Highly efficient immobilization of laminarinases from marine - - PowerPoint PPT Presentation

Highly efficient immobilization of laminarinases from marine mollusks in novel hybrid polysaccharide-silica nanocomposites

Burtseva Y.V., Shchipunov Yu.A., Karpenko T.Yu., Shevchenko N.M., T.N. Zvyagintseva

Pacific Institute of Bioorganic Chemistry and Institute of Chemistry Far East Department Russian Academy of Sciences

The sol-gel technology is presently believed to be one of the most promising approaches for the immobilization of

• enzymes. Its main advantage lies in the fact that the

entrapment of proteins proceeds without formation of covalent linkages between biomolecules and matrix. As a results, the enzymes are in their intact state after the

• immobilization. This is the reason why they hold

functionality that is supplemented by a substantial increase in their long-term and thermal stability. The aim of this study was to extend our method for the immobilization of a highly labile enzymes, laminarinases.

1,3-β-D-glucanases – laminarinases (EC 3.2.1.6) from marine mollusks Spisula sachalinensis and Chlamys albidus belonging to O-glycoside hydrolyses (EC 3.2.1) Tetrakis(2-hydroxyethyl) orthosilicate (THEOS) was taken as a

• precursor. Silica nanocomposites were synthesized in aqueous

solutions containing xanthan, locust bean gum or cationic derivative

• f hydroxyethylcellulose (KAT-HEC)

The immobilized enzymes demonstrated activity at low

concentration comparable to their content in the living cells.

is due to advantages of the new precursor and synthesized biocatalysts

The entrapment conditions are dictated by the enzyme, but not the sol-gel processes; It can be performed at pH and temperature suitable for the enzyme functioning. The organic solvents are not used to solubilize the precursor; Catalysts for the promotion of the sol-gel process are not added because of the catalytic action of the polysaccharides inside the matrix; A biocatalyst can be prepared at reduced concentrations of THEOS that reduces the heat release in the course of the precursor hydrolysis; The porous structure of hybrid nanocomposite provides the accessibility of immobilized enzyme by the enzymatic substrate and proper functioning, whereas the protein molecules are not easily washed out of the matrix;

The control experiment is the activity of laminarinases in the aqueous solution and immobilized state in matrices with various polysaccharides (0.3 wt. %) is experiment. The biocatalysts were prepared in the initial solution with 10-20 wt. % of THEOS

20 40 60 80 100 120 140 160 200 400 600 Laminarinase from Spisula sachalinensis

Enzyme activity, % relative to the initial activity of control samples

Days elapsed after immobilization Control Locust bean gum Xanthan Cat-HEC 20 40 60 80 100 120 140 160 200 400 600 Laminarinase from Chylamys albidus

Enzyme activity, % relative to the initial activity of control samples

Days elapsed after immobilization Control Xanthan Cat-HEC

40 80 120 160 20 40 60 80 100 200 220 240

control immobilizied state

40 80 120 160 40 80 120 160 200 240

immobilizied state control

The comparison

• f

both laminarinases functioning in the immobilized state makes it obvious that they exhibit various sensitivity to the composition

• f

hybrid nanocomposites

A further study of glucanases was performed to characterize some details

• f their functioning in the immobilized state. When comparing them with

similar parameters determined for the free enzymes in solution, one does not find notable differences. It is only necessary to add that there was extending of temperature and optimal region pH.

3,5 4,0 4,5 5,0 5,5 6,0 6,5 7,0 7,5

0,0 0,2 0,4 0,6 0,8 1,0

3,5 4,0 4,5 5,0 5,5 6,0 6,5 7,0 7,5 0,0 0,2 0,4 0,6 0,8 1,0 LO in the immobilized state LO in solution pH А 750

10 20 30 40 50 60 0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6 A750 t,

0C

LO in solution; LO in the immobilized state.

10 20 30 40 50 60 70 0,0 0,2 0,4 0,6 0,8 1,0 1,2 LIV in the immobilized state LIV in solution

A750

t,

0C

3,5 4,0 4,5 5,0 5,5 6,0 0,0 0,4 0,8 1,2 1,6 LIV in the immobilized state LIV in solution А 750

pH

1 2 3 4 5 6

1 2 3 4 5 6 0,0 0,2 0,4 0,6 0,8 1,0 1,2

b b b a a b a a А 750 t, h

25 C 37 C 50 C 60 C

10 15 20 25 30 35 40 1 2 3 4 5 6 7

b a T,

0C

τ1/2, h

The immobilization gave prominent rise to the temperature stability of glucanase Lo in comparison with that in the solution. The former demonstrates a time dependence of the concentration of sugars released in the course of an enzymatic hydrolysis at 37°C. The latter represents the half-life times of enzyme at various temperatures. It was determined on the example of enzymatic reaction performed at 37°C.

a – in solution b – in the immobilized state

1 2 3 4 5 6 0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8

1 2 3 4 5 6 0,0 0,2 0,4 0,6 0,8

a a b b 10-15 C 25 C 37 C

a t, h

b A 750

LIV LO

Characteristics of laminarinases in solution and immobilized state

55 5.6 3.0

Immobili zed state

0.01-0.3 50 5.6 0.25 Solution 22-39 Glc -1→3

LIV, Spisula sachalinensis

37 6.0 4.0

Immobili zed state

0.1-0.25 36 6.0 0.7 Solution 20a/38b Glc -1→3

LO, Chlamys albidus

NaCl, M Т оС рН Optimum conditions Km (mg/ml) Location Mw (kDa) Type of hydrolysing bond Enzyme, source

a determined by the gel filtration - b determined by the electrophoresis (SDS-PAGE)

