Stabilization of Bacillus Licheniformis ATCC Alkaline Protease by Immobilization and Modification

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1 Australian Jurnal f Basic and Applied Sciences, 1(3): , 2007 ISSN Stabilizatin f Bacillus Lichenifrmis ATCC Alkaline Prtease by Immbilizatin and Mdificatin Samia A. Ahmed, Shireen A. Saleh and Ahmed F. Abdel-Fattah Department f Chemistry f Natural and Micrbial Prducts Natinal Research Center, Cair. Abstract: Alkaline prtease frm Bacillus lichenifrmis ATCC21415 was partially purified by fractinal precipitatin at 70% ethanl which shwed 6.2-fld purificatin. Immbilizatin f the enzyme by physical adsrptin n lfa (as a new carrier) had the highest immbilizatin yield (70.5%).Chemical mdificatin f the enzyme by cvalent cupling with sdium -peridate activated amylpectin retained (78.3%) f the riginal activity. Immbilized and mdified enzymes retained 59.9 and 76.1%, respectively f the riginal activity after heating at 60 C fr 60 min while the native enzyme retained 19.4%. The calculated half-life values (t 1/2) f heat inactivatin at 60 C fr mdified, immbilized and native prtease were 115, 100and 25 min, respectively. Activatin energy (E A) f the native enzyme was 23.6Kcal/ml which higher than thse f immbilized and mdified enzymes (18.9 and 19.9Kcal/ml, respectively). The immbilized and mdified frms exhibited lwer Vmax and higher K m values cmpared t that f the native frm. Immbilized and mdified frms were mre stable in presence f EDTA than the native frm. The crude enzyme digested sme natural prteins and was able t extract cllagen frm chicken skin. Keywrds: B. lichenifrmis ATCC 21415, alkaline prtease, immbilizatin, mdificatin INTRODUCTION Prteases execute a large variety f functins and have imprtant bitechnlgical applicatins. They represent ne f the three largest grups f industrial enzymes and find applicatins in detergents, leather, fd, pharmaceutical industries and biremediatin prcesses (Gupta et al., 2002). Prbably the largest applicatin f prteases is in laundry detergents, where they help remving prtein based stains frm clthing (Banerjee et al., 1999). The enzyme shuld be stable and active in the presence f typical detergent ingredients fr use in detergent (Najafi et al., 2005). Stabilizatin f enzymes has received much attentin in recent years. Stabilizatin against thermal inactivatin can be perfrmed in several ways such as crss-linking t a water insluble carrier with a bifunctinal reagent r cvalent cupling t natural and synthetic plymers and entrapment in gels (Najafi et al., 2005). Chemical mdificatin with lw mlecular weight mnfunctinl reagents crss-linking with bifunctinal reagents plymer attachment has been reprted by Ben Ammar et al. (2002) and Fernandez et al. (2002). Many prteins cntaining carbhydrate residues exhibit increased thermal stability twards heat and strage, which in many cases seems t be due t the carbhydrate part f the mlecule. Mst glycprteins exhibit high water slubility, and thus it was cnsidered prmising t be stabilized water-sluble enzymes thrugh cvalent attachment t carbhydrates (Abdel-Naby 1999). The mechanism invlved in the carbhydrate-induced stability f glycsylated prteins by rigidificatin f the cnfrmatin was reprted by Klibanv (1983). On the ther hand, Srivastava (1991) argued that the hydratin effect f the attached carbhydrate may be respnsible fr imprving the stability f cnjugated enzymes. Hydrgen bnding between the plysaccharide and prtein surface has been suggested as causes f thermal stabilizatin f the synthetic glycprteins (Lendewrs et al., 1984). Fr industrial applicatins, the immbilizatin f prtease n a slid supprt can ffer several advantages, including repeated usage f enzyme, ease f prduct separatin, imprvement f enzyme stability and cntinuus peratin in packed-bed reactrs (Abdel-Naby et al., 1998). Enzyme stabilizatin will thus cntinue t be a key issue in bitechnlgy. The present study deals with partial purificatin f B. lichenifrmis ATCC alkaline prtease and its stabilizatin by immbilizatin and chemical mdificatin. The catalytic prperties and the stability f the immbilized and mdified enzymes were cmpared t thse f the native enzyme. Crrespnding Authr: Samia A. Ahmed, Department f Chemistry f Natural and Micrbial Prducts, Natinal Research Center, Cair, Dkki, Egypt. dr_sa_ahmed@yah.cm 313

