International Journal of

Molecular Sciences Article

Enhancement of Alkaline Protease Activity and Stability via Covalent Immobilization onto Hollow Core-Mesoporous Shell Silica Nanospheres Abdelnasser Salah Shebl Ibrahim 1,2, *, Ali A. Al-Salamah 1 , Ahmed M. El-Toni 3,4 , Khalid S. Almaary 1 , Mohamed A. El-Tayeb 1 , Yahya B. Elbadawi 1 and Garabed Antranikian 5 1

2 3 4 5

*

Department of Botany and Microbiology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia; [email protected] (A.A.A.-S.); [email protected] (K.S.A.); [email protected] (M.A.E.-T.); [email protected] (Y.B.E.) Department of Chemistry of Natural and Microbial Products, Pharmaceutical Industries Research Division, National Research Center, El-Buhouth St., Dokki, Cairo 12311, Egypt King Abdullah Institute for Nanotechnology, King Saud University, Riyadh 11451, Saudi Arabia; [email protected] Central Metallurgical Research and Development Institute, Helwan, Cairo 11421, Egypt Institute of Technical Microbiology, Hamburg University of Technology, Hamburg 21073, Germany; [email protected] Corresponding: [email protected]; Tel.: +966-5973-5914

Academic Editor: Vladimír Kˇren Received: 8 December 2015; Accepted: 25 January 2016; Published: 29 January 2016

Abstract: The stability and reusability of soluble enzymes are of major concerns, which limit their industrial applications. Herein, alkaline protease from Bacillus sp. NPST-AK15 was immobilized onto hollow core-mesoporous shell silica (HCMSS) nanospheres. Subsequently, the properties of immobilized proteases were evaluated. Non-, ethane- and amino-functionalized HCMSS nanospheres were synthesized and characterized. NPST-AK15 was immobilized onto the synthesized nano-supports by physical and covalent immobilization approaches. However, protease immobilization by covalent attachment onto the activated HCMSS–NH2 nanospheres showed highest immobilization yield (75.6%) and loading capacity (88.1 µg protein/mg carrier) and was applied in the further studies. In comparison to free enzyme, the covalently immobilized protease exhibited a slight shift in the optimal pH from 10.5 to 11.0, respectively. The optimum temperature for catalytic activity of both free and immobilized enzyme was seen at 60 ˝ C. However, while the free enzyme was completely inactivated when treated at 60 ˝ C for 1 h the immobilized enzyme still retained 63.6% of its initial activity. The immobilized protease showed higher Vmax , kcat and kcat /Km , than soluble enzyme by 1.6-, 1.6- and 2.4-fold, respectively. In addition, the immobilized protease affinity to the substrate increased by about 1.5-fold. Furthermore, the enzyme stability in various organic solvents was significantly enhanced upon immobilization. Interestingly, the immobilized enzyme exhibited much higher stability in several commercial detergents including OMO, Tide, Ariel, Bonux and Xra by up to 5.2-fold. Finally, the immobilized protease maintained significant catalytic efficiency for twelve consecutive reaction cycles. These results suggest the effectiveness of the developed nanobiocatalyst as a candidate for detergent formulation and peptide synthesis in non-aqueous media. Keywords: alkaline protease; immobilization; hollow core-mesoporous shell silica nanospheres; nanotechnology; alkaliphiles; detergents

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1. Introduction Currently enzymes are widely used as alternative of chemical catalysts in various industries; and playing a significant role in development of ecofriendly technology, owing to their unique properties including high specificity, mild reaction conditions, low toxicity and selectivity [1,2]. Proteases (EC 3.4.21-24 and 99) represent one of the most important classes of industrial enzymes, that account for up to 60% of the total enzyme sales worldwide [3]. Proteases have long been used to catalyze the hydrolysis of various proteins in aqueous solutions, in addition to peptides synthesis in non-aqueous media [4]. Alkaline proteases, accounting alone for approximately 40% of the total global enzymes market, proved particularly suitable for several industrial applications such as detergent, pharmaceutical, leather, dairy, silk, soy processing, brewery, meat tenderization, and waste management [5,6]. This is attributed mainly to significant activity and operational stability under harsh operational conditions, including high pH and temperature and in the presence of surfactants [4,7,8]. Enzyme immobilization on solid supports represents an efficient approach to improve enzyme stability as well as offering unique merits over the soluble biocatalyst such as operational stability, reusability of the enzyme, bioprocess control, and simplifying product separation [9,10]. Recent advance in nanotechnology have provided diverse nanostructured materials that are more effective for biocatalyst immobilization. It was shown that nanostructured materials can enhance the efficiency of the immobilized enzymes by providing the maximum limits in balancing the key parameters that determine the effectiveness of immobilized enzymes such as large surface area, minimum mass transfer resistance, and high enzyme loading [11]. However, selection of suitable enzyme carriers and the applied immobilization approach is of significant importance for successful immobilization processes. Among inorganic materials, mesoporous silica nanoparticles (MPS) are of special interest for enzyme immobilization technology, which is characterized by large surface areas, easily functionalizable surfaces and confined nanospace for enzyme inclusion, in addition to high biocompatibility and low toxicity [12]. MPS nanoparticles with different morphologies have been fabricated and applied in enzyme immobilization [13,14]. However, hollow mesoporous silica nanoparticles are characterized by several unique features that make them promising candidates for biocatalyst immobilization such as inner voids suitable for inclusion of proteins with various sizes, having surfaces (inner and outer) that can be easily chemically modified and showing high diffusion rate within the mesostructure of the silica shells [15]. In the present study, a recently characterized alkaline protease from alkaliphilic Bacillus sp. NPST-AK15 [16,17] was immobilized onto hollow core-mesoporous shell silica (HCMSS) nanospheres by two approaches including covalent and physical immobilization. Subsequently, the properties of immobilized protease were investigated. 2. Results and Discussion 2.1. Fabrication and Characterization of Hollow Core-Mesoporous Shell Silica Nanospheres Hollow core-mesoporous shell silica (HCMSS) nanospheres were synthesized by anionic surfactant through a soft-templating route assisted by ultrasonic waves. In this route, negatively charged silica nuclei are reacted with anionic surfactant through a co-structure directing agent (3-aminopropyltrimethoxysilane (APMS)) to produce mesoporous silica nanospheres. On the other hand, hollow core structures are obtained through assembling of the anionic surfactant micelles on the bubbles created in the solution by ultrasonic waves and then the silica nuclei are precipitated to form a hollow core structure. The template was removed using solvent extraction to maintain the functionalization of HCMSS spheres with amino groups. Figure 1 shows amino functionalized hollow core-mesoporous shell silica spheres (HCMSS–NH2 ) using anionic surfactant. HCMSS–NH2 possessed spheres with sizes ranged from 200–300 nm while the shell thickness was around 30 nm. The non-functionalized hollow core-mesoporous shell spheres (HCMSS-non) were produced by calcination at 550 ˝ C of amino-functionalized ones. Ethane functionalization was conducted on

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HCMSS-non HCMSS-non spheres spheres through through post-synthesis post-synthesis approach. approach. Both Both calcination calcination and and ethane ethane functionalization functionalization HCMSS-non spheres through post-synthesis approach. Both calcination and ethane functionalization steps did not affect the morphology of hollow core nanostrucures. steps did not affect the morphology of hollow core nanostrucures. steps did not affect the morphology of hollow core nanostrucures.

Figure 1. TEM images of amino-functionalized hollow core-mesoporous silica (HCMSS–NH2)

