Co-immobilization of lipase, glycerol kinase, glycerol-3 ...nopr.niscair.res.in/bitstream/123456789/15211/1/IJBB 39(5) 342-346.pdf · Co-immobilization of lipase, glycerol kinase,

Indian Journal of Biochemistry & Biophysics Vol. 39. October 2002, pp. 342-346

Co-immobilization of lipase, glycerol kinase, glycerol-3-phosphate oxidase and peroxidase onto alkylamine glass beads through glutaraldehyde coupling

Van dana Kalia and C S Pundir*

Biochemistry Research Laboratory, Dept of Bio-Sciences, M.D. University, Rohtak 124001 (Haryana), India

Received 28 January 2002; revised alld accepted 12 June 2002

A method for co-immobilizing lipase from porcine pancreas, glycerol kinase (GK) from Ceiluiomollas spp. , glycerol -3-phosphate oxidase (GPO) from Aerococcus viridans and peroxidase from horseradish onto zirconia-coated alky lamine glass beads through glutaraldehyde coupling has been described. The co-immobilized enzymes retained 71.4% of initial specific activity with a conjugation yield of 43 .6 mg/g support. The optimum pH and Km for triolein increased, while Vnm was decreased slightly, but incubation temperature for maximum activity remained unaltered after co-immobilizat ion. The co­immobilized enzymes showed increased thermal and storage stabilities in cold, compared to their native form. Among the various metal salts 'tested, only CUS04 caused inhibition of both free and co-immobilized enzymes. The co-immobilized en­zymes showed better suitability over mixture of individually immobilized enzymes in determination of serum triglyceride.

Total triglyceride (TG) levels in the serum are valuable indicators of abnormalities in lipid metabolism, atherosclerosis and hypertension I .

Increased triglyceride levels may be a risk factor for coronary artery disease (CAD)2. Although an enzymic colorimetric method employing lipase, glycerol kinase, glycerol-3-JPhosphate and peroxidase is commercially available for TG determination3

, the high cost of enzymes limits its wide spread use for routine determination. The immobilization of enzyme to some insoluble support serves the primary purpose of facilitating its recovery from the reaction mixture, permits its reuse and thus renders it economical. Among the various insoluble supports available, zirconia coated alkylamine glass, an inorganic carrier is generally resistant to microbial attack and is stable over a wide pH range or various solvents such as ethanol or acetone4

. Earlier, we have immobilized commercial porcine liver lipase and horseradish peroxidase onto alkylamine glass beads through glu­taraldehyde coupling5

,6. Glycerol kinase and glycerol-3-phosphate oxidase have also been immobilized onto non-porous glass beads and polymeric support and different membranes 7,8,9 . However, co-immobilized systems are likely to be more efficient than the separately immobilized enzymes, since intermediates can diffuse between enzymes much more easilylO. The

*Corresponding author Tel: (01262) 72012 Email: pundircs@ rediffmail.com

present report describes the co-immobilization of li­pase, glycerol kinase, glycerol-3-phosphate oxidase and peroxidase onto alkylamine glass beads through glutaraldehyde coupling and changes in their kinetic properties after co-immobilization. The superiority of co-immobilized enzymes over individually immobi­lized enzymes for determination of serum triglycerides is demonstrated.

Materials and Methods Zirconia coated alkylamine glass beads (pore

diameter, 55 nm) were a gift from Dr H H Weetall , Environment Protection group, Las Vegas, USA . Porcine pancreas lipase, Cellulomonas spp. glycerol kinase, Aerococcus viridans glycerol-3-phosphate oxidase, horseradish peroxidase (RZ= 1.0), 4-aminophenazone, L-a-glycerophosphate and triton X-100 were from Sigma Chemical Co. , USA; 3,5-dichloro-2-hydroxybenzene sulfonate was from E. Merck, Germany and glycerol, triolein and A TP were from SISCO Research Laboratories Pvt. Ltd .. Mumbai . All other chemicals were of analytical reagent grade. The kit for enzymic colorimetric determination of triglyceride was from Miles India Ltd., Baroda.

