Exuberant Immobilization of Urease on an Inorganic Support SiO2

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Exuberant Immobilization of Urease on an Inorganic Support SiO2 enhancing the Enzymatic Activities by Threefold for Perennial Utilization Sneha Mondal, Susanta Malik, Rimi Sarkar, Dipika Roy, Sanchari Saha, Shailja Mishra, Anindya Sarkar, Mousumi Chatterjee, and Bhabatosh Mandal Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00796 • Publication Date (Web): 19 Dec 2018 Downloaded from http://pubs.acs.org on December 20, 2018

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Bioconjugate Chemistry

Exuberant Immobilization of Urease on an Inorganic Support SiO2 enhancing the Enzymatic Activities by Threefold for Perennial Utilization Sneha Mondal, Susanta Malik, Rimi Sarkar, Dipika Roy, Sanchari Saha, Shailja Mishra, Anindya Sarkar, Mousumi Chatterjee and Bhabatosh Mandal* * To whom correspondence should be addressed. E-mail: [email protected] and [email protected] Department of Chemistry, Visva-Bharati, Santiniketan 731235, India

Abstract: Urease has been covalently immobilized on‘3-D networking silica gel (SG)’ using dimethyldichlorosilane (DMDCS) as second generation silane coupling reagent and mnitroaniline as linker component in a robust methodology and subsequently characterized as [{Si(OSi)4(H2O)0.05}205.2]n=4{OSi(CH3)2-NH-C6H4-N═N-urease}  282.5H2O (molecular mass: 2,63,445 g or 263.4 kDa). Selective coupling of tyrosine residue with an identifiable ‘mnitroaniline modified SG unit’ prevents enzyme-enzyme cross-linking leading to enhancement of enzymatic activity. The material worked at room temperature and its activity (luminescent and ammonia releasing efficiency) was enhanced by threefold (for both synthetic and real sample) to native enzyme values at neutral pH. Up to 30 days and 30 cycles this threefold activity remains as such but turns down gradually to native enzyme level after 60 days and 60 cycles of reuse.

Keywords: Jack Bean Urease; selective coupling of tyrosine-silica gel; threefold activity enhancement; high level reusability/durability; human blood/urine. 1

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Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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INTRODUCTION Enzymes, the biological catalysts, make enzyme-substrate transition state complex (E●S) of lower activation energy (ΔG‡) to facilitate conversion of substrates into its products and so, the ‘biocatalytic reactions’ operate at mild reaction conditions.1 The single critical advantage, the unique selectivity of enzymes2 for their natural substrates makes ‘enzymatic reactions’ simple (do not need sophisticated/or expensive instrumental set up) and so, it does require little/or no sample pretreatment that is obvious to classical reactions.3 Additionally, the enzyme-catalyzed reactions are found to be dramatically faster (hydrolysis of urea: kurease=5.0×106/kacid= 7.4×10-7; disproportionation of H2O2: kcatalse=3.5×107/KFe2+=56) over chemical catalyst.4 Exploiting these exquisite selectivity (chemo-, regio- and stereo1), mild reaction conditions,1 faster kinetics,5-8 and the advantages’ of homogeneous catalytic activities soluble enzymes are employed (1) for substrate quantitation at trace/ultra-trace level amidst in complex matrices like blood or fermentation broths,9 (2) as bio-sensors, (3) in sample purification, (4) in selective synthesis of an analyte of high purity and (5) also in bio-analytical assays.7,8 However, enzymes are produced within living cell in minute quantity, lose their ‘activities and functioning’ with time, temperature and intracellular solvent qualities; as a result of which enzymes are recovered as high cost material for extracellular usage. Moreover, in in-vitro analysis, because of denaturing at prevailing artificial enzymatic reaction conditions and time, the soluble enzymes are difficult to reuse

or

recycle

unless

it

justifies

the

cost

of

re-purification

process.10

The

reusability/recyclability can be accomplished by employing membrane reactors, special solvent systems or immobilization techniques.11-13 In case of immobilized enzymes’, because of instinctive toughness the geometry of operating part of enzyme affected a little bit with time, temperature and reaction environment;1 and enhanced enzymatic durability by restoring its 2


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reaction specificity.14 Secondly, immobilizate itself acts as a filter and is retained as such at necessary purified state, needs no additional separation for significant recycle/reuse15 and after reaction is over, as an integral part of the process, products are filtered off. Immobilization is able to provide possible modulation of catalytic properties 16 and the desired ‘reaction product’ does not face any problem from ‘protein or any microbial contamination’.1,8 A continuous or semicontinuous routine analysis using immobilizates would require a very minute amount, quantities considerably lower than can be reasonably supplied, and that would not represent a prohibitive expenditure in many cases.17 Exploiting these stability, reusability, specificity and obviously the cost effectiveness, immobilizates would have been used in several biotechnological applications, viz., production of chiral products from nonchiral substrates,18 construction of artificial organ systems, bioreactors19 or biosensors20-23 and in drug delivery system.24 Use of enzyme in analytical assay and in bio sensor in the immobilized condition have been brilliantly pioneered by Asher et. al., utilizing a class of photonic sensing motif, based on polymerized crystalline colloidal array (PCCA) technology.20-23 Non-covalent adsorption; entrapment in polymeric gel, or membrane, or capsule; cross-linking of enzyme (no need of inert support) and covalent attachment on an inert support are four most frequently used strategies for enzyme immobilization.1 The population of active enzymes on commonly used adsorbent were not much convincing as in adsorption process the ‘surface enzyme concentration’ was found to be poor and urgently required irreversibility makes ‘the major part of sorbed enzymes’ denatured.25 Sol-gel ‘first generation encapsulation’26, 27 [using tetraalkoxysilanes Si(OR)4 for a number of enzymes] and followed by its ‘second generation’ [using mixture of Si(OR)4 and alkylsilanes RSi(OCH3)3] for an enhanced activity (by 100 folds) and ‘restoring enantio-selectivity’ of bio-catalysts28 have attracted considerable interest also. Cross-linked enzyme crystals (CLECs) or its easy alternative 3