Figure 1. TEM images of amino-functionalized hollow core-mesoporous silica (HCMSS–NH2 ) nanospheres nanospheres by anionic surfactant. Figure 1. TEM prepared images of amino-functionalized hollow core-mesoporous silica (HCMSS–NH2) prepared by anionic surfactant. nanospheres prepared by anionic surfactant. The typical nitrogen adsorption/desorption isotherms for the hollow core-mesoporous shell silica sphere samples with various functionalities are presented in the Figure 2A. The isotherms showedshell The typical nitrogen adsorption/desorption isotherms for the hollow hollow core-mesoporous shell The typical nitrogen adsorption/desorption isotherms for core-mesoporous the type samples IV curveswith that depicted the uniform mesoporous featureinofFigure hollow2A. core-mesoporous shell silica sphere various functionalities are presented The isotherms showed silica sphere samples with various functionalities are presented in Figure 2A. The isotherms showed silicaIVnanospheres The Brunauer-Emmett-Teller (BET)feature surface of area and total pore volume of shell the type type curves that that[18]. depicted the uniform uniform mesoporous mesoporous hollow core-mesoporous the IV curves depicted the feature of hollow core-mesoporous shell HCMSS–NH2 nanospheres were 307.13 cm2·g−1 and 0.576 cc·g−1, respectively. Removal of amino silica nanospheres [18]. The Brunauer-Emmett-Teller (BET) surface area and total pore volume of silica groups nanospheres [18]. The Brunauer-Emmett-Teller (BET) area porevolume volume of 2·g−1 and by calcination resulted in increment2 of´surface area tosurface 394.27 cm whiletotal the pore 1 ´ 1 2 −1 −1 HCMSS–NH22 nanospheres nanospheres were were 307.13 ¨·g g and and 0.576 0.576 cc¨ g , respectively. , respectively.Removal Removalof of amino amino HCMSS–NH 307.13 cm cmsample −1). These was almost closer to amino-functionalized (0.545 cc·gcc·g results are expected because 2 ¨2 g´ 1 while the pore volume −1 while groups by calcination resulted in increment of surface area to 394.27 cm groups by calcination resulted in increment of surface area to 394.27 cm ·g the pore volume the calcination would remove the amino groups which lead to improvement of surface area of 1 ). These results are expected because −1post-synthesis was almost almost to sample cc¨ g´ was closer to amino-functionalized amino-functionalized sample (0.545 (0.545 cc·g ). These results are expected hollow closer core nanostructures. Ethane functionalization through approach resultedbecause in 2·g the calcination remove the amino groups which lead to−1improvement surface areaThis ofarea hollow decrease ofwould both surface area and pore volume to 270.42 cmlead and cc·g−1of , respectively. is of the calcination would remove the amino groups which to 0.438 improvement of surface due to blockage of surface sites and mesopores with ethane groups. Pore size distribution for the core nanostructures. Ethane functionalization through post-synthesis approach resulted in decrease hollow core nanostructures. Ethane functionalization through post-synthesis approach resulted in 2¨ g ´1 and hollow core-mesoporous shell silica samples with different illustrated in 2·g−1 −1, are of both surface area andarea pore volume to 270.42 0.438 cc¨ g´1cc·g , respectively. ThisThis is due decrease of both surface and pore sphere volume tocm 270.42 cm andfunctionalities 0.438 respectively. is Figure 2B. All samples showed an identical pore size distribution profile, which centered around to blockage of surface sites and with ethane groups. Pore size distribution for the for hollow due to blockage of surface sitesmesopores and mesopores with ethane groups. Pore size distribution the 3.6 nm. In thatshell regard, reduction of the totalwith pore different volume upon functionalization without reduction core-mesoporous silica sphere functionalities are illustrated in Figure 2B. hollow core-mesoporous shell silicasamples sphere samples with different functionalities are illustrated in of the mesopores size suggested that the ethane groups were concentrated on the bottom or the All samples showed an identical pore size distribution profile, whichprofile, centered around 3.6 nm.around In that Figure 2B. All samples showed an identical pore size distribution which centered entrance of mesopores rather than homogenously distributed all over the channels and therefore the regard, reduction of thereduction total poreof volume upon functionalization without reduction of the mesopores 3.6 nm. Involume that regard, the size total pore pore was reduced while pore was notvolume affected.upon functionalization without reduction

size suggested thatsize the suggested ethane groups concentrated the bottom or the entrance of mesopores of the mesopores thatwere the ethane groupsonwere concentrated on the bottom or the rather than distributed all over the channels andall therefore the pore volume was reduced entrance of homogenously mesopores rather than homogenously distributed over the channels and therefore the while pore size was not affected. pore volume was reduced while pore size was not affected.

Figure 2. (A) N2 adsorption-desorption isotherms and (B) pore size distribution of (a) non-functionalized, (b) amino-functionalized and (c) ethane-functionalized of hollow core-mesoporous shell silica nanospheres.

Figure N2 adsorption-desorption isotherms andsize (B)distribution pore size distribution of (a) Figure 2.2.(A)(A) N2 adsorption-desorption isotherms and (B) pore of (a) non-functionalized, non-functionalized, (b) amino-functionalized and (c) ethane-functionalized of hollow (b) amino-functionalized and (c) ethane-functionalized of hollow core-mesoporous shell silica nanospheres. core-mesoporous shell silica nanospheres.

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FT-IR that different functional groups existed in the FT-IR measurements measurementswere weremade madeand anddemonstrated demonstrated that different functional groups existed in hollow structure as shown in Figure 3. Non-functionalized hollow silica spheres (Figure 3a) showed the hollow structure as shown in Figure 3. Non-functionalized hollow silica spheres (Figure 3a) main Si–O peak which is characteristic for silica atfor 1050–1250 cm´1 . Amino of hollow showed main Si–O peak which is characteristic silica at 1050–1250 cm−1functionalization . Amino functionalization nanospheres (Figure 3b) through using APMS can be confirmed from the presence of a N–H of hollow nanospheres (Figure 3b) through using APMS can be confirmed from the presencepeak of a ´1 [19]. Functionalization at 1637 cmat of hollow silica spheres with ethane groupgroup (CH2 –CH ) was N–H peak 1637 cm−1 [19]. Functionalization of hollow silica spheres with ethane (CH22–CH 2) ´1 −1together with a C–H stretching vibrations peak shown by the of aofpeak at 1415 cmcm was shown by appearance the appearance a peak at 1415 together with a C–H stretching vibrations peak ´1 which confirmed the presence of hydrophobic ethane groups within mesochannels at 2862 cm cm−1 at 2862 which confirmed the presence of hydrophobic ethane groups within mesochannels as ´1 as shown in Figure 3c [20]. However, the appearance of anpeak N–Hatpeak cm C–H −1 and −1 shown in Figure 3c [20]. However, the appearance of an N–H 1637 at cm1637 C–H and at 2862 cmat ´ 1 2862 cm (from APMS functionalization) as seen in Figure 3a (non-functionalized) could indicate (from APMS functionalization) as seen in Figure 3a (non-functionalized) could indicate the presence the presence of some residual amino and ethoxy groups APMS functionlization were not of some residual amino and ethoxy groups from APMSfrom functionlization that were that not removed removed completely during the calcination However, intensity of N–H peaks completely during the calcination process.process. However, weak weak intensity of N–H and and C–HC–H peaks in in Figure 3a suggest low content of these functional moieties due to the effect of heat treatment. Figure 3a suggest low content of these functional moieties due to the effect of heat treatment.

Figure 3. (a) non-functionalized; non-functionalized; (b) (b) amino-functionalized amino-functionalized and and (c) (c) ethane-functionalized ethane-functionalized Figure 3. FT-IR FT-IR spectra spectra of of (a) hollow core-mesoporous core-mesoporous shell shell silica silica nanospheres. nanospheres. hollow

2.2. Protease 2.2. Protease Immobilization Immobilization Purification of of alkaline alkaline protease protease from from alkaliphilic alkaliphilic Bacillus Bacillus sp. sp. NPST-AK15 NPST-AK15 culture culture supernatant supernatant Purification was carried out through a combination of ammonium sulfate precipitation and anion exchange was carried out through a combination of ammonium sulfate precipitation and anion exchange chromatography as aswe wedescribed describedinin a previous work [17]. purified enzyme immobilized chromatography a previous work [17]. TheThe purified enzyme was was immobilized onto onto the synthesized HCMSS nanospheres by covalent attachment and physical adsorption. Three the synthesized HCMSS nanospheres by covalent attachment and physical adsorption. Three samples samples used for physical immobilization included HCMSS–C HCMSS-non, HCMSS–C2H5, and were usedwere for physical immobilization that includedthat HCMSS-non, 2 H5 , and HCMSS–NH2 . HCMSS–NH 2. For covalent immobilization, the amino functionalized hollow core-mesoporous silica For covalent immobilization, the amino functionalized hollow core-mesoporous silica (HCMSS–NH2 ) (HCMSS–NH 2) nanospheres were first activated by glutaraldehyde as a bifunctional cross linking nanospheres were first activated by glutaraldehyde as a bifunctional cross linking agent prior to agent prior to proteaseThe conjugation. The of the matrix with glutaraldehyde takes place protease conjugation. crosslinking ofcrosslinking the matrix with glutaraldehyde takes place in two steps in in two the steps in amino which the free within amino groups within the andofhollow core of HCMSS–NH 2 which free groups the mesopores andmesopores hollow core HCMSS–NH 2 nanospheres nanospheres react with terminal aldehyde groups of glutaraldehyde molecules to form a Schiff-base react with terminal aldehyde groups of glutaraldehyde molecules to form a Schiff-base linkage and linkage the groups free amino groups in protease condense moleculeswith condense withfree the terminal other freealdehyde terminal then theand freethen amino in protease molecules the other aldehyde groups of glutaraldehyde to form a second Schiff-base bonding [21]. The results presented groups of glutaraldehyde to form a second Schiff-base bonding [21]. The results presented in in Table 1 revealed that NPST-AK15protease proteasewas wasimmobilized immobilizedsuccessfully successfully onto onto different different hollow hollow Table 1 revealed that NPST-AK15 core-mesoporous shell core-mesoporous shell silica silica (HCMSS) (HCMSS) nanospheres nanospheres either either by by covalent covalent or or physical physical immobilization. immobilization. However, immobilization of NPST-AK15 protease by covalent conjugation was more effective exhibiting much higher immobilization yield (75.6%) and loading efficiency (73.6%) relative to