2 MATERIALS AND METHODS Strain: Bacillus lichenifrmis ATCC was btained frm the American Type Culture Cllectin, USA. Prductin Media: The culture medium fr enzyme prductin was defined as fllws (g/l): KH2PO 4, 0.5; MgSO 4.7H2O, 1.0; CaCO 3, 1.0; NaCl, 1.0; dextrin, 35; peptne, 1.0; (NH 4) 2SO 4, 1.0 The cmpnents were disslved in wheat bran extract (5%) and ph 7.0. The Erlenmeyer flasks (250) cntaining 25ml f sterile medium. The flasks were inculated and then incubated at 37 C fr 96h in a rtary shaker regulated at 180 rpm. The cells harvested by centrifugatin at 5000 g fr 20min in a refrigerated centrifuge at 4 C. Prtein Assay: Prtein was measured by the methd f Lwry et al. (1951) with bvine serum albumin (BSA) as a standard. Enzyme Assay: Prtease activity was determined as reprted earlier Bergkvist (1963) by determinatin f the amunt f casein hydrlyzed. One unit (U) f enzyme activity crrespnded t the amunt f enzyme which liberated ne μml tyrsine per min. Thermal Stability: Thermal stability f free, immbilized and mdified prtease were tested by incubating the enzymes in glycine-naoh buffer (0.1M, ph 9.0) at a designated temperature (30-90min) befre activity assay. ph Stability: The enzyme was incubated at 30 C in 0.1M glycine-naoh buffer f different ph values (ph 6-11) fr 60min. Digestin f Natural Prteins: The crude enzyme (10U) was incubated with human bld clt, cagulated egg white and chicken skin in glycine-naoh buffer (0.1M, ph 9.5) at 35 C /10h. Alkaline Prtease Mdificatin: The mdificatin was carried ut accrding t Ben Ammar (2002). Oxidatin f Plysaccharides: 250 mg f each plysaccharide were disslved int 10ml f 0.1M sdium peridate slutin and allwed t stand at 30 C fr 6h. After that, 0.3ml f ethylene glycl were added and allwed t react fr 1h. The reactin mixture was dialyzed against water at 4 C vernight, and then lyphilized. Enzyme Cupling with Activated Plysaccharides: 0.4mg f alkaline prtease and 100mg f xidized plysaccharides were cmbined in glycine-naoh buffer (0.05M, ph 9.0). The cnjugates were precipitated at 50% ethanl and lyphilized. Immbilizatin Methds: Physical Adsrptin: One gram f the carriers were incubated with 1ml f enzyme slutin (220U) at 4 C vernight. (Abdel- Naby et al., 1998). Cvalent Binding: One gram f the carriers were treated with 2ml f 2.5% (v/v) glutaraldehyde fr 2h at 30 C. Then washed with distilled water t remve the excess glutaraldehyde. The wet carriers were mixed with 1ml f enzyme slutin (220U) and incubated fr 6h at 30 C. (Abdel-Naby et al., 1998). 314

3 Inic Binding: 0.5gm f the catin r anin exchanger was equilibrated with acetate buffer (0.1M, ph 6.0) r Tris-HC buffer (0.1M, ph 9.0). The carriers were incubated with 1ml f enzyme slutin (100U) at 4 C fr 12h. (Abdel-Naby et al., 1998). Entrapment: In Agar: 10ml f different cncentratin f agar slutin (2, 4, 6%) at 45 C were mixed with enzyme 3 slutin(220u). The mixture was quickly cled t 4 C, cut int 1x1x1cm fragments. (Cheetham et al., 1985). In Ca-alginate: 100ml f different cncentratins f sdium alginate slutin (2, 4, 6%) were mixed with enzyme slutin(220u). The entrapment was carried ut by drpping the alginate slutin in 0.1M CaCl 2. The resulting beads ( mm diameter) were cllected. (Abdel-Naby 1993). RESULTS AND DISCUSSIONS The results f a typical prcedure fr the partial purificatin f B. lichenifrmis ATCC21415 alkaline prtease are summarized in Table (1) pinted t the mst active fractins prduced by ethanl, acetne and ammnium sulphate. The highest specific activity was achieved by precipitatin at 70% ethanl (16.4U/mg prtein) and shwed 6.2-fld purificatin. This fractin was used fr the succeeding part f wrk. Ammnium sulphate at 60 and 70% saturatin recvered and 13.73% f initial activity with 3.6 and 3.9-fld purificatin. Pseudmnas aeruginsa prtease shwed 3.6-fld purificatin by using ammnium sulphate 70% (Najafi et al., 2005). The lwest recvered activity btained at 50% acetne (1.4-fld purificatin), this result was similar t that btained by Thangam and Rajkumar (2002). Table 1: Partial purificatin f B. lichenifrmis ATCC21415 alkaline prtease. Purificatin Ttal prtein Ttal Recvered Specific activity Purificatin (mg/fractin) activities(u) activity (%) (U/mg prtein) (-fld) Crude Enzyme Ethanl 70% Acetne 50% (NH 4) 2SO4 60% % Ethanl fractin precipitated at 70% cncentratin was used fr enzyme immbilizatin. Enzyme immbilizatin by physical adsrptin (Figure 1) indicated that the lwest immbilizatin yield (36.4%) was fund with chitsan. The highest yield was btained by adsrptin n lfa (70.5%) with highest immbilized enzyme (81.4U/g carrier). It was therefre selected in the fllwing experiments and its prperties were cmpared with thse f the free enzyme. Fig. 1: Immbilizatin f B. lichenifrmis ATCC21415 alkaline prtease by physical adsrptin 315