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cross-linked enzyme aggregates (CLEA), obtained by precipitation of proteins followed by crosslinking with glutaraldehyde, give an increased enantio-selectivity, but the process urgently required the removal of less selective isoenzymes.29,30 Enzyme adsorption on magnetic nanoparticles31, 32 or covalently linked enzyme on selective size (20±10 nm instead of the usual 75-100 nm) thiophene-functionalized nanoparticle exhibits significantly higher stability (over a period of almost one month)33,34 but, it needs magnetic set up for their separation and reusability. Covalent attachment solely depends on two major accomplishments: (1) inert support and (2) inherent character of enzyme relating to its conformation and configuration. Inert support is guided by its physical (viz., size, shape, porosity, pore size) and chemical (composition of its inner core, indispensable spatial surface functionality and non functional groups if any) states.35 Inorganic supports in general, and glass in particular, are normally resistant to microbial attack. They are able to retain their configuration over a wide range of pH, remain as such under various solvent conditions and have got a higher modulus of elasticity over organic polymer supports.36 SG has an instinctive toughness and thermal resistivity;

37

immobilized enzyme within pore

channels is well protected from microbial attack.38 Moreover, usually enzymatic reactions occur in aqueous solution and so, a hydrophilic polymer like SG is preferred as enzyme carrier.39 Enzyme immobilization on SG through stable covalent linkage was carried out for the first time by ‘Weetall’

36, 40-43

in 1969-1970 utilizing γ-aminopropyltriethoxysilane as a ‘silane coupling

reagent’ and is being utilized till date.20, 42, 43 Though this pioneering work is able to sort out the problem of must needed covalent linking between enzyme and SG, but it does not overcome the applicability relating to stability, selectivity, durability and reusability of immobilizate; moreover, the three to four step reaction sequences [surface -OH group of SG remarkably coupled with γaminopropyltriethoxysilane to introduce propylamine fragment through {Si-O}3Si-(CH2)3-NH2 4


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bonding (compound X; scheme 1). This primary amine derivative is the key compound and was utilized in three different ways for the relevant enzyme immobilization. In first method compound X was converted to its isothiocyanate derivative (compound Y) for enzyme immobilization through sulfonamide linkage (scheme 1; path a).44 In the second method, compound X was functionalized with p-nitrobenzoic acid to obtain an arylnitro derivative (compound Z),45 which one was reduced to amine and subsequently diazotized to effect immobilization of enzyme(s) through diazo linkage (scheme 1; path b) or in ‘path c’ carboxyl group of the enzyme was activated first with a water soluble 1-Cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-ptoluenesulfonate to obtain its corresponding O-acylisourea derivative (compound Y3), which one was reacted with ‘aminofunctional-SG’46 (i.e., compound X) to effect amide bond formation required an urgent and stringent reaction conditions viz., prolonged refluxing ≥ 90-120 hours, repetitive filtrations, temperature control, vacuum/or inert atmosphere with a prolonged period of mechanical shaking. It also required a large number of costly and hazardous solvent/chemicals. In addition to that constraints ‘path a’ and ‘path c’ suffer from selectivity (because all of the 18 different amino acid residues of urease contain -NH2 and -COOH groups for chemical immobilization; table S1) to effect enzyme-enzyme cross-linking leading to loss of enzymatic activities.

5



Scheme 1: Chemical immobilization of enzyme on silica gel path (a) through sulfonamide linkage [Step I: X→Y1 (Drop wise addition of 10% CSCl2 solution in CCl4 into KHPO4 suspension of X with constant stirring for 20 minutes); Step II: Y1→Y1•Enzyme (Y1 was thoroughly washed with H2O, acetone and 3.5M NaHCO3, then in suspension of Y1 1.5 fold excess enzyme was added with constant stirring for 24h)]; path (b) through diazo linkage [Step 6


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I: X→Y2 (p-nitrobenzoic acid was added in the toluene-suspension of X and warmed the mixture for 18h); Step II: Y2→Y2 •Enzyme (Y2 was reduced to amine derivative with aq. Na2S2O4 under boiling condition, 0.5M ice cold NaNO2 solution was added to amine suspension in ice cold 0.7M CH3COOH with constant stirring till KI-Starch test found positive after 15 minutes of addition of last portion, diazo derivative of Y2 and enzyme in borate buffer at 0-4°C was reacted for diazo coupling)] and path (c) through amide linkage [Step I: (carboxyl groups of the enzyme was activated with a water soluble 1-Cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-ptoluenesulfonate to obtain O-acylisourea derivative of enzyme, Y3); Step II: Y3→X•Y3 (suspension of X and Y3 in aq. HCl at pH 4.0 was kept overnight at 6°C)].