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Int. J. Mol. Sci. 2016, 17, 184 5 of 18 However, immobilization of NPST-AK15 protease by covalent conjugation was more effective exhibiting much higher immobilization yield (75.6%) and loading efficiency (73.6%) relative to physical physical immobilization, which can be attributed to the strong covalent bonds formed between immobilization, which can be attributed to the strong covalent bonds formed between the protease the protease molecules and the activated HCMSS-NH2 nanospheres through the cross linking agent. molecules and the activated HCMSS–NH2 nanospheres through the cross linking agent. On the other On the other hand, immobilization of NPST-AK15 protease by physical adsorption gave low hand, immobilization of NPST-AK15 protease by physical adsorption gave low immobilization yield immobilization yield and loading efficiency in all tested supports, suggesting weak electrostatic and and loading efficiency in all tested supports, suggesting weak electrostatic and hydrophobic interaction hydrophobic interaction between the NPST-AK15 protease molecules and HCMSS nanospheres [22]. between the NPST-AK15 protease molecules and HCMSS nanospheres [22].

Table 1. Immobilization of NPST-AK15 alkaline protease onto hollow core-mesoporous shell silica Table 1. Immobilization of NPST-AK15 alkaline protease onto hollow core-mesoporous shell silica (HCMSS) nanospheres. The standard deviations were in the range of 1%–3%. (HCMSS) nanospheres. The standard deviations were in the range of 1%–3%.

Matrix Matrix

HCMSS-non HCMSS–NH HCMSS-non2 HCMSS–NH HCMSS–C 2H52 HCMSS–C2 H2 5 HCMSS–NH

Immobilization Immobilization Method Yield * (%) Immobilization Immobilization Method Yield Physical adsorption 44.6* (%) Physical adsorption 47.3 Physical adsorption 44.6 Physical adsorption 47.3 Physical adsorption 19.3 Physical adsorption 19.3 Covalent attachment 75.6

Activity Yield ** (%) Activity Yield ** (%) 40.0 42.3 40.0 42.3 16.3 16.3 72.2

Loading Efficiency *** (%) Loading Efficiency *** (%) 41.2 44.7 41.2 44.7 18.0 18.0 73.6

HCMSS–NH2 Covalent attachment 75.6 72.2 73.6 * Immobilization yield (%) = [(A − B)/A] × 100, where A is the total activity of the enzyme added in *the Immobilization yield (%) =solution [(A ´ B)/A] A is activity of the enzyme added in the initial immobilization and Bˆis100, thewhere activity ofthe thetotal unbound protease; ** Activity yield = initial immobilization solution and B is the activity of the unbound protease; ** Activity yield = (C/A) ˆ 100, (C/A) × 100, where A is the total activity of the enzyme added in the initial immobilization solution, where A is the total activity of the enzyme added in the initial immobilization solution, and C is activity of and C is activity ofprotease; the immobilization protease;=*** efficiency [(Pi − Piunb )/PP i]unb × 100, Pi the immobilization *** Loading efficiency [(PLoading and are where the initial i ´ Punb )/P i ] ˆ 100,=where protein subjected immobilization, and theto unbound protein, respectively. and Punb are theto initial protein subjected immobilization, and the unbound protein, respectively.

FT-IR Free and ImmobilizedProtease Protease 2.3.2.3. FT-IR of of Free and Immobilized The binding of the NPST-AK15 protease to HCMSS–NH was further confirmed by analysis. FT-IR The binding of the NPST-AK15 protease to HCMSS–NH confirmed by FT-IR 2 was2 further analysis. spectra for pure free NPST-AK15 protease, HCMSS–NH 2 immobilized nanospheres and FT-IR spectraFT-IR for pure free NPST-AK15 protease, HCMSS–NH nanospheres and enzyme 2 immobilized enzyme are illustrated in Figure 4. The spectrum of protease (Figure 4a) showed a are illustrated in Figure 4. The spectrum of protease (Figure 4a) showed a strong peak at range −1 which corresponds to the amide I and amide II groups [10]. ´ 1 strong peak at range of 1640–1650 cm of 1640–1650 cm which corresponds to the amide I and amide II groups [10]. The strong broad Theappearing strong broad appearing 3100–3600 cm−1 canto be N–H attributed to N–Hofstretching protease 1 can band at band 3100–3600 cm´at be attributed stretching protease of amides [10]. amides [10]. FT-IR spectrum of HCMSS–NH 2 nanospheres is shown in Figure 4b. The strong peak at´1 FT-IR spectrum of HCMSS–NH2 nanospheres is shown in Figure 4b. The strong peak at 1050–1250 cm 1050–1250 cm−1 is attributed to Si–O bonding [19]. The amino functionalization of HCMSS spheres is attributed to Si–O bonding [19]. The amino functionalization of HCMSS spheres was demonstrated was demonstrated from the appearance of an N–H, peak at 1637 cm−1 as well as OCH2CH3 and C–H from the appearance of an N–H, peak at 1637 cm´1−1as well as OCH2 CH3 and C–H stretching vibrations stretching vibrations peaks at 2930 and 2862 cm , respectively [19]. The FT-IR of the immobilized peaks at 2930 and 2862 cm´1 , respectively [19]. The FT-IR of the immobilized protease shown in protease shown in Figure 4c indicated the presence of a strong peak at´1640–1650 cm−1 for amide, and 1 for amide, Figure 4c indicated the presence of a strong peak at 1640–1650 cm and a broad one a broad one at ´ 3100–3600 cm−1 for the N–H group of the protease, together with the main peaks of 1 at hollow 3100–3600 cm for the N–H group of the protease, together with the main peaks of hollow core-mesoporous shell silica spheres as Si–O bond at 1050–1250 cm−1. These results core-mesoporous shell silicasuccessful spheres asimmobilization Si–O bond at 1050–1250 cm´1 . These results demonstrate and confirm of the NPST-AK15 protease ontodemonstrate HCMSS–NHand 2 confirm successful immobilization of the NPST-AK15 protease onto HCMSS–NH2 nanospheres. nanospheres.

Figure 4. FTIR spectra of Free (a) Free NPST-AK15 protease enzyme (balck); (b) amino-functionalized Figure 4. FTIR spectra of (a) NPST-AK15 protease enzyme (balck); (b) amino-functionalized hollow hollow core-mesoporous silica (HCMSS–NH 2) nanospheres (blue) and (c) NPST-AK15 protease core-mesoporous silica (HCMSS–NH2 ) nanospheres (blue) and (c) NPST-AK15 protease immobilized 2) nanospheres immobilized onto amino-functionalized hollow core-mesoporous silica (HCMSS–NH onto amino-functionalized hollow core-mesoporous silica (HCMSS–NH 2 ) nanospheres (red). (red).

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2.4. Loading Capacity

800

100

700

80

600 500

60

400 40

300 200

20

100 0

Protease activity [Unit]

Protein (µg)/ nanospheres [mg]

2.4. Loading Capacity capacity of Ac–HCMSS–NH2 nanospheres was estimated by covalent The loading immobilization variousofamounts of NPST-AK15 protease and ranged from 50 to 200 μg protein/ The loadingofcapacity Ac–HCMSS–NH was estimated by covalent immobilization 2 nanospheres 10 mg nanoparticles. The results shown Figure 5 indicated amount ofmg loaded protease of various amounts of NPST-AK15 proteaseinand ranged from 50 tothat 200 the µg protein/10 nanoparticles. increased with increasing enzyme concentration giving maximum loading of 88.1 μg protein/mg The results shown in Figure 5 indicated that the amount of loaded protease increased with increasing carrier, then remained This resultofshows that Ac–HCMSS–NH –NHthen 2 nanospheres enzymeand concentration givingconstant. maximum loading 88.1 µg protein/mg carrier,2and remained have highThis loading capacity for covalent immobilization of NPST-AK15 protease [23–25], which can constant. result shows that Ac–HCMSS–NH –NH nanospheres have high loading capacity for 2 2 2·g−1) and pore volume of the nanoparticles (0.576 be attributed to the high surface area (307.13 cm covalent immobilization of NPST-AK15 protease [23–25], which can be attributed to the high surface 1 ) and pore 1 , respectively) available cc·g , respectively) forvolume the enzyme Furthermore, of HCMSS area−1(307.13 cm2 ¨ g´available of theconjugation. nanoparticles (0.576 cc¨ g´functionalization for nanospheres with amino Furthermore, groups through in situ approachofrather than post-synthesis method the enzyme conjugation. functionalization HCMSS nanospheres with aminoresulted groups in integration a higher rather densitythan of amino groups method availableresulted for protease conjugation a through in situofapproach post-synthesis in integration of a[26,27]. higher Thus, density protein of 88.1 μg per mg Ac–HCMSS–NH 2–NH 2 nanospheres in all of aminoloading groups available forprotein protease conjugation [26,27]. Thus, a protein loading ofwas 88.1used µg protein subsequent investigations. per mg Ac–HCMSS–NH2 –NH2 nanospheres was used in all subsequent investigations.

