are important important parameters parameters for for exploring exploring potential potential The pH ˝ C. In industrial applications. ST2570 is a thermophilic protein that is stable at 70 °C. In this this part part of of the the study, study, ˝C for the thermal stability was investigated investigated by by incubating incubatingeither eitherthe thefree freeor orimmobilized immobilizedenzyme enzymeatat9090°C fora immobilized aperiod periodofoftime. time.The Theremaining remainingactivity activitywas wasmonitored monitored after after each each incubation incubation time. All the immobilized were prepared prepared with with the the optimal optimal conditions conditions described described at at the theend endof ofSection Section2.4. 2.4. enzymes used were As shown in Figure 4A, when immobilized ST2570CQ was incubated for 2 h, it still retained 60% its activity, activity, which which was was still still three three times times higher higher than than that that of of free free enzyme. enzyme. The The stability stability of of various various of its inorganic supports supports because because inorganic inorganic supports supports immobilized enzymes can be frequently improved by inorganic dimensional stabilities stabilities [24]. Inorganic supports always are capable of forming forming manageable manageable have greater dimensional particles with with aa high high surface surface area, area, are are reactive reactivetowards towardsderivatizing/functionalizing derivatizing/functionalizing agents and present particles chemical resistance resistance excellent thermal and microbial resistance, reasonable mechanical strength and chemical under the operational conditions [20]. The thermal stability of enzymes was speculated to drastically ofof a relatively rigid support [25,26]. TheThe pH increase if they they are areattached attachedtotoaacomplementary complementarysurface surface a relatively rigid support [25,26]. stability of free and immobilized enzymes is illustrated in Figure 4B. When the enzymes were pH stability of free and immobilized enzymes is illustrated in Figure 4B. When the free enzyme enzyme and incubated in buffers with different pH values for 24 h, it was found that both the free immobilized enzyme were stable when the pH value is higher than 7, indicating that no severe the immobilized conformational change, which affects the pH stability of ST2570, occurred during immobilization [26]. [26]. it with with the the support did not As for the free enzyme that already had the best pH stability, combining it the pH pH stability stability appreciably appreciably anymore. anymore. improve the
Figure 4. 4. The stability of immobilized ST2570CQ: ST2570CQ: (A) (A) The The thermal thermal stability stability of of Figure The thermal thermal and and pH pH stability of free free and and immobilized free and site-specific immobilized ST2570CQ; (B) The pH stability of free and immobilized ST2570CQ. ST2570CQ. ■)) free ( free enzyme enzyme (●) ( ) immobilized immobilized enzyme; enzyme; 50 50 mM mM PB PB buffer buffer (pH (pH 6.0–9.0) and 50 mM Gly-NaOH buffer (pH 8.6–10.5). All the immobilized enzymes used were prepared with the optimal conditions described (pH at the end conditions forfor thethe activity assay werewere constant stirring at 110atrpm 5 min end of ofSection Section2.4. 2.4.The The conditions activity assay constant stirring 110for rpm for ˝ 70 °C constant temperature vibrator. ThreeThree trials trials were were performed for each test. test. 5inmin in water-bathing 70 C water-bathing constant temperature vibrator. performed for each
Batch and and Semi-Continuous Semi-Continuous Reactors Reactors 2.5.2. Operational Stabilities of Immobilized Enzyme in Batch assess the the feasibility feasibility of of immobilized immobilized Operational stability is the most important factor used to assess enzymes for industrial productions. A number of investigations were previously carried out in this area [21,27,28]. The operational stability of immobilized enzyme was determined in the batch and flow reactors. reactors. The results were compared compared in terms of activity and cycling times of the semi-continuous flow immobilized enzyme. To eliminate the influence of storage time on enzyme activity, all measurements carried out in the the same same day. day. In the the batch batch reaction reaction operation operation (Figure 5A), the enzyme activity were carried remained above 95% of the original activity after five cycles in the reactors loaded with 10 mg of immobilized enzyme. The result in this work shows a significant improvement in the operation