Our group, during the synthesis of a class of ‘chromatographic extractors’ by immobilizing ligands on SG;47-51 very efficiently introduces the nitrobenzoyl fragment on SG surface (to compound Z of scheme 1) through an instantaneous reaction using dimethyldichlorosilane (DMDCS) as a ‘second generation silane coupling reagent’. It is successfully able to replace the ‘time consuming and stringent’ (refluxing ≥ 90-120 hours) first and second step of ‘Weetall reaction’ by an ‘instantaneous one pot single step straightforward methodology’ (scheme 2).

7



Scheme 2:

Functionalization of SG with nitrobenzoyl fragments using DMDCS and

immobilization of urease through diazo-linkages.

Here, in the present investigation, this ‘nitro derivative (compound A of scheme 2)’ has been utilized for covalent immobilization of urease- a model enzyme system; an enzyme having selective amino acid residue, tyrosine for efficient diazo coupling reaction.45 This reaction 8


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methodology (scheme 2) not only undergoes a selective diazo coupling reaction with tyrosine but also avoids ‘enzyme-enzyme cross-linking’.46 Urease (EC 3.5.1.5, the only known Ni containing enzyme in plants) was the first enzyme to be crystallized from a leguminous plant source, jackbean seeds (Canavalia ensiformis).52 It hydrolyses urea to unstable carbonic acid, which undergoes an immediate conversion to ammonia and carbon dioxide.1 Immobilized urease has a wide variety of applications; viz., utilities in biosensors

22, 53-55

for quantitation of urea and Hg2+,

bioreactors in artificial kidney machines,56 and in purification of foods and beverages.57 In present research, urease has been covalently immobilized on ‘3-D networking SG’ in a robust methodology at an ambient temperature. Subsequently, its full proof characterization and stability is investigated; and finally the enzymatic activity and application in some real sample analysis have also been systematically carried out. Results and Discussion Composition and characterization: ‘SG unit cell’ (pm dimension) undergoes its surface modification to produce ‘immobilizate particles’ of a definite dimension, {Si(OSi)4 (H2O)0.16}n{OSi(CH3)2-NH-C6H4-N═N-urease}4 (scheme 2 and discussed in experimental section). Four urease monomers are found to be appeared at four hands of tetrahedra. This sort of surface modification is rationalized as follows: From DLS measurement for the distribution of particle size, the optimum value of immobilizate is appeared at 320 nm (Fig.1a). The size is 16 fold to its precursor ‘nano SG’ (18-20 nm) and is a clear indication of surface modification. Numerous number of such ‘immobilizate particles’ having surface active groups are in aggregate to produce a bulk of particle sizes 4.0-5.0 μm (Fig.1c). SEM photograph of immobilizates [synthesized from normal sized SG; particle size: 60 μm (average)] is shown in Fig.1b. The irregular shaped outshining ‘bulk particles’ of different dimensions [300±50 μm; again an 9



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enhancement nearly by 16 flod] and rough surfaces are found to be hard, and are disorderly distributed within matrix. On chemical modification with enzyme, surface properties of pure silica gel [BET surface area: 420 m2g-1; particle size: 60 μm (average); pore size: 80 Å average pore diameter] were found to be considerably shifted in {SiO2}@Urease (BET surface area: 184 m2g-1; particle size: 300±50 μm; pore size: 53 Å average pore diameter) (Fig.1d). (b)

(a)

(c)

(d)

Fig.1: (a) Dynamic light scattering (DLS) of nano FSG (Functionalized Silica Gel)-Urease material; (b)SEM image of immobilized urease; (c) SEAD (left) and TEM (right) image of nano FSG-Urease; (d) Nitrogen adsorption-desorption BET isotherm of immobilized urease.

10


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On the other hand, retention of amorphous identity (a single broad peak at 22.26° in XRD spectrum of {SiO2}@Urease; Fig.2a) clearly indicates that all sorts of chemical reactions for surface modification occur at the surface of SG only keeping intact the core identity of ‘SG unit cell’ {Si(OSi)4(H2O)0.16}n (as per scheme 2).46-50 The full XPS survey spectrum (Fig.2b) and high resolution peak differentiation and imitating spectrum for differerent elements (Fig.2c-e) of the as-prepared material were obtained by employing 1486.7 eV radiation as line source. These were closely analyzed for better understanding of elemental composition and formulation of the synthesized material. The XPS peaks for O-2s (23.12 eV), Si-2p (102.65 eV), Si-2s (152.2eV), C-1s (284.92 eV), O-1s (532.09 eV) and a small hump near 399.65 eV for N-1s agree the standard values58 and clearly confirm the presence of silicon, oxygen, carbon and nitrogen as elemental composition of the synthesized immobilizate. Practically, both the O-1s (532.09 eV and 535.8 eV) and Si-2s (152.2 eV and 154.8 eV) peaks appear as doublet (Fig.2b). It justifies the presence of two different environments for the corresponding elements; O-Si (SG) / O-C (enzyme) and Si-CH3 (DMDCS) / Si-O (SG) and thereby, agrees the proposed path of synthesis (scheme 2). High resolution N-1s peak appears as multiples (Fig.2c) and exemplifies the different amino acid residues of immobilized urease. XPS peak at 399.66 eV attributes to the presence of NH2 (primary amine of Aspartic Acid, Threonine, Serine, Glutamic Acid, Glycine, Alanine), >NH (guanidinum group of Arginine), ═NH (guanidinum group of Arginine), acyclic -NH (pyrrolidine side chain of Proline) and XPS peak at 405.33 eV executes the presence of aromatic -NH (imidazole side chain of Histidine), aromatic -N [indole side chain of Tryptophan (Table S1) and N═N- appears in -N═N-enzyme linkage; scheme 2]. Ni-2p3/2 (850.8 eV) and Ni-2p1/2 (871.0 eV) low intense peaks appear as multiples in the XPS signature (Fig.2d) of {SiO2}@Urease. Here, the enzyme in its very high molecular mass (90,000 g) contains only two nickel centers and within 11