0 1

2

3

4

5

6

7

8

9

10

11

12

13

14

Protease protein content [mg] Protease activity activity

µg protein/mg carrier

Figure 5. forfor immobilization of Figure 5. Loading Loading efficiency efficiencyof ofhollow hollowcore-mesoporous core-mesoporousshell shellsilica silicananospheres nanospheres immobilization NPST-AK15 protease. of NPST-AK15 protease.

2.5. Properties Properties of of the the Immobilized Immobilized NPST-AK15 NPST-AK15 Protease 2.5. Protease 2.5.1. Influence of pH and Temperature 2.5.1. Influence of pH and Temperature The results of the effect of pH on the catalytic activity of both free and immobilized NPST-AK15 The results of the effect of pH on the catalytic activity of both free and immobilized NPST-AK15 protease show that the catalytic activity was increasing as pH increased, reaching maximum activity protease show that the catalytic activity was increasing as pH increased, reaching maximum activity at pH pH 10.5 protease, respectively. respectively. Hence, Hence, the the optimum optimum pH pH of of at 10.5 and and 11.0 11.0 for for soluble soluble and and immobilized immobilized protease, alkaline protease immobilized on Ac–HCMSS–NH nanospheres was shifted toward the alkaline alkaline protease immobilized on Ac–HCMSS–NH22 nanospheres was shifted toward the alkaline range by by 0.5 0.5 units units in in comparison with the the free In addition there was range comparison with free protease protease (Figure (Figure 6). 6). In addition there was significant significant increment of the relative activities of the immobilized NPST-AK15 protease at a wide pH range increment of the relative activities of the immobilized NPST-AK15 protease at a wide pH range (pH 5–13) in comparison with free enzyme. The slight shift of optimal pH value to the alkaline region (pH 5–13) in comparison with free enzyme. The slight shift of optimal pH value to the alkaline was probably due to the of its microenvironment caused by the immobilization of the enzyme region was probably duechange to the change of its microenvironment caused by the immobilization of the on the carrier. An increase of the optimum pH was observed for the other enzymes immobilized on enzyme on the carrier. An increase of the optimum pH was observed for the other enzymes the solid matrix immobilized on [25,28]. the solid matrix [25,28].

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110 100 110 90 100 80 7090 6080 5070 4060 3050 2040 1030 020 10 0

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Relative activity [%]

Relative activity [%]

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5 5

6 6

7 7

8 8

9 10 9 pH 10 pH

Free enzyme Free enzyme

10.5 10.5

11 11

12 12

13 13

Immobilized enzyme Immobilized enzyme

Figure 6. Effect of pH on the activity of the free and immobilized NPST-AK15 protease. Enzyme Figure 6. Effect of pH on the activity of the free and immobilized NPST-AK15 protease. Enzyme activity was measured at on 55 ˝ C. The results represent theimmobilized mean of threeNPST-AK15 separate experiments, and error Figurewas 6. Effect of pH of the free and protease. Enzyme activity measured at 55 the °C. activity The results represent the mean of three separate experiments, and bars are indicated. activity was measured at 55 °C. The results represent the mean of three separate experiments, and error bars are indicated. error bars are indicated.

The results results of of the the influence influence of of reaction reaction temperature temperature on on the the catalytic catalytic activity activity of of soluble soluble and and The The results of the influence of reaction temperature on the catalytic activity of soluble and immobilizedenzyme enzymerevealed revealedthat thatthe theoptimum optimumtemperature temperaturefor forboth bothfree freeand andimmobilized immobilizedenzyme enzyme immobilized immobilized enzyme revealed that the optimum temperature for both free and immobilized enzyme ˝ C (Figure was 60 60 °C 7A). However, However, the the immobilized immobilized protease protease exhibited exhibited higher higher relative relative activities activities in in was (Figure 7A). was 60 °C (Figure 7A). However, the immobilized protease exhibited higher activities in ˝ C). Inrelative comparison with soluble enzyme, especially at high temperatures (65–75 addition, the thermal comparison temperatures (65–75 (65–75°C). °C).InInaddition, addition,thethe comparison withsoluble solubleenzyme, enzyme, especially especially at at high high temperatures stability of free and immobilized protease was investigated at various temperatures (50–60 ˝ C). thermal stability of free and immobilized protease was investigated at various temperatures thermal stability of free and immobilized protease was investigated at various temperatures As(50–60 shown in 7Binthe protease immobilized onto the nanosphere much better much thermal (50–60 °C). AsFigure shown 7B onto the theshowed nanosphere showed much °C). As shown inFigure Figure 7Bthe the protease protease immobilized immobilized onto nanosphere showed ˝ stability compared to free enzyme. After treatment fortreatment 1 h at 55 for C,11the immobilized enzyme still better thermal stability compared totofree After treatment for 55°C, °C,the theimmobilized immobilized better thermal stability compared freeenzyme. enzyme. After hhatat 55 retained 73.5% of its initial activity, while the free protease maintained only 30%. Moreover, while enzyme still retained 73.5% of its initial activity, while the free protease maintained only30%. 30%. enzyme still retained 73.5% of its initial activity, while the free protease maintained only ˝ C the immobilized theMoreover, solublewhile enzyme was completely inactivated when treated for 1 h at 60 Moreover, the soluble enzyme was completely inactivated when treated for 1 h at 60 °C while the soluble enzyme was completely inactivated when treated for 1 h at 60 °C thethe enzyme still enzyme retained 63.6% and 29.3% ofand its initial after treatment for 1 and 2for h,for respectively. immobilized still 63.6% 29.3% of its initial initial activity, after treatment 1 and 2 h, immobilized enzyme stillretained retained 63.6% and 29.3%activity, activity, after treatment 1 and 2 h, The significant improvement of the immobilized NPST-AK15 protease protease thermal stability isstability mostly respectively. The significantimprovement improvement ofthe the immobilized immobilized NPST-AK15 stability respectively. The significant of NPST-AK15 proteasethermal thermal is mostly multipoint covalent attachment attachment of protease totothe matrix that toattributed multipoint covalent attachment of protease to the matrix that protect the isattributed mostly attributed totomultipoint covalent ofmolecules protease molecules molecules the matrix that protect the protease tertiary structure and prevent preventtransition conformation ofofthe enzyme upon protease tertiary structure andstructure prevent conformation of thetransition enzyme upon heating, leading to protect the protease tertiary and conformation transition the enzyme upon leading improved thermalstability stability [9,29]. [9,29]. anheating, improved thermal stability [9,29]. heating, leading toto ananimproved thermal

Relative acitivity [%] Relative acitivity [%]

110 110 100 100 9090 80 8070 7060 6050 5040 4030 3020 2010 10 0 0

30 30

35 35

40 40

45 50 55 60 45 Temperature 50 55 [°C] 60

65

65

Temperature [°C] Free enzyme

Immobilized enzyme Immobilized enzyme

Free enzyme (A) (A)

Figure 7. Cont. Figure 7. Cont.

Figure 7. Cont.

70

70

75

75

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100 90 Residual activity [%]

80 70

50 °C Free

60

50 °C Immo

50

55 °C Free

40

55 °C Immo

30 20

60 °C Free

10

60 °C Immo

0 0

20

40

60

80

100

120

Time [min] (B) Figure 7. Effect of temperature on activity (A) and stability (B) of free and immobilized NPST-AK15 Figure 7. Effect of temperature on activity (A) and stability (B) of free and immobilized NPST-AK15 protease. Protease activity was measured under the standard assay conditions at various protease. Protease activity was measured under the standard assay conditions at various temperatures temperatures (30–75 °C) at pH 10. For thermal stability, the enzyme was pre-incubated at different (30–75 ˝ C) at pH 10. For thermal stability, the enzyme was pre-incubated at different temperatures for temperatures for 2 h at pH 11.0, and the residual enzyme activities were estimated at regular 2 h at pH 11.0, and the residual enzyme activities were estimated at regular intervals under standard intervals under standard assay conditions. The non-heated enzymes were taken as 100%. The results assay conditions. Theofnon-heated enzymes were taken 100%. represent the mean of three represent the mean three separate experiments, andas error barsThe areresults indicated. separate experiments, and error bars are indicated.