enzymes in other reports [26–31]. The stability of immobilized ST2570CQ on SBA-15 by physical adsorption was also examined in the batch wise reaction (Figure 5B); which turned out the physically immobilized enzyme lost 40% of its original activity when used for the second time; and only retained less than 20% of its original activity in the fifth use; which may be ascribed to significant deprival of the enzyme from the support during the reaction agitation and recovery process. On the contrary, Molecules 2016, 21, 895 8 of 13 the activity loss of the chemical immobilized ST2570CQ on SBA-15 may be due to the deactivation of enzyme by strong shaking during washing operation. The results demonstrate that the multi-point chemical of ST2570CQ to SBA-15 provide a much stronger linkage loaded and confinement forof remainedbinding above 95% of the original activity after five cycles in the reactors with 10 mg site-specific immobilized than the shows immobilized ST2570CQ by physical adsorption immobilized enzyme. TheST2570CQ result in this work a significant improvement in the operation [32]. stability In a semi-continuous reactor, thebatch immobilized ST2570CQwith retained almost 100% of its of immobilized ST2570CQ flow on SBA-15 in the reaction compared the immobilized enzymes original being used 10 times in the columnST2570CQ (Figure 5C). operational stability in the in otheractivity reportsafter [26–31]. The stability of immobilized on The SBA-15 by physical adsorption semi-continuous flow reactor better than the operational stability thethe batch type reactor. The was also examined in the batchiswise reaction (Figure 5B); which turnedinout physically immobilized difference in outcome was most likely because the for gradual loss of time; immobilized enzymes the enzyme lost 40% of its original activity whenof used the second and only retainedwhen less than enzymes separated byincentrifugation duringmay eachberound in the batch reactor. This probably did 20% of itswere original activity the fifth use; which ascribed to significant deprival of the enzyme not occur the semi-continuous flow reactor, which the immobilized were the notactivity separated. from the in support during the reaction agitationinand recovery process. Onenzymes the contrary, loss Operational stability improved when a on semi-continuous reactor was utilized, in contrast with of the chemical immobilized ST2570CQ SBA-15 may beflow due to the deactivation of enzyme by strong the resultduring of Bachas–Daunert [8]. Thus, highdemonstrate operationalthat stabilities obtained chemical with immobilized shaking washing operation. The the results the multi-point binding of ST2570CQ preparations indicate that these immobilized enzyme preparations can successfully be ST2570CQ to SBA-15 provide a much stronger linkage and confinement for site-specific immobilized used for continuous [28]. ST2570CQ than the production immobilized ST2570CQ by physical adsorption [32].
Figure Figure5.5. The Theeffect effectofofreuse reuseon on immobilized immobilized enzymes enzymes in in reactors. reactors. The Theactivity activityofofsite-specific site-specific covalently-immobilized enzymes in a batch reactor (A); the activity of enzyme immobilized by covalently-immobilized enzymes in a batch reactor (A); the activity of enzyme immobilized by physical physical adsorption in a batch reactors (B); and the activity of site-specific covalently-immobilized adsorption in a batch reactors (B); and the activity of site-specific covalently-immobilized enzymes in a enzymes in a semi-continuous flowThe reactor The tests were out at room temperature. semi-continuous flow reactor (C). tests(C). were carried out atcarried room temperature.