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the molecular network of ‘immobilized urease’, these two Ni centers are found to be present at two different electronic environments.51 Poor population density of nickel lowers the peak intensity while their different electronic environments cause the multiplicity of Ni-2p spectral signature. (a)

(c)

(b)

(e)

(d)

Fig. 2: (a) XRD spectra; XPS (b) survey and (c-e) peak-differentiation-imitating analysis of different elements of immobilizate. To find out ‘weight average participation’ and true composition of synthesized material, reliable TGA was carried out. The immobilizate,{Si(OSi)4(H2O)x}n{OSi(CH3)2-NH-C6H4-N═N-urease}4  yH2O undergoes a three stages of weight loss (eq.1-eq.3); a rapid weight loss (1.93%) at 100°C

for its surface water (yH2O), followed by a steady weight loss at 100-145°C for release of internal water (xH2O) and finally, at 900°C (with 4.4% weight loss) the material leaves a residue of an inorganic ash, {SiO2}5n+4 (Fig.3a). Applying eq.4, the numerical values of n (3283), x (0.05) and y (1130) were computed from the respective weight loss equations (eq.3, eq.2 and eq.1). It, thus 12


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results the molecular composition and molecular mass of the immobilizate respectively as, {Si(OSi)4(H2O)0.05}n=3283{OSi(CH3)2-NH-C6H4-N═N-urease}4  1130 H2O and 1,05,3739 g (1053.7 kDa).  y . H O ; (1.93%; 40 100 0 C )

2   {Si(OSi)4 (H2O)x}nR {Si(OSi)4 (H2O)x}nR  yH2O  

 xn. H O ; ( 0.3%; 100 145 0 C )

 {Si(OSi)4}nR {Si(OSi)4 (H2O)x}nR  2     

(eq.2)

 CO ;  SO ;  NO ;....etc. ( 4.4%; 145  900 0 C )

2 x  x       (5n+4){SiO2} {Si(OSi)4}nR  

Winitial  W final Winitial

(eq.1)

(eq.3)

 100 = Weight loss (%)

(eq. 4)

Where, R = {OSi(CH3)2-NH-C6H4-N═N-urease}4 = 3,60,712 g (urease contains 840 aa residues and has the molecular mass of 90 kDa;59). (a)

(b)

(c)

Fig. 3: (a) TGA; (b) EDX-spectra; (c) FT-IR spectra of synthesized material. 13



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But, out of 18 different amino acid residues only tyrosine contains phenolic -OH group and is selectively able to participate in diazo coupling reaction (scheme 2). Urease in its molecular network contains 16 tyrosine residues (Table S1). It requires four ‘SG unit cells’ (as every unit cell possesses tetrahedrally disposed four -N═N- groups; scheme 2) to covalently immobilize an enzyme molecule. So, the true molecular dimension of ‘SG unit cell’ would become {Si(OSi)4 (H2O)0.05}n=205.2 and molecular composition of the immobilizate would be [{Si(OSi)4 (H2O)0.05}205.2]n=4{OSi(CH3)2-NH-C6H4-N═N-urease}  282.5H2O (molecular mass: 2,63,445 g or 263.4 kDa) instead of the previous one, {Si(OSi)4 (H2O)0.05}n=3283{OSi(CH3)2-NH-C6H4-N═Nurease}4  1130 H2O. Now, considering molecular mass of dried material as 2,57,621g and EDX elemental composition (C: 0.22%; O: 0.52%, N: 0.028%; Fig.3b), the amount of carbon, nitrogen and oxygen in free enzyme was computed (eq.5-eq.7) [C: 56580 g (39% w/w), O: 81418 g (56% w/w), N: 7171.4 g (5% w/w)] and found to be comparable to XPS (Table S2) quantification (C: 45%, O: 50% and N: 3.4% excluding Si). The nitrogen (% w/w) would become 9.6% (reported 16.6% w/w)

60

for a

molecular weight of 75,000 g (Bailey & Boulter value).61 It, thus conveys that the ‘probability sequence of synthesis’ (scheme 2) and molecular formulation are nearly close to the real picture of happening.