2.5.2. Kinetics Studies 2.5.2. Kinetics Studies Investigation of the kinetic parameters (Km and Vmax) is also a critical feature for investigating kinetic parameters (Km Kinetics and V max ) is also a critical investigating theInvestigation effectiveness of the an immobilization process. parameters of both feature free andfor immobilized protease were estimated using Lineweaver–Burk plot by using casein as a substrate at pH 11 and the effectiveness of an immobilization process. Kinetics parameters of both free and immobilized 60 °C. The Michaelis–Menten (Km) of free and NPST-AK15 proteaseat were protease were estimated usingconstant Lineweaver–Burk plotimmobilized by using casein as a substrate pH0.109 11 and ˝ C. 0.074 mM, respectively, which clearly increase of the enzyme affinity protease toward the 60and The Michaelis–Menten constant (Km indicated ) of free and immobilized NPST-AK15 were substrate by about 1.5-fold up on immobilization onto Ac–HCMSS–NH 2 nanospheres (Figure 8; 0.109 and 0.074 mM, respectively, which clearly indicated increase of the enzyme affinity toward Table 2). This is mostly due to high mass transport of reactants and products within the carrier the substrate by about 1.5-fold up on immobilization onto Ac–HCMSS–NH2 nanospheres (Figure 8; which is a isunique of themass nanostructure Alsoproducts the Brownian of which the Table 2). This mostlyfeature due to high transport ofmaterials. reactants and within motion the carrier couldof result in enhanced substrate-enzyme [30]. motion This behavior protease is nanospheres a unique feature the nanostructure materials. Alsointeraction the Brownian of theofnanospheres immobilized onto Ac–HCMSS–NH2 nanospheres was superior to several nanoparticles bound to could result in enhanced substrate-enzyme interaction [30]. This behavior of protease immobilized enzyme [25,31]. In addition, the maximum activity (Vmax), enzyme turnover number (kcat), and onto Ac–HCMSS–NH2 nanospheres was superior to several nanoparticles bound to enzyme [25,31]. catalytic efficiency (kcat/Km) values of NPST-AK15 protease were increased by about 1.6-, 1.6-, and In addition, the maximum activity (V max ), enzyme turnover number (kcat ), and catalytic efficiency 2.4-fold, respectively, upon immobilization on to HCMSS–NH2 nanospheres. This may be due to (kcat /Km ) values of NPST-AK15 protease were increased by about 1.6-, 1.6-, and 2.4-fold, respectively, more efficient conformation of immobilized protease within the nanoscale spaces of the hollow upon immobilization on to nanospheres. This may be due to moreand efficient conformation nanospheres in respect to HCMSS–NH free enzyme 2[28]. The high affinity toward substrate catalytic activity ofof immobilized protease within the nanoscale spaces of the hollow nanospheres in respect NPST-AK15 alkaline protease immobilized onto Ac–HCMSS–NH2 nanospheres indicate to thefree enzyme [28]. The high affinity toward substrate and catalytic activity of NPST-AK15 alkaline protease efficiency and effectiveness of applied support and immobilization approach. immobilized onto Ac–HCMSS–NH2 nanospheres indicate the efficiency and effectiveness of applied support and 2.immobilization approach. Table Kinetic parameters of free and immobilized NPST-AK15 alkaline protease. The standard deviations were in the range of 1.5%–3.1%. Table 2. Kinetic parameters of free and immobilized NPST-AK15 alkaline protease. The standard Kinetic Parameters Free Protease Immobilized Protease deviations were in the range of 1.5%–3.1%.

Km (mM) VKinetic max (μM·min−1·mg−1) Parameters kcat (s−1) K (mM)−1 −1 kcat/Kmm (mM ´1 ·s ) ´1

V max (µM¨ min ¨ mg ) kcat (s´1 ) kcat /Km (mM´1 ¨ s´1 )

0.109 41.2 Free Protease 1156.5 0.109 10.6 × 103 41.2 1156.5 10.6 ˆ 103

0.074 66.1 Immobilized Protease 1855.4 0.074 25.1 × 103 66.1 1855.4 25.1 ˆ 103

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0.0018 0.0016

[1/V]

0.0014 0.0012 0.001 0.0008 0.0006 0.0004 0.0002 0 -1

-0.5

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

[1/S] Immobilized enzyme

Free enzyme

Figure 8. Estimation of kinetic parameters of the free and immobilized NPST-AK15 protease. The enzyme Figure 8. Estimation of kinetic parameters of the free and immobilized NPST-AK15 protease. The activity was measured at various casein concentrations (1.0–10.0 mg/mL) at pH 11 and 60 ˝ C. The K enzyme activity was measured at various casein concentrations (1.0–10.0 mg/mL) at pH 11 and 60 °C.m and Vmax values were determined using linearized Lineweaver-Burk plot. S: Substrate concentration; The Km and Vmax values were determined using linearized Lineweaver-Burk plot. S: Substrate V: Protease specific activity. concentration; V: Protease specific activity

2.5.3.Effect EffectofofInhibitors Inhibitorsand andOrganic OrganicSolvents Solvents 2.5.3. Theresults resultsofofthe theinfluence influenceofofsome someinhibitors inhibitorson oncatalytic catalyticactivity activityofoffree freeand andimmobilized immobilized The NPST-AK15 protease are summarized in Table 3. Phenylmethanesulfonyl fluoride (PMSF) NPST-AK15 protease are summarized in Table 3. Phenylmethanesulfonyl fluoridecompletely (PMSF) inhibited the free protease at 5.0 mM, at suggesting it belongs toit serine protease family [3,17]. completely inhibited the freeactivity protease activity 5.0 mM, suggesting belongs to serine protease However, theHowever, immobilized enzyme retained 86.3% of its initial In addition, the free family [3,17]. the immobilized enzyme retained 86.3%activity. of its initial activity. while In addition, protease lost about 75% of its initial activity after treatment with 5 mM dithiothreitol (DTT), the while the free protease lost about 75% of its initial activity after treatment with 5 mM dithiothreitol immobilized protease maintained 97.5% at the same conditions. The metal chelating agent, EDTA, (DTT), the immobilized protease maintained 97.5% at the same conditions. The metal chelating causedEDTA, inhibition of the soluble enzyme the extent of 60.2% andextent 30.5% at of 2 and agent, caused inhibition of the to soluble enzyme to the of concentrations 60.2% and 30.5% at 5.0 mM, respectively, suggesting that some metals may play a role in the activity/stability the concentrations of 2 and 5.0 mM, respectively, suggesting that some metals may play a role inofthe enzyme [17], whereas at the same the immobilized protease retained 95% and 81.5% activity/stability of the enzyme [17],conditions whereas at the same conditions the still immobilized protease still of its initial values, respectively. However, there was no significant difference between the effect of retained 95% and 81.5% of its initial values, respectively. However, there was no significant β-mercaptoethanol (β-ME) and immobilized NPST-AK15 Protection of protease difference between the effecton of free β-mercaptoethanol (β-ME) on free protease. and immobilized NPST-AK15 against such inhibitors upon immobilization HCMSS–NH explained 2in 2 nanospheres could protease. Protection of protease against such onto inhibitors upon immobilization onto be HCMSS–NH two different ways: (i) In case of allosteric inhibitor in which it interacts with the enzyme at site other nanospheres could be explained in two different ways: (i) In case of allosteric inhibitor inawhich it than the catalytic center, it is likely immobilization of the enzyme through that niche leads to distortion interacts with the enzyme at a site other than the catalytic center, it is likely immobilization of the of the allosteric andniche subsequently significant reduction of the (ii) Alternatively, enzyme throughsite that leads tocauses distortion of the allosteric siteinhibition; and subsequently causesif the inhibitor interacts directly with the active center, the process of enzyme immobilization may significant reduction of the inhibition; (ii) Alternatively, if the inhibitor interacts directly withcause the a slight change in the enzyme active site that affects the inhibitor binding to enzyme with less effect on active center, the process of enzyme immobilization may cause a slight change in the enzyme active the substrate-enzyme binding especially in the case of high molecular weight substrates [9]. site that affects the inhibitor binding to enzyme with less effect on the substrate-enzyme binding As shown in Figure 9, the stabilityweight of the substrates NPST-AK15 especially in the case of high molecular [9].protease in various organic solvents was greatly improved upon covalent immobilization onto the Ac–HCMSS–NH The results 2 nanospheres. As shown in Figure 9, the stability of the NPST-AK15 protease in various organic solvents was revealed that the stability of the immobilized protease in butanol, isopropanol, chloroform and ethanol greatly improved upon covalent immobilization onto the Ac–HCMSS–NH2 nanospheres. The results was significantly increased 2.5-, 2.2-,protease 1.8- and in 1.8-fold respectively, in respect to theand free revealed that the stability ofby theabout immobilized butanol, isopropanol, chloroform enzyme. addition, its stabilityby inabout methanol, toluene, and1.8-fold chloroform was increased bytoabout ethanol wasInsignificantly increased 2.5-, 2.2-, 1.8- and respectively, in respect the 1.1-fold. in acetone, toluene, methanol, amyl alcohol and methanol by about 1.7-, 1.5-, 1.4-, and free enzyme. In addition, its stability in methanol, toluene, and chloroform was increased by1.4-fold, about in comparison to free enzyme, respectively The significant of stability 1.1-fold. in acetone, toluene, methanol, amyl(Figure alcohol9). and methanol by enhancement about 1.7-, 1.5-, 1.4-, andin solvent for NPST-AK15 protease immobilized onto Ac–HCMSS–NH nanospheres can be attributed 2 1.4-fold, in comparison to free enzyme, respectively (Figure 9). The significant enhancement of to maintaining the active structural conformation of the enzyme through the multipoint stability in solvent for NPST-AK15 protease immobilized onto Ac–HCMSS–NH2 nanospherescovalent can be

attributed to maintaining the active structural conformation of the enzyme through the multipoint