2.6. Catalytic Ability of Immobilized ST2570CQ In a semi-continuous flow reactor, the immobilized ST2570CQ retained almost 100% of its ST2570 was first in 10 2009, andinitthe cancolumn selectively catalyze L-2-halogenated acid intoin original activity after reported being used times (Figure 5C). The operational stability Dthe -2-hydroxy alkyl acid flow (Scheme 2) [18]. The immobilized and free ST2570 exhibited different catalytic semi-continuous reactor is better than the operational stability in the batch type reactor. abilities when L-2-haloalkanoic used as abecause substrate. Thegradual highest loss conversion rate of L-2-haloalkanoic The difference in outcome waswas most likely of the of immobilized enzymes when the enzymes were separated by centrifugation during each round in the batch reactor. This probably did not occur in the semi-continuous flow reactor, in which the immobilized enzymes were not separated. Operational stability improved when a semi-continuous flow reactor was utilized, in contrast with the result of Bachas–Daunert [8]. Thus, the high operational stabilities obtained with immobilized ST2570CQ preparations indicate that these immobilized enzyme preparations can successfully be used for continuous production [28].
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2.6. Catalytic Ability of Immobilized ST2570CQ ST2570 was first reported in 2009, and it can selectively catalyze L-2-halogenated acid into 9 of 13 (Scheme 2) [18]. The immobilized and free ST2570 exhibited different catalytic abilities when L-2-haloalkanoic was used as a substrate. The highest conversion rate of L-2-haloalkanoic catalyzed by by the the immobilized immobilized and and free free ST2570 ST2570 were were 49.74% 49.74% and and 40.94%, 40.94%, respectively. respectively. The The improvement improvement catalyzed of catalytic ability is attributed to the better thermal and operational stability of immobilized of catalytic ability is attributed to the better thermal and operational stability of immobilized ST2570, ST2570, which are the most important factors during reactions performed with high temperatures and long which are the most important factors during reactions performed with high temperatures and long reaction times. reaction times. Molecules 2016, 21, 895 acid D -2-hydroxy alkyl
Scheme 2. 2. Hydrolysis Hydrolysis reaction reaction catalyzed catalyzed by by LL-2-dehalogenase. -2-dehalogenase. Scheme
3. Experimental 3. ExperimentalSection Section 3.1. Materials 3.1. The LL-2-haloacid from thermophile archaeon Sulfolobus tokodaiistr.7 (JCM The -2-haloaciddehalogenase dehalogenase(ST2570) (ST2570) from thermophile archaeon Sulfolobus tokodaiistr.7 10545),10545), plasmids pET28a,pET28a, myc-his A Rv0712A(FGE) plasmid (add gene plasmid 16132), Escherichia coli (JCM plasmids myc-his Rv0712 (FGE) plasmid (add gene plasmid 16132), BL21(DE3) pLysS (Novagen,pLysS Madison, WI, USA), mesoporous Support SBA15 (550–600 m2·g−1, Escherichia coli BL21(DE3) (Novagen, Madison, WI, Silica USA), mesoporous Silica Support 2 ¨3·g 7–9 nm,(550–600 0.65–0.67m cm Co., Ltd., Nanjing,Materials China), (3-Aminopropyl) SBA15 g´−11, ,Nanjing 7–9 nm,XFNANO 0.65–0.67Materials cm3 ¨ g´1Tech , Nanjing XFNANO Tech Co., Ltd., triethoxysilane (APTES, 98%, Aladdin, New York, NY, USA),98%, L-2-chloropropionic acid (Alfa Nanjing, China), (3-Aminopropyl)triethoxysilane (APTES, Aladdin, New York, NY, Aesar, USA), Hill, MA, USA), Grade, Waltham, MA, USA), LWard -2-chloropropionic acidacetonitrile (Alfa Aesar,(HPLC Ward Hill, MA,Fisher USA),Scientific, acetonitrile (HPLC Grade, Fisherninhydrin Scientific, reagent (2% (w/w) ethanol solution) and all reagents were of analytical Waltham, MA, USA), ninhydrin reagent (2%other (w/w) ethanol solution) and allgrade. other reagents were of analytical grade. 