4 Enzyme immbilizatin by cvalent binding thrugh a spacer grup (glutaraldehyde) shwed cnsiderable bund (gd lading efficiency) and immbilizatin yield (Figure 2). This gd lading efficiency fr the immbilizatin by cvalent binding might have been due t the frmatin f stable crss linking between the carrier and the enzyme thrugh a spacer grup. In additin, cvalent binding thrugh a spacer grup prbably increased the lcal surface area f the carrier and cnsequently reduced the steric hindrance arund the active site f the enzyme mlecule Sis et al. (1990). The enzymes cvalently bund t ceramic shwed the highest immbilizatin yield (60.9%). Fig. 2: Immbilizatin f B. lichenifrmis ATCC21415 alkaline prtease by cvalent binding. Sme in exchangers were used fr the immbilizatin by inic binding (Figure 3). Inic binding shwed lwer enzyme binding cmpared t ther immbilizatin methds. DEAE-Cellulse DE-52 was the mst suitable in exchanger fr enzyme immbilizatin which gave the highest activity (25.4U/g carrier) with the highest immbilizatin yield (44.6%). Immbilizatin f Bacillus mycides alkaline prtease by inic binding using Amberlite IR-120 gave 28.1%U/g carrier with immbilizatin yield f 15% Abdel-Naby et al. (1998). Entrapment f enzyme in agar and Ca-alginate with different cncentratins were examined. The results in figure 4 indicated that agar (4%) was sufficient fr reaching maximal bund enzyme (68.9U/10mlgel) and Fig. 3: Immbilizatin f B. lichenifrmis ATCC21415 alkaline prtease by inic binding. 316

5 Fig. 4: Immbilizatin f B. lichenifrmis ATCC21415 alkaline prtease by entrapment. immbilizatin yield (35.2%). Sdium alginate (2%) had the highest immbilizatin yield (23.2%). The decrease in yield with increase in carrier cncentratin might be due t the decrease in prsity f the gel matrix which caused diffusin limitatin f the substrate Sis et al. (1990). Stabilizatin f enzyme by chemical mdificatin was reprted in Table (2). Cvalent cupling f enzymes t sluble plysaccharides has been reprted as a cmmn technique fr imprving their prperties especially thermal stability fr enzyme technlgy Farqi et al. (1997). Amng all the preparatins tested, the enzyme cupled t activated amylpectin shwed the highest recvered activity (78.3%) with the highest specific activity (12.8U/mg prtein). This recvered activity was higher than that reprted by Yamagata et al. (1994) when cupled an alkaline prtease frm Bacillus sp. t activated dextran (56%). In general, the specific activity f free enzyme was higher than the immbilized r mdified enzyme this may be due t the rigidificatin f the enzyme prtein cnfrmatin and decrease in the flexibility f the enzyme mlecule Rbertsn et al. (1996). Thermal stability f the immbilized and mdified prtease cmpared t the free enzyme (Table 3) shwed that bth immbilized and mdified enzymes were mre resistant t thermal inactivatin. Table 2: Cvalent cupling f B. lichenifrmis ATCC21415 alkaline prtease t activated plysaccharides. Activated * Cupled enzyme Specific activity Recvered plysaccharides f cnjugates (U/mg prtein) activity (%) Prtein (mg) Activity (U) Amylpectin (10 : 10 ) Pectin (3x10 : 10 ) Dextran (4x10 ) Dextran (7x10 ) Dextran (2x10 ) * Enzyme added t ne gram activated plysaccharides in 10 mg prtein cntaining 163U. The immbilized and mdified enzymes retained 59.9 and 76.1%, respectively f the riginal activity after heating at 60 C fr 60min, hwever the free enzyme retained nly 19.4%. Afaq and Iqbal (2001) demnstrated that immbilized papain exhibited a marked increase in thermstability and retained 87% f its riginal activity after 1h incubatin at 65 C while the free papain lst almst 75% f its riginal activity. The high stability f the immbilized enzyme culd be due t the diminished autlysis f the enzyme fixed t the carrier. The 317