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attachment to the matrix entrapment of the protease molecules within within the nanospaces of the covalent attachment to theand matrix and entrapment of the protease molecules the nanospaces hollow core ofcore Ac–HCMSS–NH nanospheres. Generally, the biotechnological applications of various of the hollow of Ac–HCMSS–NH 2 nanospheres. Generally, the biotechnological applications of 2 enzymes could be significantly increased by their in organic mediamedia ratherrather than than in their various enzymes could be significantly increased byutilization their utilization in organic in natural aqueous conditions. In theInlast the utility various proteases in organic their natural aqueous conditions. thedecade last decade the of utility of various proteases in solvents organic gained much importance owing to potential of proteasesofinproteases synthesis in of synthesis ester and peptides solvents gained much importance owing toapplications potential applications of ester in non-aqueous media [32]. media [32]. and peptides in non-aqueous Table 3. 3. Influence Influence of of some some inhibitors inhibitors on on the the catalytic catalytic activity activity of of free free and and immobilized immobilized NPST-AK15 NPST-AK15 Table protease. The The standard standard deviations deviations were were in in the the range range of of 1.5%–3.2%. 1.5%–3.2%. protease.

Inhibitor Inhibitor None None

Concentration Concentration

β-mercaptoethanol β-mercaptoethanol

1 55

1

PMSF

1 55

DTT DTT

1 55

EDTA EDTA

22 55

PMSF

Residual protease activity [%]

1

1

FreeEnzyme Enzyme Free

Immobilized Enzyme Immobilized Enzyme

100 100 94.3 ± 2.4 94.3 ˘ 2.4 88.0˘±2.2 2.2 88.0 15.5 ± 0.16 15.5 ˘ 0.16 00 90.2 ± 1.9 90.2 ˘ 1.9 22.60˘±0.8 0.8 22.60 62.2 ± 2.1 62.2 ˘ 2.1 30.1˘±1.5 1.5 30.1

100 94.0 94.0 89.0 89.0 92.9 92.9 86.3 86.3 101.8 101.8 97.5 97.5 95.0 95.0 81.5 81.5 100

120 100 80 60 40 20 0

Solvent (25%) Free enzyme

Immobilized enzyme

Figure9.9.Effect Effectof oforganic organicsolvents solventson onstability stabilityof offree freeand andimmobilized immobilizedNPST-AK15 NPST-AK15 protease. protease. Figure

2.5.4. Influence Influenceof ofSurfactants Surfactants and and Commercial Commercial Laundry Laundry Detergents Detergents 2.5.4. The results results presented ofof some surfactants on on thethe stability of free and The presentedin inFigure Figure10A 10Ashow showofofthe theeffect effect some surfactants stability of free immobilized NPST-AK15 alkaline protease. It was found that the stability of the immobilized protease and immobilized NPST-AK15 alkaline protease. It was found that the stability of the immobilized exhibited higher stability in 5%stability lauryl glucoside, sulfate (SDS), cetyltrimethylammonium protease exhibited higher in 5% sodium lauryl dodecyl glucoside, sodium dodecyl sulfate (SDS), bromide (CTAB) and Triton X-100 by 9.4-, 7.5-, 3.4-, and 1.6-fold, respectively, comparison to free cetyltrimethylammonium bromide (CTAB) and Triton X-100 by 9.4-, 7.5-,in 3.4-, and 1.6-fold, enzyme. In addition, the stability of immobilized protease in wasofimproved by about 1.1-fold. respectively, in comparison to free enzyme. In addition, theTween stability immobilized protease in Finally, to assess the compatibility and feasibility of the enzyme immobilized onto Ac–HCMSS–NH Tween was improved by about 1.1-fold. Finally, to assess the compatibility and feasibility of the2 nanospheres as laundryonto detergents additive, its2 stability towardassome commercial laundry detergents enzyme immobilized Ac–HCMSS–NH nanospheres laundry detergents additive, its was investigated. The commercial results illustrated in Figure 10B revealed that the immobilized protease was stability toward some laundry detergents was investigated. The results illustrated in stable and compatible in most tested commercial laundry detergents. In comparison to free protease, Figure 10B revealed that the immobilized protease was stable and compatible in most tested the immobilized enzyme exhibited higher stability in 5% of OMO, Ariel, Bonux and X-TRA by commercial laundry detergents. In comparison to free protease, theTide, immobilized enzyme exhibited about 5.2-, 4.9-, 4.7-, 4.5-, 4.2-fold In addition, the stability immobilized in higher stability in 5% ofand OMO, Tide,respectively. Ariel, Bonux and X-TRA by aboutof5.2-, 4.9-, 4.7-,protease 4.5-, and

4.2-fold respectively. In addition, the stability of immobilized protease in 5% Ayam, Persil, and REX as improved by about 2.1-, 1.8-, and 1.8-fold, respectively. The noteworthy stability of NPST-AK15

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Residual protease activity [%]

5% Ayam, Persil, and REX as improved by about 2.1-, 1.8-, and 1.8-fold, respectively. The noteworthy alkaline covalently immobilized activatedimmobilized Ac–HCMSS–NH 2 nanospheres in various2 stability protease of NPST-AK15 alkaline protease on covalently on activated Ac–HCMSS–NH commercial laundry detergents suggested the effectiveness of the developed nanobiocatalyst as a nanospheres in various commercial laundry detergents suggested the effectiveness of the developed candidate for detergents formulation. nanobiocatalyst as a candidate for detergents formulation.

120 100 80 60 40 20 0

Surfactant Free enzyme

Immobilized enzyme (A)

Residual activity [%]

120 100 80 60 40 20 0

Commercial detergent Free enzyme

Immobilized enzyme (B)

Figure 10. Effect of surfactants (A) and commercial detergents (B) on stability of free and Figure 10. Effect of surfactants (A) and commercial detergents (B) on stability of free and immobilized immobilized NPST-AK15 protease stability. SDS: sodium dodecyl sulfate; CTAB: NPST-AK15 protease stability. SDS: sodium dodecyl sulfate; CTAB: cetyltrimethylammonium bromide. cetyltrimethylammonium bromide.

2.5.5. Operational Stability of Immobilized Protease 2.5.5. Operational Stability of Immobilized Protease The operational stability of immobilized biocatalysts is considered as one of the most important feature The operational stability of immobilized biocatalysts is considered as one of the most important that affects their applications in various bioprocesses. Generally, enzyme leakage from the carriers and feature that affects their applications in various bioprocesses. Generally, enzyme leakage from the enzyme inactivation represent one of the main obstacles for immobilized enzyme applications particularly carriers and enzyme inactivation represent one of the main obstacles for immobilized enzyme applications particularly in repeated batch and continuous process [33]. The reuse of NPST-AK15 protease immobilized onto Ac–HCMSS–NH2 nanospheres was studied in several batches for

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12 onto of 18 continuous process [33]. The reuse of NPST-AK15 protease immobilized Ac–HCMSS–NH2 nanospheres was studied in several batches for substrate hydrolysis that performed substrate hydrolysis at 40 °C and pHreutilizations 11. The resultsfor indicated after reutilizations at 40 ˝ C and pH 11. that The performed results indicated that after ten and that twelve repeated cycles, for ten and twelve repeated cycles, the immobilized protease could retain up 65.3% and 55.6% of its the immobilized protease could retain up 65.3% and 55.6% of its initial activity, respectively, revealing initial activity, respectively, revealing a good operational stability of the developed nanobiocatalyst a good operational stability of the developed nanobiocatalyst (Figure 11). The operational stability (Figure 11). The operational of immobilized biocatalyst is anof immobilized essential factor for of immobilized biocatalyst is an stability essential factor for cost-effective application enzyme cost-effective application of immobilized enzyme system and development of novel biotechnology. system and development of novel biotechnology.