8  12  p  12  0.22 257621

eq.5

3284 16  q 16  0.52 257621

eq.6

3 14  r 14  0.028 257621

eq.7

14


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[Where, respectively p, q and r are the total number of carbon, oxygen and nitrogen atoms present in immobilized enzyme. The molecular mass of the dried material is 2,57,611 g.] To find out bonding interactions and a 2-D structure of immobilizate, FT-IR was carried out (Fig.3c). The ‘smooth broad peak’ at 2500-3750 cm-1 in pure SG for silanol -OH was found to be replaced by a multiple containing few ‘shoulder peaks’ for the presence

62

of N-H peak at 3661

cm-1, C-H peak for -CH3 and -CH2 respectively appears at 2967 cm-1 and 2902 cm-1. Methyl C-H appears from DMDCS, N-H peak indicates bonding between m-nitroaniline and DMDCS, while the -CH2 group may indicate the presence of immobilized enzyme. The 13C and 1H NMR of the nano material were carried out for further verification and confirmation of immobilization of urease in the synthesized molecule. In its molecular network urease contains 18 different amino acid residues (Table S1) and out of these amino acids only four (Tyrosine, Phenylalanine, Tryptophan and Histidine) contain aromatic groups. Due to similarity reason out of several, only 8-10 aromatic carbons are found to be identifiable (Table S1) and appear in

13C

(110-150 ppm)

and 1H NMR spectra [Fig.S1(a,b)]. These aromatic carbon atoms appear along with several aliphatic congeners at XPS C-1s peak (Fig.2e). All aliphatic -CH3 and -CH2 of the amino acids along with S-CH3 of methionine residue appear as an intense peak at 284.0 eV, acyclic -CH2 of proline along with -COOH of all the amino acids are found to be present at 286.1 eV and aromatic carbons of tyrosine, phenylalanine, tryptophan, histidine residues are well executed at 287.31 eV. Now, up to this, the covalent immobilization of urease on SG surface and molecular composition of the corresponding immobilizate have been highlighted through authenticated experimental results. But, these experimental findings cannot give any insight into the retention of conformation and configuration of urease molecule on its immobilization.

15



Fluorescence is an intrinsic and inherent signature of molecular properties. It is able to convey the change (at any degree) of enzymatic behavior (relating to its conformation and configuration) appeared at immobilizate. Three amino acid residues tyrosine, tryptophan and phenylalanine have intrinsic fluorescence.63 But, being present in enzyme, only tryptophan is found to be fluorescence active.64 Because of cross-linking, interaction with other active groups and molecular vibrations, tyrosine has got poor ‘fluorescence quantum yield’. The fluorescence pattern of both pure enzyme (0.5 ppm solution in dichloromethane) and immobilizate (0.5-2.5 ppm solution in dichloromethane) are shown in Fig.4a and Fig.4b. An intense peak at 390 nm [I390(trp): 371 units] for tryptophan residue (because of environment polarity of protein, the peak is slightly shifted from normal values at 333-334 nm63) and a very low intense peak at 475 nm [I475(tyr): 53 units] for tyrosine residue are found to be present in pure urease (Fig.4a). On immobilization, intensity for both the peaks are found to be increased at noticeable degrees [I390(trp): 1900 units ; I475(tyr): 680 units] (Fig.4b). It is also to be noted that with increase in concentration of immobilizate (0.5 ppm2.5 ppm) the tryptophan peak intensity decreases while for the tyrosine peak it is found to be increased (Fig.4b). At an excitation at 330 nm, the electronically singlet excited state (S1) so appeared returns back to its singlet ground state (S0) through three different ‘meta states’ (S0', S0'' and S0'''; scheme 3b). Among these, S0''' is obtained by employing a transition at higher wave length (of minimum band gap) and is entirely non-radiative. In reality, the singlet ground state (S0) is a combination of S0' and S0''. For free enzyme, a major part of excited molecules make radiationless transition to reach at S0'' ‘meta state’ (case II; ground state of tyrosine) while a considerable number of excited molecules return to S0' state (case I; ground state of tryptophan) through a radiative path. In the immobilizate, tyrosine (being bonded at long peptide chain) is covalently linked with SG through -N═N- coupling (scheme 3a); so, its movement gets totally 16


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restricted, avoids cross linking and chemical interactions with other parts of molecule. In such cases, a large number of excited molecules come back at S0'' ‘meta state’ through radiative path (case II). It results a good fluorescence quantum yield (peak intensity at 475 nm becomes 700 units; increased by 13 times), which increases with increase in concentration (Fig.4b). On the other hand, tryptophan is not directly bonded to SG; it’s ‘movement restriction’ appears only because of the restricted movement of tyrosine residue and still it swims (scheme 3a), has got the chance of cross linking and molecular interactions leading to radiation less transition. This partial restriction in movement enhances the fluorescence intensity of tryptophan abruptly [I390(trp): 1900 units, the enhancement is 5 times; immobilizate concentration: 0.5 ppm] in comparison to pure enzyme [I390(trp): 371 units ; enzyme concentration: 0.5 ppm]; which one is decreased for further increase in concentration (Fig.4b). Because of molecular crowding in concentrated solution, the monomers get cross-linked and it is continued. At a solution concentration of 3.72 ppm (extrapolated; concentration vs. intensity; (Fig.4c) the fluorescence intensity of immobilizate would attain at 371 units, the intensity of pure enzyme (Fig.4a). That is, at that very condition the enzyme in immobilizate got the ‘native enzyme properties’ (relating to its conformation and configuration). This luminescence behavior of the immobilizate of varying concentration (0.52.75 ppm) was examined (Fig.4d) in presence of urea of fixed concentration (800 µmol in 50 mL). The tryptophan peak was found to be absent while tyrosine peak intensity was found to be increased (650-1020 units) along with the increase in immobilizate concentration and this peak enhancement was remain as such as it was (Fig.4b) in absence of urea. The tyrosine moiety is covalently attached to SG unit cell and its configuration remains unchanged during enzymesubstrate reaction. But, being in free movement the electronic behavior of tryptophan residue gets changed; which in turn makes it radiationless. 17