Assay of mixture of free lipase, glycerol kinase, glycerol-3-phosphate oxidase and peroxidase

Assay was carried out in a 15 ml conical fl ask wrapped in a black paper. The reaction mixture was prepared as described by Fossati and Prencipe3 and

KAllA & PUNDIR: CO- IMOBILIZATION OF ENZYMES THROUGH GLUTARALDEHYDE COUPLING 343

cOIl sisted of 0.54 flmol MgC,", 0.63 flmol ATP, 1.0 iJmol potassium ferrocyanide, 0.27 J.lmol 4-:1 minophenazone, 1.53 J.lmol 3,5-dicholoro-2-hydroxybenzene sulfonic :1cid (DHBS) and 1.0 f..lmol Triton X-IOO per litre of 0. 1 M sodium phosphate buffer, pH 7.0. Mixture of all the four enzy mes was prepared by dissolvi ng in 0.1 M sodium phosphate buffer. pH 7.0, in a unit activity ratio of 100:50:20: I, at \vhich opti mum act ivity for enzyme mixture was obtained. To 900 f..ll reaction mixture, 50 f..ll of enzyme solution was added and preincubated at 37°C for 5 min. The reaction was started by adding 50 f..ll of 2.26 mmol triolein . After incubati on at 37°C for IS min , A 5 10 was read. H~O~ generated in the reaction mixture was ex trapolated from standard curve between H 20~

and A5 10

Co-illllllobilizat ion of enZYllles The mixture of lipase, glycerol kinase, glycerol-3

phosphate ox idase and peroxidase in 0.1 M sod ium phosphate bulTer. p H 7.0, was co-immobilized onto 250 mg alkylamine glass beads through glutaraldehyde coupling using the method of Lynn " . In a 25 ml conical flask, 250 mg alkylamine glass beads were activated by adding 4.0 ml of 2.5% glutara ldehyde in 0.1 M sodium phosphate buffer, pH 7.0 (v /v) with occasional stirring for 2 hr at room temperature (30±2°C). The excess of glutaraldehyde was decanted followed by many washings of beads with 0.05 M sodium phosphate buffer (p H 7.0) until pH of the washing discard was 7.0. Enzyme solution (J ml ) was added to the activated beads and kept for 48 hr at 4°C with occasional st irring. The unbound enzy me was decanted and tested for ac ti vity and protein '2 . The beads were washed with 1.0 ml of react ion buffer (0. 1 M, sodi um phosphate buffer, pH 7.0) repeatedly until no activity was detected in the washings.

All the four enzymes were also immobilized individ ually on lo alkylamine glass beads (50 mg) as described above and mixed.

Assav (~l co-illllilobiliz.ed enZYllles

It was carried out in same manner as described for mixture of free enzy mes except that 250 mg alky lamine glass beads bound to all the four enzy mes in place of free enzyme mixture and additional 50 111 reac ti on buffer were used and the reaction mixture was kept under continuous stirring during incubation. After incubation at 37°C under continuous stirring, the reacti on mi xture was carefu ll y withdrawn, lea ving

the glass beads inside the flask. with the help of an Eppendorf pipette and transferred it to the cuve tte. AslO was read. The assay of mi xture of immobi lizcd enzymes was al so carried out as dcscribed for co­i mmobilized enzymes.

Deterlllination of Kill, VIIICIX and Eo To study the stability , enzymes preparations were

kept overnight in reaction buffer of varying p H from 5.0 to 10.5 using the followin g buffer each at a fina l concentration of 0.05 M: p H 5.0 to 6.0- sod ium succinate, pH 6.5 to 7.5- sod ium phosphate buffer. p H 8.0 to 9.0- glycine NaOH buffer and pH 9.5 to 10.5-sodium carbonate and bicarbonate; and tested for enzy me acti vity next day under standard assay conditions. For thermal stabi I ity, co-i mmobil ized enzy mes were kept at 70°C fo r 30 min . After cooling it in ice bath for 5 min , the act ivity was assayed. Kill and Villax were calculated from Lineweaver Burk plot by plotting 11v vs. lIS (tri olein) , whil e Ea was determined from Arrhenius plot by plotting log v \' .1'.

lIT.

Rel/se and storage ofco- illllnobiliz.ed enz.Ylll es To reuse the co-immobili zed enzy mes. the glass

beads were washed with di still ed water 5-6 times and finally with reaction buffer prior to their use in the next assay. The washing di scard was withdrawn each time using an eppendorf pipette. The glass beads were stored in di stilled water at 4°C when not in use.

Deterlllination ofserulll triglyceride bv eiliploving c()­illllllobilized enZYlll es

It was carried out as described for the assay of co­immobilized enzy mes except th at triolein so lut ion was replaced by 50 111 serum. Triglyceride in serum was calculated from standard curve prepared by plotting triolein concentrati on in the range 0.5-11 .0 mM )IS. A 5 10

Results and Discussion Co- illllno/Jilization and kin etic proper/ies ~l en::'Ylll es

Commercially ava ilab le lipase from porcine li ve r. glycerol kinase fro m Celll/loll lOnas spp. glycerol-3-phosphate oxidase from AerococclIS \Iirido ll s and peroxidase from horseradish have been co-immobilized onto alkylamine glass through glutaraldehyde coupling with a conj L~ga ti o n yield 'Of 43.68 mg/g support and 7 1.4% retenti on of ini tial specifi c activity (Table I). A comparison of kinetic properties of co-immobi li zed cnzy mc and mi xture or individually immobili zed enzy mes with those of free