Residual activity [%]

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100 80 60 40 20 0 1

2

3

4

5

6

7

8

9

10

11

12

Cycle numbers Figure 11. Reusability of NPST-AK15 protease immobilized within hollow core-mesoporous shell silica Figure 11. Reusability of NPST-AK15 protease immobilized within hollow core-mesoporous shell (HCMSS–NH2 ) nanospheres. silica (HCMSS–NH2) nanospheres.

3. Experimental Section 3. Experimental Section All chemicals were of the analytical grade and, unless specified, obtained from Sigma Chemical Co. All chemicals were of the analytical grade and, unless specified, obtained from Sigma Chemical (St. Louis, MO, USA). Co. (St. Louis, MO, USA). 3.1. Alkaline Protease Production and Purification 3.1. Alkaline Protease Production and Purification Alkaliphilic alkaline protease producing Bacillus sp. strain NPST-AK15 was previously isolated Alkaliphilic alkaline protease producing Bacillus strain NPST-AK15 was[16,17]. previously from Wadi El-Natrun valley hypersaline soda lakes sp. (Wadi El-Natrun, Egypt) The isolated enzyme from Wadi was El-Natrun valley hypersaline soda (Wadi El-Natrun, Egypt)and [16,17]. The enzyme production previously optimized [16] and thelakes alkaline protease was purified characterized [17]. production previously optimized [16]inand the alkaline protease was purified and characterized The enzymewas production was carried out production medium of following composition (pH 10.5): [17]. enzyme production carried out in(1.0 production of (0.2 following composition yeastThe extract (7.5 g/L), fructose was (20 g/L), K2 HPO g/L), Mg2medium SO4 ¨ 7H2 O g/L), NaCl (50 g/L), 4 (pH yeast extract (7.5 g/L), fructose (20 g/L), K2HPO ˝ 4 (1.0 g/L), Mg2SO4·7H2O (0.2 g/L), NaCl and 10.5): Na2 CO 3 (10 g/L). An overnight culture grown at 40 C and 150 rpm was inoculated into 500-mL (50 g/L), andflasks Na2CO 3 (10 g/L). An overnight culture grown at 40 °C and 150 rpm was inoculated into Erlenmeyer containing 100 mL of the production medium and incubated for about 36 h at 40 ˝ C. 500-mL Erlenmeyer flasks containing 100 mL of the medium and incubated forrpm about After the incubation period, cell-free supernatant wasproduction recovered by centrifugation at 10,000 for 36 h at 40 °C. After the incubation period, cell-free supernatant was recovered by centrifugation at ˝ 10 min at 4 C and assayed for protease activity and protein content. Thereafter, NPST-AK15 protease 10,000 rpm for 10 min at 4 °C and assayed for protease activity and protein content. Thereafter, was purified from cell-free supernatant by combination of ammonium sulfate precipitation and anion NPST-AK15 protease was purified from supernatant bywork combination of ammonium sulfate exchange column chromatography as wecell-free described in previous [17]. precipitation and anion exchange column chromatography as we described in previous work [17]. 3.2. Fabrication of Hollow Core-Mesoporous Shell Silica Nanospheres 3.2. Fabrication of Hollow Core-Mesoporous Shell Silica Nanospheres 3.2.1. Amino-Functionalized Hollow Core-Mesoporous Shell Silica Nanospheres 3.2.1. Amino-Functionalized Hollow Core-Mesoporous Shell Silica Nanospheres Amino-functionalized hollow core-mesoporous shell silica (HCMSS–NH2 ) nanospheres were Amino-functionalized hollow core-mesoporous nanospheres synthesized according to previously reported methodsshell withsilica some(HCMSS–NH modifications2)[34,35]. Briefly, were 4 mL synthesized according reported methods withsolution some modifications [34,35]. Briefly, 0.1 M HCl was added toto35previously mL of N-lauroylsarcosine sodium (1 mM) prepared in deionized 4water, mL 0.1 M HCl added 35mL mLofof3-aminopropyltrimethoxysilane N-lauroylsarcosine sodium solution (1 mM) prepared in stirred for was 1 h and thento0.1 (APMS) was added to the deionized water, stirred for 1 for h and then Thereafter 0.1 mL of 1.5 3-aminopropyltrimethoxysilane solution with further stirring 10 min. mL of tetraethyl orthosilicate(APMS) (TEOS) was was added solution stirred with further stirring for 10 to min. Thereafter 1.5 for mL4 of tetraethyl added to the reaction, for 10 min, subjected ultrasonic waves min at 750 W,orthosilicate and 20 KHz (TEOS) was added to the reaction, stirred forthen 10 min, waveswas forheated 4 min for at (Sonic vibro cell, Connecticut, CT, USA), and kept subjected to relax forto1 ultrasonic h. The mixture ˝ C20 750 and KHz vibro Connecticut, CT, USA), and then keptwashed to relax fordeionized 1 h. The 18 hW, at 80 and the (Sonic obtained solidcell, materials was collected by centrifugation, with mixture was heated for 18 h at 80 °C and the obtained solid materials was collected by centrifugation, washed with deionized water and dried for 12 h at 60 °C. The template was removed by acid extraction to keep the hollow core-mesoporous shell silica spheres functionalized with the amino moieties. For removal of anionic surfactant, the final material was suspended in 100 mL of

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water and dried for 12 h at 60 ˝ C. The template was removed by acid extraction to keep the hollow core-mesoporous shell silica spheres functionalized with the amino moieties. For removal of anionic surfactant, the final material was suspended in 100 mL of ammonium acetate solution (8%) prepared in ethanol:H2 O (4:1) and refluxed for 12 h at 90 ˝ C. In order to remove amino groups from the formed hollow core-mesoporous shell silica spheres (HCMSS–NH2 ) to convert it to non-functionalized form, HCMSS–NH2 were calcined for 6 h in air stream at 550 ˝ C to give HCMSS nanospheres. 3.2.2. Hydrophobic Ethane-Functionalized Hollow Core-Mesoporous Shell Silica Nanospheres Ethane-functionalized hollow core-mesoporous shell silica (HCMSS–C2 H5 ) nanospheres were prepared using post-synthesis functionalization approach according to the method of Brunel [36]. Briefly, 1 mL of 1,2-bis(trimethoxysilyl)ethane (BTME) was added to suspensions of freshly evacuated HCMSS dispersed in toluene (2 g/34 mL), and heated at 120 ˝ C with stirring for 3 h. The solid materials were collected by filtration and washed twice with a diethyl ether–dichloromethane mixture (1:1). 3.3. Characterization of the Synthesized Mesoporous Silica Based Nanospheres A JEOL JSM-2100F electron microscope (Tokyo, Japan) operated at 200 kV was used to obtain Transmission electron microscopy (TEM) images of the synthesized nanomaterials. A Quantachrome NOVA 4200 analyzer (Boynton Beach, FL, USA) was used for measurement of Nitrogen sorption isotherms at 77 K for various nanospheres, which were degassed in a vacuum for 18 h at 200 ˝ C before the measurements. The specific surface area of the nanospheres was calculated using the Brunauer-Emmett-Teller (BET) method at relative pressure range of 0.02–0.20. The pore volumes and size distributions of different silica nanospheres were derived from the adsorption branches of isotherms using the Barrett–Joyner–Halenda (BJH) model. In addition, the total pore volumes (V t ) were calculated from the adsorbed amount at a relative pressure (P/P0 ) of 0.995. Finally, Bruker Vertex-80 spectrometer (Bruker, Massachusetts, MA, USA) was used for recording Fourier transform infrared (FT-IR) spectra. 3.4. Alkaline Protease Immobilization 3.4.1. Physical Adsorption NPST-AK15 protease immobilization by physical adsorption was carried using HCMSS-non, HCMSS–C2 H5 and HCMSS–NH2 according to Ibrahim et al. [37]. Briefly, 50 mg of carriers were dispersed in 1 mL of glycine buffer (50 mM, pH 8) that contained the purified NPST-AK15 protease and the suspensions were kept for overnight at 4 ˝ C with gentle shaking. The HCMSS–enzyme nanoparticles were recovered by centrifugation for 10 min at 10,000 rpm, and washed twice with glycine buffer to remove any unbound enzyme. The wash solution was collected for protein content and protease activity measurement. 3.4.2. Covalent Attachment Alkaline protease was covalently immobilized onto amino-functionalized hollow core-mesoporous shell silica (HCMSS–NH2 ) nanospheres according to [38] with some modifications. The matrix was first activated by glutaraldehyde (OCHCH2 CH2 CHO) as bifunctional cross linking agent and then conjugation of NPST-AK15 protease to the activated nanospheres. Briefly, 50 mg of the HCMSS–NH2 nanospheres were dispersed in 5 mL of glutaraldehyde solution (5%, v/v), prepared in deionized water and stirred for 2 h at room temperature. The activated matrix (Ac–HCMSS–NH2 ) was recovered from the suspension by centrifugation, and washed with large amounts of deionized water to remove any excess glutaraldehyde. Then, the Ac–HCMSS–NH2 nanopartciles (50 mg) was suspended in 1 mL of glycine buffer (50 mM, pH 8) containing the enzyme and maintained overnight at 4 ˝ C with gentle shaking. The Ac–HCMSS–NH2 –protease nanospheres were recovered by centrifugation at