Page 18 of 39

(b)

(a)

(c)

(d)

Fig. 4: Florescence spectra of (a) pure enzyme (0.5 ppm solution) ; (b) immobilizate (0.5-2.5 ppm solution) ; (c) plot of concentration vs. fluorescence intensity ; (d) immobilizate (0.5-2.75 ppm solution) in presence of 800 μmol. urea.

18


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Scheme 3: (a) Binding pattern of tryptophan (trp; projected outside) and tyrosine (tyr; placed inside) in immobilizate; (b) Energy level diagram- case I: fluorescence peak at 390 nm [I390(trp): 371 units in pure enzyme and I390(trp): 1900 units in immobilized enzyme] for tryptophan residue; case II: fluorescence peak at 475 nm [I475(tyr): 53 units in pure enzyme and I475(tyr): 680 units in immobilized enzyme] for tyrosine residue; case III: radiationless transition. 19



Analytical applications: Thus, the foregoing photo physical results (Fig.4a and Fig.4b) enlighten the closeness in chemical behavior (peak position at 390 and 475 nm) of both pure enzyme and immobilizate. It encouraged us to carry out enzymatic process in synthetic and some real samples (human blood and urine) to judge analytical applicability of synthesized material. Choice of substrate concentration: Pure urea makes a stable complex with αisonitrosopropiophenone and gives a distinct absorbance peak at 540 nm (Fig.5a), which follows Beer’s law in the concentration range 1.5×103 - 6.0×103 µmol.L-1 (i.e., 50 mL contains 75-300 µmol urea) (inset of Fig.5a). So, 300 µmol 50 mL urea solution was employed as substrate for further steps of experiments. On enzymatic hydrolysis, 300 µmol urea should have been able to generate 600 µmol ammonia.1 So, a standard solution comprising 600 µmol ammonium chloride in 50 mL distilled water was prepared and employing ‘Riegler’s Reagent’ its UV-visible absorbance at 340-380 nm was assessed (which is found to be 0.21 units) to ascertain the enzymatic activity of the immobilizate (Fig.5b). Optimization of immobilizate dose: To find out the optimum dose, a varying amount of immobilizate (0.25-1.5 g) was added to the substrate solution (300 µmol urea in 50 mL); when 0.5 g immobilizate was found to be enough for complete hydrolysis of substrate into product (i.e., 600 µmol ammonia/absorbance ~0.20 at 340-380 nm) (Fig.5b and Fig.5c).

20


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(c)

(b)

Fig. 5: Absorbance at 540 nm (a) for pure urea solution; Absorbance at 340-380 nm for (b) 600 µmol standard ammonium chloride solution and (c) pure urea in presence of immobilizate of varying amount (0.25-1.5 g).

Studies on activity: This hydrolyzing activity of immobilizate was compared with pure enzyme. In such cases, two sets of 50 mL aqueous solution of urea (containing 300 µmol) were taken and to incorporate the same amount of enzyme, respectively 0.5 g immobilizate (containing 0.3 mL 21



urease) and 0.3 mL pure enzyme (as control) were added. At an interval of 2 minutes, the liberated ammonia was estimated spectrophotometrically (Fig.6a) using ‘Riegler’s Reagent’. Remarkably, the activity (absorbance at 340 nm) of the immobilizate was found to be threefold higher in comparison to the pure enzyme (immobilizate: 0.22 units; pure enzyme: 0.07 units) and thereby, agrees with the foregoing photo physical process (Fig.4a and Fig.4b). For its further verification, a set of experiments were made by employing an addition of varying amount of pure enzyme (0.3 mL-0.9 mL) to the substrate of same concentration (300 µmol urea in 50 mL water; Fig.6b). The absorbance for 0.9 mL pure enzyme was superimposed with the immobilizate (0.5 g) signature. It depicts that the activity of 0.9 mL pure enzyme is equivalent to 0.3 mL urease (present in 0.5 g immobilizate). Practically urease in its pure form exists as a hexamer.45 But in the present methodology, out of eighteen different amino acid residues of urease, only the tyrosine residue (containing phenolic -OH group) is selectively coupled to an identifiable ‘SG unit cell’ through diazo coupling reaction and thereby avoids inherent enzymatic cross-linking. Being present on two different ‘SG units’ urease exists as a dimer [SG-en…en-SG], which in turn enhances the enzymatic activity by three fold.46 Reusability and recycling: Now, turning to the essence of perennial utilities, inevitably the studies on reusability and recycling of the immobilizate at an ambient temperature were taken into account. In the column operation, at every cycle the immobilized enzyme after converting the substrate (urea) into product (ammonia) remains as such on the filter bed. If so facto, 50 mL aqueous solution (pH 7.0 ± 0.10) containing 300 µmol urea in phosphate buffer was continuously passed through a definite amount of immobilizate (0.5 g), packed into a glass column (experimental details are discussed in experimental section) at room temperature and the amount of ammonia so produced by immobilized enzyme in the column eluate was estimated 22