344 INDI AN 1. BIOCHEM. BIOPHYS., VOL. 39, OCTO BER 2002

Table I-Co-i mmobili za tion of lipase, glycerol kinase, glycerol-3-phosphate ox idase and perox id ase onto alkylam ine glass beads

(pore diam .. 55 nm)

Enzy me protein (mg) Enzyme units added to Enzy me coupled (%) Conjugation yield (mg Retenti on of sp. ac ti vity ('70)

added to beads (250 beads to glass beads (250 enzy mel g support ) mg) ( ~lmo I /H202/min ) mg)

15. 12 4 13 72.2 43.68 7 1.4

Tab le 2- A comparison of kineti c properties of mi xture of free and ind ividually immobili zed and co- immobi lized lipase. glycero l

kinase, glycerol-3 -phosphate ox idase and perox idase

ITo determ ine the optimum pH . the pH of the reaction buffer was va ried from 5.0 to 10.0 using the follow in g bufTers. eac h al a fi II ;,] I

concentral ion of 0. 1 M : p H 5.0 to 6.0, sod ium succ inate; pH 6.5 to 7.5, sodium phosphate; pH 8.0 to 10.0, glyc ine- NaO H. E" was

calc ulated from Arrhenius plot by plotting logv 11.1' lIT . Km and Vrna., were ca lcul ated from Lineweaver-B urk plot by plolli ng

I lv \ '.1 1/ [511

Optimum pH Opt imum temp. (OC)

E" (Kca l/mol) Thermal stability at 70°C fo r 30 min . (ac ti vity retained)

Time of linearity

Kl11 (mM)

\I""" (nmol /min. ) Siorage in distilled water at 4°C for 6 months (% loss)

enzy me in mi xture is given in Table 2. The pH optima for co-immobilized enzymes was 8.5 , which is hi gher than that of mi xture of free enzymes (pH 7.0). The hydrogen ion concentration experienced by the en­zy me in the locality of polycationic matrix is pre­dicted to be lower than that of the bulk solu ti on and hence pH max imum ex hibited bi', an ~nzyme. is di splaced towards higher pH values -. The lIlCUbatlon temperature (3 7°C) and time required (15 min) for max imum acti vity were unaltered after co-im­mobi lization. The KIl1 for tri olein fo r co-immobi lized enzy mes was 30.2 mM, which is higher than that for mi xture of free enzy mes (2 1.1 mM) . Vll1a .x was de­creased from 19.6 nmol/min to 13.8 nmol/min after co-immobi li zati on. The changes in kinetics of co­immobili zed enzymes are controlled by four factors: change in the enzy me concentration , steric effect, micro-environmental effects and bu lk and di ffusional efrects l4

.

The mi xture of individuall y immobilized enzymes showed max imum acti vity at p H 6.5 when incubated at 37°C fo r 15 min .

p H. Th erl11al and storage stability of co-illlmobilized en-;r/1/es

The co-immobil ized enzy mes were fairly stable over a pH range of 6.0-9.0, which is higher than that

Mi xture of free enzy mes Co- immobili zed

enzymes 7.2

37

9.8

30 15

2 1.1

19.6

42

8.5

37

5.45

53 15

30.18 13.8

20

120 r----------------,

100

60

40

20

0

~ Co- immobilized enzymes

-O-- Individually immobilized enzY llles

\

I I

I

j 5 6 7 8 9 10 11

pH

Fig. I-pH Stability curve for co-immobili zed an d th mix ture of individuall y im mobil ized lipase. glycero l kinase. glycerol-3-phosphate ox idase and peroxidase [Enzy ml: preparations wc re kept overni ght in the reac tion buffers of varying pH from 5 to 10.5, each at a final concentration of 0.05 M and tested for enzyme aC li vity nex t day under standard assay -:oild iti ons I

fo r mixture of indi viduall y immobilized enzy mes (pH stability range 6.0 to 8.0) (Fig. 1). Thermal stab ility and storage stabiliti es of the enzy rnes in co ld were

KALlA & PUNDIR: CO-IMOBILIZATION OF ENZYMES THROUG H GLUTARALDEHYDE COUPLI NG 345

11 0 r---------------,

100 k--_e_

90

60

50

o

---- Mixture of Free enzymes

2

-e-/lldividually immobilized

3 4

Time (in months)