6 Table 3: Thermal stability f B. lichenifrmis ATCC21415 alkaline prtease. Temperature (ºC) Residual activity (%) Free enzyme Immbilized enzyme Mdified enzyme Time f heating (min) The immbilized and mdified enzymes retained 59.9 and 76.1%, respectively f the riginal activity after heating at 60 C fr 60min, hwever the free enzyme retained nly 19.4%. Afaq and Iqbal (2001) demnstrated that immbilized papain exhibited a marked increase in thermstability and retained 87% f its riginal activity after 1h incubatin at 65 C while the free papain lst almst 75% f its riginal activity. The high stability f the immbilized enzyme culd be due t the diminished autlysis f the enzyme fixed t the carrier. The secnd pssible explanatin may be related t the rigidity f the cnfrmatin f the enzyme mlecules resulting frm binding t the carrier Afaq and Iqbal (2001). On the ther hand, plymerized sucrse was evaluated as therm prtectant additive fr trypsin enzyme by mdificatin Fernandez et al. (2004). The enzyme thermstability was increased (22-fld mre stable) against thermal incubatin at 50 C after mdificatin prcesses. The stability against thermal denaturatin induced by the attachment f the highly hydrphilic ligsaccharide mieties t the prtease culd be mainly due t the preventin f interactin between hydrphbic clusters lcated n the prtein surface and surrunding water mlecules Fernandez et al. (2002). Half-life time f immbilized and mdified enzymes were always higher than that crrespnding t the free enzyme. At 60 C the t ½ f immbilized and mdified enzymes were 100 and 115min, while it was 25min fr the free enzyme. Althugh t ½ f immbilized, mdified and free enzymes at 70 C were 25, 27.3 and 7.1min, respectively. Similarly Wehidy (2005) calculated the t ½ f immbilized, mdified and free Bacillus stearthermphilus alkaline prtease at 60 C which were 46.5, 27.9 and 7.3 min, respectively. As shwn in Table 4, immbilizatin and mdificatin resulted in a nticeable stabilizatin f enzyme against denaturatin at alkaline ph. Immbilized and mdified enzymes retained 69.8 and 98.1%, respectively f its riginal activity when pre-incubated at ph11 as cmpared t 38.5% inactivatin seed fr free enzyme. Fernandez et al. (2002) fund that mdified á-chymtrypsin was 7-fld mre stabile than the free enzyme against incubatin at ph 9.0. Table 4: ph stability f B. lichenifrmis ATCC21415 alkaline prtease. Relative activity (%) ph Free enzyme Immbilized enzyme Mdified enzyme Cntrl Specific activities at 100% fr : Free enzyme = 16.3 U/mg prtein Immbilized enzyme = 15.4 U/mg prtein Mdified enzyme = 12.8 U/mg prtein The results in figure (5), recrded that bth immbilized and mdified enzymes were ptimally at a higher temperature f 80 C than the free enzyme (70 C). Tanaksal et al. (2001) reprted that the ptimum temperature fr Cnidiblus macrsprus alkaline prtease was increased frm 40 t 50 C after immbilizatin. The ptimum temperature fr trypsin activity was increased by 10 C when adding the â-cycldextrin cntaining plysaccharide Fernandez et al. (2004). This fact suggests that the cnfrmatinal rigidity t the prtease was increased after the attachment t the ligsaccharide mieties, then requiring mre temperature t express its maximum catalytic activity. Arrhenius plts f temperature data f the three frms f enzyme were linear. The calculated f activatin energy (E ) by the relatinship: A 318