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spectrophotometrically at an interval of two hours for four consecutive experiments per day. The column activities (absorbances at 340-380 nm were near about 0.22 units) at the fourth week of the prepared immobilizate were found to be remaining as such for the four consecutive experiments (Fig.6c). The column was thoroughly washed with phosphate buffer and makes it ready for the subsequent cycles after every usage. Thus, the material may be used for 30 cycles (4 experiments per day; at an interval of seven days). Durability of the synthesized immobilizate: In order to assess the durability of material, 1 g immobilizate (having 0.6 mL urease) was incorporated into the column containing 600 μmol urea and the column experiment was continued for 60 days employing the analysis of the data at an interval of every seven days (Fig.6d). The column activity at an ambient temperature was found to be remaining as such up to 30 days and after its gradual decrease reached 45% at 60 days (Fig.6d). The activity-time relationship (Fig.6d inset) follows a first order kinetics (kobs = 1.12×10-2 day-1) with an agreeable t1/2 value, 62 days. These results suggest that the material may be recycled and reused up to 60 days with its 45% activity (i.e., the absorbance value reaches at ~0.18 units, still higher than pure enzyme (0.15 units)). At 60 days the absorbance of 1 g immobilizate (having 0.6 mL urease) is equivalent to 0.75 mL pure urease. The aqueous micro environment being present inside the 3-D networking SG involved in enzyme catalysed substrate hydrolysis

65

and also provides suitable physiological conditions to minimize the denaturation of

the immobilized enzyme, 66 which eventually, prolongates enzymatic function up to 30 days.

23



(a)

(b)

Page 24 of 39

(c)

Fig. 6: At 340 nm absorbance (a) obtained from immobilizate (0.5 g containing 0.3 mL urease) and pure urease (0.3 mL); (b) obtained from immobilizate (0.5 g containing 0.3 mL urease) and pure urease (0.3-0.9 mL); (c) at fourth week for column eluates (amount of immobilizate: 0.5 g and amount of substrate: 300 μmol urea in 50 mL distilled water) at an interval of two hours; (d) for column eluate (amount of immobilizate: 1.0 g and amount of substrate: 600 μmol urea in 50 mL distilled water) at an interval of 30-60 days.

Application to real samples: In human liver, on hydrolysis of protein free ammonia is produced; which is immediately converted into urea, a harmless waste product and through blood stream it reaches to kidney. After glomerular filtration, this blood urea ultimately excretes through urine. 24


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So, its amount in blood (serum and plasma) and urine samples is an effective indicator of kidney functioning. Keeping this in mind, a known amount of urea (300 µmol) is taken in 1 mL analyte sample (urine, serum and plasma) containing 0.5 g synthesized immobilizate and the amount of ammonia was estimated spectrophotometrically using ‘Riegler’s Reagent’. The same experiment was carried out by employing an equivalent amount (0.3 mL) of pure urease (in place of 0.5 g immobilizate which contains 0.3 mL urease) to compare the activity of the synthesized material [Fig.7(a-c)]. The immobilizate in urine was found to be equally active as in aqueous synthetic solution (i.e., absorbance value attains at 0.20 units; already found in synthetic solution containing 300 µmol urea); however, the experimental absorbance value was enhanced by 0.20 units for both standard (pure urease) and immobilizate in plasma sample (pure enzyme: 0.27 and immobilizate: 0.37; Fig.7b). For human blood serum the enhancement was 0.5 units in standard and 1.25 units in immobilizate (Fig.7c). It briefs the presence of an additional ammonia releasing factor.67 Thus the synthesized material was found to be active in different classes of real sample analysis. (b)

(a)

(c)

Fig.7: Activity of immobilizate in human (a) urine; blood (b) plasma and (c) serum.

Comparison with materials: Among the reported materials (Table S3)

68-81,

the reported as-

prepared immobilizate75 possesses 7-13.5 folds activity; but it drops down to native enzyme values only after 50 hours and its activity remains 90% with respect to native enzyme after 8 25