5 6

Fig. 2-Storage stability curve of mi xture of free . indi vidually immob il ized alld co- immo-bili zed lipase, glycerol kinase. glycerol-3- phosphate ox idase and peroxidase [Standard assay condit ions we re used for eac h assay]

increased afte r co-immobili zation. Co-immobilized enzymes retained 53% of their initial activity on ex posure to 70°C for 30 min which is hi gher than 48% and 30% retentions of initi al activity for mi xture of individually immobili zed and free enzy mes respec ti vely. The co-immobili zed enzymes showed 20% loss of initial activity after 6 months of regul ar use when stored at 4°C in distilled water, whil e free enzy mes mixture showed 42% loss of activity (Fig. 2) under similar conditi ons. A mi xture of individuall y immobili zed enzymes showed 22% loss of initial act ivity. Stability of co-immobi li zed and immob ili zed enzy mes was improved due to the greater di mensional stabiliti es of the inorgan ic supports and stabi li zing etlect of the mi croenviro nment rendered by the sup­porti O.

Effects (~l llJ eta/ .w lts on co- illllllo/Jili::,ed en::')'lIJes To study the interference by metal ions normall y

fou nd in the serum, the followi ng metal salts were added in the reacti on mixture indi viduall y, each at the final conce ntration of I mM: NaCI, KCI, MnCI 2,

MgCl 2• CuSO~ . CaSO~ . FeSO~, ZnSO~ and NaL Of these, only CuSO~ caused 46% inhibition of co­immobili zed enzymes, wh ich is almost similar to th at fo r mi xture of free enzymes (40%).

400

350

300

250

200

'C 150

0 ..r;:

100 Q; E c: 50 ., VI

~ 0 Q.

,., 400 D

'0 350

'" E ;; 300 >-E 250 2 ., en 200

150

100

50

0

0

(A) ( r = 0 .984086 )

<Do

(B) ~ ___ ~

( r = 0.937486 1

0'6 o 00

o

00

0° 0

°

o

o o

o o

50 100 150 200 250 300 350 400

Senllll TG (mg/ut) by enzo-kil Illcl hou

Fig. 3--Correlati on between serum triglyceride (TG ) \ a lu ~ s

determined by commercial enzo- kit method empl oy ing so luble enzy mes (x -ax is) and the present method (y-axis): I (A ). using co­immobili zed lipase, glycero l kinase. glycero l-3-phosphate ox id ­ase and peroxidase; (B). usin g mi xture of indi viduall y immobilized enzymes].

Deter/llination ol serullJ triglvcerides \\'itli co­ill/mobilized enZYlIJe

The tri glyceride concentrati on tn the serum samples of apparent ly healthy adults was determi ned by co-immobili zed lipase, glycerol kinase, glycerol-3-phos phate ox idase and perox idase and found to be in the range 78-200 mg/dl for health y individuals with a mean of 155 mg/dl (n=20). The va lues of serum tri glycerides determined by our method were in the similar range with those obtained by free enzyme method (70-200 mg/dl)l .

To evaluate the accuracy of the present method. serum triglycerides values were determined by the present method employ ing co- immobili zed enzymcs (Y I), mi xture of indi viduall y immobi li zed enzymes (Y2) and commercially avail abl e enzy mic colorimetric method (x). The serum triglycerides va lues of 20 individuals ( 12 healthy and 8 di seased persons suf­fering from hypertriglyceridemia) obtained by the present method showed good correla tions (1'1= 0.98-1-and 1'2=0.937) with those of commercial en zyme co lorimetri c kit method using free enzy me; the regress ion equati on bei ng y 1= 1.0 1594x + 2.56 and Y2 = 0.906x + l8.95 (Fi g. 3A and 8 ). These results show

346 IND IAN J. BIOCHEM . BIOPH YS. , VO L. 39. OCTOBER 2002

the better suitability of co-immobili zed enzy mes over mi xture of indi vidually immobili zed enzy mes for determinati on of serum tri glycerides .

To sum up, the present work provides a technique for co- immobili zati on of lipase, glycerol kinase, glycerol-3-phosphate ox idase and peroxidase, onto gl utaraldehyde ac ti vated alky lamine glass beads. The co-immobili zed enzy mes were acti ve even after their regul ar use for 6 months when stored at 4°C in di still ed water. Compared to the mi xture of native enzy mes, they possessed higher pH and thermal and sto rage stabiliti es. Also, the enzy mes co-immobili zed to glass beads are more easil y recoverable and reusable th an the mi xtu re of indi vidually immobilized enzymes. The present technique of co-immobili zat ion of enzymes can be of wide use in simplifying and economi zing commerciall y ava ilable enzy me kits and bio-. ensors along with certain automated instruments for tri glyceride determinati on.

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