3.2. Methods 3.2. Methods 3.2.1. Expression and Purification 3.2.1. Expression and Purification To obtain sufficient amounts of target protein for the analysis, enzymes were produced by E. coli obtain sufficient amounts ofbased targeton protein for the analysis, werepLysS, produced by BL21To (DE3) pLysS. The method was a two-plasmid system inenzymes BL21 (DE3) the first E. coli BL21 pLysS. The method was based a two-plasmid BL21 (DE3) pLysS, plasmid was(DE3) the reconstructed pET28a with the on gene encoding forsystem the S.intokodaii L-2-haloacid the first plasmid was the reconstructed pET28a with the gene encoding for the S. tokodaii L -2-haloacid dehalogenase (ST2570), and the other plasmid co-expressed the formylglycine-generating enzyme dehalogenase (ST2570),cysteine and the in other plasmid co-expressed formylglycine-generating enzyme (FGE), which converts the polypeptide fusion tagthe of LCTPSR to formylglycine (FGly) to (FGE), which converts cysteine in the polypeptide fusion tag of LCTPSR to formylglycine (FGly) to obtain ST2570 with or without the aldehyde groups in the right place. obtain ST2570 with or without aldehyde in the right place. by site-directed mutation to The aldehyde tag gene the sequence wasgroups introduced to pET28a Thethe aldehyde gene sequence was introduced to pET28a by site-directed mutation to replace replace His-tagtag sequence. The plasmid pET28aDH was constructed by deleting the His-tag at the the His-tag sequence. The plasmid pET28aDH was constructed by deleting the His-tag at the N-terminal of N-terminal of the multiple cloning site in pET28a. The plasmid pET28aCQ was constructed by the multiple cloning site in pET28a. The plasmid pET28aCQ was constructed by replacing the His-tag replacing the His-tag at the C-terminal of the Multiple Cloning Site in pET28aDH with the gene at the C-terminal of the The Multiple Cloning Site in was pET28aDH with by the replacing gene encoding for LCTPSR. encoding for LCTPSR. plasmid pET28aNQ constructed the His-tag at the The plasmid pET28aNQ was constructed by replacing the His-tag at the N-terminal of the Multiple N-terminal of the Multiple Cloning Site in pET28a with the sequence encoding LCTPSR. Lastly, the Cloning Site in pET28a with the sequence encoding LCTPSR. Lastly, the plasmid pET28aDQ was plasmid pET28aDQ was constructed by replacing the His-tag at the C-terminal of the Multiple constructed thewith His-tag the C-terminal of the Multiple Cloning pLysS Site in cells pET28aNQ with Cloning Siteby inreplacing pET28aNQ the at sequence encoding LCTPSR.BL21(DE3) that carried the sequence encoding LCTPSR.BL21(DE3) pLysS cells that carried plasmids pET28a (DH, CQ, NQ plasmids pET28a (DH, CQ, NQ or DQ)-st2570 and myc-his A Rv0712 [33] were cultured in LB medium ˝ or DQ)-st2570 and myc-his Rv0712 [33] were cultured LB medium 37 C until an OD600toofa at 37 °C until an OD600 of 0.8Awas obtained. The cells wereininduced by theataddition of L-pectinose 0.8 was obtained. The cells were induced by the addition of L -pectinose to a final concentration of final concentration of 0.2% for 30 min, induced further for 16 h by 0.5 mM IPTG at 18 °C then ˝ C then harvested. The cell pellet was 0.2% for 30 min, induced further for 16 h by 0.5 mM IPTG at 18 harvested. The cell pellet was resuspended in 50 mM PB at pH 6.0, disrupted by ultrasonication in an resuspended in 50 mM PB pH 6.0, disrupted by at ultrasonication in extract an ice bath centrifuged at ice bath and centrifuged atat 12,000 rpm for 20 min 4 °C. The crude was and incubated at 80 °C for 30 min and centrifuged again. The freeze-dried protein powder was stored at −20 °C. Protein concentrations were determined using the Bradford method.