days. On the other hand, the activity of the present material was found to be threefold and remains as such up to 30 days and can be reused for 30 cycles (4 experiments per day; at an interval of seven days). In addition to that, its activity turns down to native enzyme level after 60 days at an ambient temperature and the material may be successfully applied both for human blood and urine samples. A full proof molecular characterization (molecular composition and mass: 2,63,445 g or 263.4 kDa) has been addressed in the present material only. Conclusion: Successfully Urease has been covalently immobilized on ‘3-D networking SG’ using DMDCS (a second generation silane coupling reagent) in a robust methodology at an ambient temperature and subsequently characterized as [{Si(OSi)4(H2O)0.05}205.2]n=4{OSi(CH3)2-NH-C6H4N═N-urease}  282.5H2O (molecular mass: 2,63,445g or 263.4kDa). The material worked at room temperature and its activity was enhanced by threefold (for both synthetic and real samples) to native enzyme values at neutral pH. Up to 30 days and 30 cycles this activity remains as such and turns down to native enzyme level after 60 days with a reuse of 60 cycles. Experimental [Instrumentation and reagents/chemicals are discussed in file S1 as supporting documents]. Immobilization of urease on silica gel (scheme 2): Synthesis of SiO2@m-nitrobenzyl: SG unit cell,82 {Si(OSi)4 (H2O)0.16}n≥11 (Wt: 2276 g) contains four surface -OH groups (1.76 mMg-1) and utilizing these surface -OH groups the unit cells are interconnected through H-bonds to produce an entire 3-D molecular network.83 So, 10 g SG was added to 200 mL m-nitroaniline (100 mM

DCM solution) for complete functionalization.

DMDCS was added (drop wise) to the reaction mixture with continuous shaking. It simultaneously couples SG with m-nitroaniline through an instantaneous reaction and each ‘SG unit cell’ appears as separate entity (scheme 2); the nitro group behaves as an end capping group 26


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and thereby the resulting nitro derivative exists as an identifiable entity, {Si(OSi)4 (H2O)0.16}n{OSi(CH3)2-NH-C6H4-NO2}4. This synthesized nitro derivative was washed with DCM and followed by several times with distilled water (20 mL in succession). The colored mass was air dried and stored in glass vessel at 27°C. Synthesis of SiO2@m-aminobenzyl: The water suspension of SiO2@ m-nitrobenzyl was taken in a reaction vessel and aqueous sodium hydrosulfite (Na2S2O4.2H2O) solution was added slowly with vigorous stirring at the boiling solution to obtain corresponding amine derivative; {Si(OSi)4 (H2O)0.16}n{OSi(CH3)2-NH-C6H4-NH2}4.84 The yellow reduced mass was filtered off, washed with cold distilled water and kept at room temperature. Synthesis of {SiO2}@Urease: To the ice-cold SiO2@m-aminobenzyl suspension (in 2N CH3COOH solution) of a definite dimension, NaNO2 (10% w/v ice-cold) was added slowly and the completion of diazotization was identified with KI-Starch paper.36,84 This as prepared diazo compound [{Si(OSi)4 (H2O)0.16}n{OSi(CH3)2-NH-C6H4-N═N-}4] was taken in borate buffer (pH 8.75 ± 0.25) and ice-cold ‘enzyme solution’ containing 6 mL ‘jack bean urease type III’ (U1875 in glycerol solution, 500-800 units mL-1) was added drop wise with constant stirring; After addition is over, the reaction admixture was kept under incessant stirring for another 30 minutes. In this ‘reaction course’ urease employing its tyrosine residue (having phenolic -OH group) gets immobilized through diazo coupling reaction resulting an immobilizate of a ‘definite dimension’, {Si(OSi)4 (H2O)0.16}n{OSi(CH3)2-NH-C6H4-N═N-urease}4. It, thereby, differs from the earlier reported immobilizates. The filtered immobilizate was washed several times with borate buffer solution and the hydrated mass, {Si(OSi)4(H2O)0.16}n{OSi(CH3)2-NH-C6H4-N═N-urease}4  yH2O was collected at 4-6°C in glass covered vessel.

27



Page 28 of 39

Characterization of {SiO2}@Urease: The chemical characterization of synthesized material was performed by a set of instrumental analysis. Surface properties by SEM and BET, particle size by TEM and DLS, probable molecular composition and molecular weight by TGA, elemental composition by EDX and XPS, retention of amorphous identity was achieved through XRD spectrum analysis, 2-D chemical structure by FT-IR and NMR and the retention of enzymatic qualities by luminescence spectral analysis. Experimental set up employed for the analytical applicability of the immobilizate: 0.5 g synthesized immobilizate (containing 0.3 mL urease; see synthesis procedure) and 0.3 mL pure enzyme (as control) were taken (both from ice-cold source) in two different reaction vessels, each containing 300 µmol urea in aqueous solution (50 mL) at room temperature. The activity of immobilizate in synthetic and real samples [human blood: serum, plasma; urine samples (1 mL)] was compared with ‘standard urease solution’ (control) by estimating the amount of ammonia liberated spectrophotometrically employing ‘Riegler’s Solution’ as chromogenic reagent.85

Conflicts of interest: There are no conflicts to declare. Acknowledgment : One of the authors Sneha Mondal acknowledges to DST-FIST, New Delhi, India for research facilities,Visva-Bharati University fellowship for financial support and Dr. Susanta Malik thanks to SERB (NPDF), New Delhi, India (PDF/2017/001690) for financial support.

Supporting Information: [Experimental details and materials; Table S1 (The Amino Acids of Jack Bean Urease); Table S2 (XPS Quantification) ; Table S3 (Comparison table); Fig. S1 (a, b) 1H and 13C

NMR spectrum of nano FSG-Urease] This material is available free of charge via the Internet

at http://pubs.acs.org. 28


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Graphical Abstract

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Exuberant Immobilization of Urease on an Inorganic Support SiO2

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