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10,000 rpm for 10 min and washed twice with the same buffer to remove any unbound enzyme. The wash solution was collected for protease activity protein content determination. 3.5. Assay of Alkaline Protease Activity The catalytic activity of soluble and immobilized NPST-AK15 protease was determined using casein as a substrate according to previous method reported with some modifications [23]. A 1.0 mL of casein solution prepared in glycine buffer (50 mM, pH 10.0) was pre-incubated for 5 min at 50 ˝ C, then 1.0 mL of diluted free protease or 1.0 mL glycine buffer containing 50 mg of immobilized enzyme was added and the reaction mixtures were incubated min at 50 ˝ C for 20. Thereafter, the enzymatic reaction was terminated by addition of 1 mL of 10% (w/v) of trichloroacetic acid (TCA). The mixture was kept at room temperature for 20 min and centrifuged at 10,000 rpm for 10 min to recover the precipitated materials. Lowry method was used to estimate the soluble materials in the mixture supernatant using tyrosine as a standard [39]. One unit of protease activity was defined as amount of enzyme required to liberate 1 µg of tyrosine/min under the experimental assay conditions. Bradford method was used to measure the protein by using bovine serum albumin (BSA) as a standard protein [40]. 3.6. Properties of Immobilized Protease 3.6.1. Influence of Temperature on Protease Activity and Stability The optimum temperature of catalytic activity for soluble and immobilized NPST-AK15 protease were determined by measuring the enzyme activity at various temperatures ranged from 30–70 ˝ C under standard assay conditions. The relative activities were calculated as percentage (%) of maximum enzyme activity and the values were plotted against the respective temperature. The thermal stability of both free and immobilized protease was investigated by calculating the residual activity after incubation of enzymes at various temperatures (40–60 ˝ C) for different incubation times (20–120 min). The activity of untreated protease was taken as control (%). 3.6.2. Influence of pH on Protease Activity The effect of pH on the catalytic activity of both soluble and immobilized NPST-AK15 protease was investigated by assaying the enzyme activity at different pH and appropriate buffers (50 mM), such as sodium acetate (pH 5.0 and 6.0), Tris–HCl (pH 7.0 and 8.0), glycine–NaOH buffer (pH range 9.0–10.5) and carbonate–bicarbonate buffer (pH 11.0–13.0). The substrate solution (1% casein) was prepared in the respective pH buffers and the enzyme preparations (soluble and immobilized) were incubated for 20 min at the optimum temperature of enzyme activity. The relative activities as percentage of maximum activity were calculated and plotted against the respective pH. 3.6.3. Influence of Solvents and Inhibitors The effects of several enzyme inhibitors on the activity of free and immobilized proteases were studied. The free and immobilized protease was pre-incubated with each inhibitor at final concentrations of 1.0 or 5.0 mM for 30 min at room temperature, and the residual protease activities were determined under the standard assay conditions. The tested inhibitors included ethylene diaminetetraacetic acid (EDTA), β-mercaptoethanol (β-ME), phenylmethanesulfonyl fluoride (PMSF), and dithiothreitol (DTT). For investigation of the stability of free NPST-AK15 protease in the presence of organic solvents, the enzyme samples were pre-treated in various organic solvents (isopropanol, ethanol, methanol, dimethyl ether, diethyl ether, benzene, toluene, chloroform, acetone, butanol, and amyl alcohol) at final concentrations of 25% (v/v) for 1 h at 40 ˝ C. Thereafter, the residual protease activities were measured. The protease activity assayed in absence of inhibitor or solvents was used as a control (%).

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3.6.4. Influence of Surfactants and Commercial Detergents The stability of soluble and immobilized NPST-AK15 protease was further investigated in the presence of several surfactants such as lauryl glucoside, sodium dodecyl sulfate (SDS), Tween 80, cetyltrimethylammonium bromide (CTAB), Brij® 93 and Triton X-100. The free and immobilized enzyme was pre-incubated with various surfactants at final concentrations of 1.0% and 5.0% for 1 h at 40 ˝ C, and then the residual enzyme activities were determined. Furthermore, in order to assess the compatibility and feasibility of the immobilized proteases as laundry detergent additives, the enzyme was mixed with various commercial laundry detergent solution including Persil, Ariel, Tide, Bonux, X-TRA, REX, OMO, and Ayam prepared in tap water, with final concentrations of 1% (w/v) and incubated for 24 h at room temperature. The endogenous proteases in the commercial laundry detergents were inactivated by heating the diluted detergents for 10 min in a boiling water bath before the addition of the NPST-AK15 protease. The protease activity measured in the absence of any detergents or surfactants was used as a control. 3.6.5. Determination of Kinetic Parameters For estimation of the maximum reaction rate (V max ) and the Michaelis–Menten constant (Km ) of free and immobilized protease, the activity assay was performed using different casein concentrations from 0.1 to 10 mg/mL in 50 mM gylcine buffer at their optimum pH and temperature for soluble and immobilized enzyme. The activity assays were performed as stated above, and then Km and V max were determined using Lineweaver–Burk plot [41]. In addition, the value of the turnover number (kcat ) which defined as the maximum number of chemical conversions of substrate molecules per second that a single catalytic site will execute for a given enzyme concentration, was calculated from the equation: kcat = V max /[E], where [E] is the enzyme molar concentration in the reaction mixture and V max is the maximal reaction rate. All calculated parameters were the mean of triplicate determinations from three independent assays. 3.6.6. Reusability of the Immobilized NPST-AK15 Protease For investigation of the reusability of immobilized NPST-AK15 protease, the immobilized enzyme was reused for 12 consecutive cycles using 2 mL of the standard reaction mixture. The reaction mixture that contained the immobilized enzyme was incubated in a shaking water bath (120 rpm) at 40 ˝ C for 20 min. At the end of each cycle, the immobilized protease was removed from the reaction medium, washed with glycine buffer and was used to start a cycle using fresh substrate solution. 3.7. Statistical Analysis Experimental results were given as mean value ˘ SD of three parallel measurements. All statistical analyses were conducted using Microsoft Excel. 4. Conclusions Alkaline protease from Bacillus sp. NPST-AK15 was immobilized onto functionalized and non-functionalized HCMSS nanospheres by physical and covalent immobilization. However, NPST-AK15 protease immobilization by covalent attachment onto activated amino-functionalized hollow core-mesoporous shell silica (Ac–HCMSS–NH2 ) nanospheres, through cross linking agent, was more effective showing much higher immobilization yield and loading efficiency in comparison with the physical adsorption approach. The immobilized NPST-AK15 protease exhibited significant improvement of thermal and pH stability in respect to free enzyme. Moreover, NPST-AK15 protease immobilization led to enzyme protection against several inhibitors. Interestingly, the immobilized protease exhibited significant improvement of protease stability in a variety of organic solvents, surfactants and commercial laundry detergents. In addition, the immobilized enzyme exhibited good operational stability up to twelve

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reaction batches. The developed immobilized alkaline protease is a promising nanobiocatalyst for laundry detergent formulations and various applications for alkaline protease. Acknowledgments: This project was funded by the National Plan for Science, Technology and Innovation (MAARIFAH), King Abdulaziz City for Science and Technology, Kingdom of Saudi Arabia, Award Number (12 BIO2899-02). Author Contributions: The work objectives were selected and designed by Abdelnasser Salah Shebl Ibrahim; The results analysis, interpretation, discussion, manuscript preparation and submission were carried out by Abdelnasser Salah Shebl Ibrahim; Ahmed M. El-Toni was responsible for synthesis and characterization of the nanostructured materials, manuscript discussion and preparation; Abdelnasser Salah Shebl Ibrahim, Mohamed A. El-Tayeb and Yahya B. Elbadawi carried out the experimental work related to microbiology and biochemistry; Ali A. Al-Salamah contributed in the data analysis, discussion, and provided administrative support; Garabed Antranikian consulted and guided the project; Khalid S. Almaary contributed in discussion, revision and editing of the manuscript. All authors have read and approved the final manuscript. Conflicts of Interest: The authors declare no conflict of interest.

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