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12,000 rpm for 20 min at 4 ˝ C. The crude extract was incubated at 80 ˝ C for 30 min and centrifuged again. The freeze-dried protein powder was stored at ´20 ˝ C. Protein concentrations were determined using the Bradford method. 3.2.2. Enzyme Assay The 3 mL reaction system consisting of 50 mM Gly–NaOH buffer (pH 9.5) and 1 mM L -2-chloropropionic acid as a model substrate was incubated in an oscillating water bath at 70 ˝ C for 5 min. The free or immobilized enzyme was added for further incubation in a water bath at 70 ˝ C and shaken with a speed of 110 rpm for 5 min. The reaction was terminated by addition of 50 µL of 1 M nitric acid and 2 mL of 6% ammonium ferric sulfate solution. The system was treated with 1 mL of 0.4% mercury thiocyanate solution to produce a color reaction after removal of the immobilized enzyme. Each sample was tested three times (p < 0.05). The OD460 value for each sample was tested using a UV-1700 spectrophotometer (Shimazu, Kyoto, Japan), and the lactic acid yield combined with the chlorine ion standard curve was calculated. One unit of activity is defined as the production of 1 µmol hydrochloric acid per min at 70 ˝ C and pH 9.5. The activity of free L-2-haloacid dehalogenase was given as U/mgenzyme , and immobilized enzyme activities were expressed as U/mgenzyme or U/gsupport . 3.2.3. Labelling of Recombinant Proteins with the Fluorescence Probe The fluorescence-labeling reaction was performed by combining recombinant proteins and C5-aminooxyacetamide [21] and the reactions were analyzed by SDS-PAGE and Odyssey Near Infrared Laser Imaging system. 3.2.4. Modification of SBA-15 SBA-15 was washed with distilled water and dried overnight at 60 ˝ C before salinisation. Different concentrations of APTES (0%, 2%, 6%, and 10%) was added to the dried 1 g SBA-15 in 50 mL of anhydrous ethanol (v/v) and then incubated in the oscillation incubator at 80 ˝ C and 200 rpm for 12 h. SBA-15 was washed thoroughly with anhydrous ethanol and distilled water on a filtered funnel until no APTES was detected in the filtrate, and the supports were dried overnight at 100 ˝ C. The amount of APTES in different solutions was determined using a ninhydrin-based monitoring system described earlier by Özlem Alptekin [34]. The conditions used to initially optimize the activation of support was 2 mg of ST2570CQ per 10 mg of SBA-15-NH2 incubated in 50 mM PB buffer (pH 6.5) at 10 ˝ C for more than 6 h. 3.2.5. Immobilization of ST2570CQ The amino-functionalized SBA-15 (SBA15-NH2 ) (10 mg) was incubated with 2 mL of phosphate buffer (50 mM, pH 6.0) containing 2 mg of the heat-treated ST2570 at 10 ˝ C for 6 h of shaking at 150 rpm and reduced with 1% (w/w) NaCNBH3 for 1 h. Aside from the target protein, other proteins without aldehyde groups cannot establish a covalent link with SBA15-NH2 . Thus, the immobilized enzyme was easily separated by centrifugation at 12,000 rpm for 10 min and washed twice with 1 M NaCl solution, saturated ammonium sulfate solution, and distilled water to remove the other physically adsorbed proteins. Immobilization efficiency (IE, %) and retention of activity (R, %) were calculated as follows: IE “
The activity of added enzyme ´ The remaining activity in the supernatat ˆ 100% The activity of added enzyme
(1)
The observed activity of immobilized enzyme ˆ 100% The theoretical activity of immobilized enzyme
(2)
R“