7 Fig. 5: Effect f temperature assay n the activity f B. lichenifrmis ATCC21415 alkaline prtease. Slpe = activatin energy/ 2.303R where R is the gas cnstant (1.976 cal/ml) Activatin energy f free, immbilized and, mdified enzymes were 23.6, 18.9 and 19.9 Kcal/ml, A respectively. Abdel-Naby et al., (1999) reprted that (E) f mdified enzyme was lwer due t theenzyme stabilizatin by glycsylatin. Mdified enzyme in cmparisn with the free enzyme (Figure 6) shwed n change in the ph ptimum (10.0), where the ph ptimum fr immbilized enzyme was Gusek et al. (1990) fund that immbilizatin shifted the ph activity prfile t mre alkaline ph values with an ptimal activity at a higher ph (9.4) cmpared t the free enzyme (9.0). The K m and V max values were determined frm Linweaver-Burk plts f the (Figure 7 and 8). K m f immbilized and mdified enzymes were 5.0 and 5.3 mg/ml respectively which were higher than the free enzyme 4.8 mg/ml. Increasing the K m value after immbilizatin abut 10- fld was reprted by Ohmiya et al. (1978). Fig. 6: Effect f ph n the activity f B. lichenifrmis ATCC21415 alkaline prtease. The increase f the Km may be due t mass transfer resistance f the substance accessibility t the enzyme active site. The calculated maximal reactin rate (V max) f the free enzyme was 22.7U/mg prtein which higher than thse f the immbilized and mdified enzymes (20.0 and16.9u/mg prtein), respectively. Decreasing the 319

8 Fig. 7: Lineweaver-Burk plt fr the free and immbilized enzyme. Fig. 8: Lineweaver-Burk plt fr the free and immbilized enzyme. V max after immbilizatin and mdificatin was similarly reprted by Wehidy (2005) and Abdel-Naby et al. (1999). This decrease might be due t the fixatin f the enzyme n the immbilizatin carrier which in turn and might be lead t decrease in the flexibility f the enzyme mlecule which is cmmnly reflected by a decrease in the catalytic activity. The enzyme activity was assayed after incubating the enzyme with varius ins, inhibitrs and detergents (10mM) at 37 C fr 15min (Table 5). Mst f the metal ins tested had a stimulatr effect (Ca, C, Mg, Mn and Na ) n the activity f free, immbilized and mdified enzymes. On the ther hand, Cu in Table 5: Effect f sme metal ins n the activity f B. lichenifrmis ATCC21415 alkaline prtease. Metal in (10mM) Relative activity (%) Free enzyme Immbilized enzyme Mdified enzyme CaCl CCl CuSO 4.5H2O MgSO 4.7H2O MnCl 2.4H2O NaCl EDTA IAA Pba SDS Nne

9 decreased the enzyme activity. This inhibitry effect was higher fr the free enzyme (65.7%) than the immbilized and mdified enzymes (26.2 and 25.2%), respectively. The free enzyme was strngly inhibited (relative activity 43.4%) by Ethylene-Diamine-Tetra- Acetic acid (EDTA) cmpared t the immbilized r mdified enzyme (87.5 and 86.8%), respectively. The results suggested that the immbilizatin and mdificatin prtected the enzyme against the inhibitry effects f sme metal ins and inhibitrs. These results are in agreement with thse reprted fr ther enzymes Abdel-Naby et al. (1999). The latter authrs reprted that the glycsylatin f enzyme frmed stabile cvalent bnd that led t achievement f resistance against chemical. The ability f crude prtease t digest sme natural prteins was tested (Figure 9) and shwed that the enzyme can cnvert the insluble frms f human clt and cagulated white egg t sluble frm. The enzyme als was able t digest chicken skin after incubatin fr a lng time with it. The results suggesting usefulness f this enzyme fr different applicatin such as extractin f cllagen frm skin fr cllagen replacement therapy, waste treatment and ther related applicatin. Fig. 9: Effect f crude enzyme n sme natural prteins. Cnclusin: Lfa is a new carrier fr B. lichenifrmis ATCC21415 alkaline prtease immbilizatin by physical adsrptin with the highest immbilizatin yield (70.5%). Chemical mdificatin f prtease was perfrmed by cvalent cupling with sdium peridate- activated amylpectin retained (78.3%). Immbilizatin and mdificatin prtect enzyme against temperature and inhibitrs. The crude enzyme can digest sme natural prteins and was able t extract cllagen frm chicken skin. ACKNOWLEDGMENT The authrs thanks the Academy f Scientific Research and Technlgy fr supprting their search thrugh prject titled "Applicatin f the recent appraches t imprve the activity and stability f sme micrbial enzymes f ecnmical imprtance REFERENCES Abdel-Naby, M.A., Immbilizatin f Aspergillus nigernrc107 xylanase and B-xylsidase, and prperties f the immbilized enzymes. Applied Bichemistry and Bitechnlgy, 38 : Abdel-Naby, M.A., A-M.S. Ismail, S.A. Ahmed and A.F. Abdel-Fattah, Prductin and immbilizatin f alkaline prtease frm Bacillus mycides. Biresurce Technlgy, 64: Abdel-Naby, M.A., Stabilizatin f cellbiase by cvalent cupling t sluble plysaccharide. Micrbilgical Research, 154:

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