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The conditions for the immobilization of ST2570CQ on SBA-15-NH2 , including pH, temperature, time and initial amount of protein per 10 mg of support, were optimized. The activity of the immobilized enzyme was determined for all the samples. 3.2.6. Characterization of Immobilized ST2570CQ In this part, the pH and thermal stabilities of the immobilized ST2570CQ were detected. The thermal stabilities of free and immobilized enzymes were investigated by incubating both the free and immobilized enzymes at 90 ˝ C for different lengths of time. The remaining enzyme activity was monitored after each incubation. After incubation in buffers with different pH values (pH 6.0–9.0 for 50 mM PB buffer; pH 8.6–10.5 for 50 mM Gly–NaOH buffer) for 24 h, the remaining activities of free and immobilized enzymes were detected according to Section 3.2.2. The operational stabilities in batch and semi-continuous reactors were also investigated. For batch reactors, all reactions were carried out in a vibrating water-bathing at a constant temperature of 70 ˝ C and shaking at 110 rpm for 5 min. The reaction mixture consists of 3 mL of 50 mM Gly–NaOH solution at pH 9.5 containing 1 mM L-2-chloropropionic and 10 mg of immobilized ST2570CQ. The enzyme was recovered by centrifugation and washed with distilled water to prepare for the next cycle. The product was then detected according to Section 3.2.2. For the semi-continuous flow reactor, 10 mg of immobilized ST2570CQ was loaded into a column (1 cm2 ˆ 10 cm). The reaction mixture (mobile phase) consisting of 3 mL of 50 mM Gly-NaOH buffers (pH 9.5) and 1 mM L-2-halogenated acid was applied to a column and incubated for 10 min at 70 ˝ C. The reaction mixture was then allowed to flow out of the column, then the product was then detected according to Section 3.2.2. The column was washed with 5 mL distilled water five times to prepare for next cycle. All the immobilized enzymes used for characterization were prepared with the optimal conditions described in Section 2.4. 3.2.7. Catalytic Ability of Immobilized ST2570CQ Rates of lactic acid production catalyzed by free and immobilized ST2570CQ were also measured using high-performance liquid chromatography (HPLC). A mixture consisting of 1 mL of 50 mM Gly-NaOH pH 9.5 containing 20 mM L-2-CPA was assigned as the control group, while 0.05 mg (9 U) and 20 mg (9 U) free and immobilized ST2570CQ were included separately in experimental groups. The conversion rate was calculated according to the following formula: conversion rate p%q “
substrate pthe control groupq´the residue of substrate pthe experiment groupq substrate pthe control groupq
(3)
The HPLC conditions are as follows: 4.6 mm ˆ 250 mm Waters Nova-pak C18 HPLC column; mobile phase: a mixture of 0.01 mol/L phosphate buffer (pH 2.0) and acetonitrile (8:2, v/v); flow rate: 0.6 mL/min; UV detector wavelength: 210 nm; column temperature 18 ˝ C; volume of sample injection: 10 µL. 4. Conclusions In this experiment, we prepared a panel of aldehyde tagged proteins using a pET28a plasmids system that was designed in our previous work. The C-terminal aldehyde-tagged protein (ST2570CQ) exhibited significant enzymatic properties, such as the generation of free aldehyde groups, a higher level of protein expression and improved enzyme activity. ST2570CQ was covalently immobilized to SBA-15 modified with 2% APTES ethanol solution (v/v) in the following optimal conditions: 2 mg of crude ST2570CQ per 10 mg of SBA15-NH2 in 50 mM PB buffer pH 6.5 and incubated at 10 ˝ C for 3 h. The immobilized ST2570CQ exhibited a 3-fold increase in thermal stability and improved operational stability and catalytic ability than free ST2570. These improved properties increase the potential of site-specific, covalently immobilized ST2570 for industrial-scale production and application. Acknowledgments: The authors are grateful to the National Natural Science Foundation of China (No. 20772046) and the Jilin Province Natural Sciences Foundation (Grant 20120943) for financial support.
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Author Contributions: Hui Jian and Renjun Gao conceived and designed the experiments; Hui Jian, Yan Bai and Rong Li performed the experiments; Renjun Gao and Yingwu Wang contributed reagents/materials/analysis tools; Hui Jian wrote the paper. Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are not available from the authors. © 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).