Endo-1,3-β-D-glucanases of marine organisms, found at first as hydrolases, catalyze three reactions running simultaneously and practically with almost equal efficiency. Endo Endo-

• 1,3

1,3-

• β

β-

• D

D-

• glucanases of marine organisms

glucanases of marine organisms, , found found at first as hydrolases at first as hydrolases, , catalyze three reactions running catalyze three reactions running simultaneously and practically with almost equal simultaneously and practically with almost equal efficiency. efficiency. S''-A ( (reaction of reaction of transglycosylation transglycosylation) ) S''-ОН ( (reaction of reaction of hydrolysis hydrolysis) )

S''-S ( (glucanosyl glucanosyl transferase transferase activity activity) ) S' S' E ES S E E+ +S S E ES'' S'' A S H2O

Biochemistry (Moscow) 1997. 62: 1300.

a e

d c

b

• 3

3, ,6 6-

• О

О-

• di

di-

• substituted

substituted Glc Glc residue residue

• nonsubstituted

nonsubstituted Glc Glc residue residue

• 3

3-

• О

О-

• substituted

substituted Glc Glc residue residue

• 6

6-

• О

О-

• substituted

substituted Glc Glc residue residue a b c

Laminaran from Laminaria cichorioides

Zvyagintseva, T.N., Elyakova, L.A., Isakov, V.V., 1995. Enzymatic conversion of laminarans into 1->3;1->6-β-D-glucans, possessing immunostimulating activity (in Russian). Bioorg. Khim. 21(2), 218-225.

1,3 1,3-

• β

β-

• D

D-

• glucanase from

glucanase from Chlamys Chlamys albidus albidus

Translam Translam

The hypothetical mechanism of the receiption of translam

Products of Enzymatic Transformation of Products of Enzymatic Transformation of laminaran laminaran from from Laminaria Laminaria cichorioides cichorioides and Their Properties and Their Properties

Laminaran

TRANSLAM immunostimulator, radioprotector, crioprotector and antitumor agent ANTIVIR Phytoimmunostimulator: potato, tobacco, tomato, soybean

Glc + Glcn Glc1 6 X = 1,2,3 are the main components 6-8 20 80:20 Antivir 8-10 25 75:25 Translam 3-5 90:10 Laminaran

M.m., kDa

1→3:1→6

Yield, %

Glucan

E (Lo)

2 4 6 8 10 0,0 0,5 1,0 1,5 2,0 2,5 3,0

T20 T40 T80 V, ml A 490 T10

translam

(10 kDa)

laminaran

(5 kDa)

Gel permeation chromotography on Superdex 75 HR 10/30

• f laminaran from L. cichorioides and translam obtained by

action laminarinase LO in the immobilized state.

13С-NMR-spectra of initial laminaran

A 1,3;1,6-β-D-glucan (laminaran) contains β-1,6-bound glucose residues (10%) and mannitol

13С-NMR-spectra of translam

A new 1,3;1,6-β-D-glucan (translam) contains β-1,6-bound glucose residues (20-25%), but no mannitol

Conclusions

The results presented in the article demonstrated that the 1,3-β- D-glucanases were successfully immobilized in the novel hybrid polysaccharide-silica nanocomposites. 1,3-β-D-Glucanases had the maximal activity at conditions (pH, temperature and ionic strength) that were optimal for them in solutions before the entrapment; provided the synthesis biologically active, branched 1,3;1,6-β-D- glucan (translam); retained or even had sometimes an increased activity, became more thermally stable and demonstrated prolonged long-term stability. These facts give evidence that the suggested immobilizing method is ideally suited for the entrapment of enzymes and development of biocatalyst for biotechnological applications.

• 1. Shchipunov
• Yu. A., Mukhaneva

O.G., Zvyagintseva T. N., Shevchenko N. M. Polyelectrolyte complexes of naturally occurring fucoidans with cationically and hydrophobically modified hydroxyethyl cellulose. //Visokomolecular. Soedin.(Russian) Ser. А,

• 2003. V. 45. P. 295-303.
• 2. Karpenko T.Yu., Shchipunov Yu.A., Bacunina I.Yu., Burtseva

Yu.V., Zvyagintseva T.N. Biocatalysts Prepared by the Immobilization of O-glycoside Hydrolases inside Polysaccharide- Silica

• Nanocomposites. In Proceedings of 18-th European

Conference on Biomaterials including Third Young Scientists’

• Forum. 2003. T099. October. Stuttgart, Germany.
• 3. Shchipunov Yu. A., Karpenko T. Yu., Bakunina, I. Yu., Burtseva,
• Yu. V., Zvaygintseva T. N. A new precursor for the immobilization
• f enzymes inside sol–gel-derived hybrid silica nanocomposites

containing polysaccharides. J. Biochem. Biophys. Meth. 2004, V.

• 58. P. 25-39.

Our articles

• 4. Burtseva
• Yu. V., Karpenko
• T. Yu., Shevchenko N. M.,

Zvyagintseva T. N., Shchipunov Yu. A. Properties and specificity endo-1,3-β-D-glucanases of marine mollusks immobilized in hybrid

• nanocomposites. Regional Science Conference. 2004. P. 75.
• November. Vladivostok, Russia.
• 5. Shchipunov Yu. A., Burtseva Yu. V., Karpenko T. Yu.,Shevchenko
• N. M., Zvyagintseva T. N. Immobilization and characterization of

laminarinases (endo-1,3-В-D-glucanases) from marine mollusks in novel hybrid polysaccharidesilica nanocomposites. Journal of Molecular Catalysis B: Enzymatic. 2006.

This work was supported by grants of Russian Science Support Foundation, President of Russian Federation, FEB RAS and RFFR № 06-04-48540-а and № 05- 04-48291-а, Molecular and cell Biology. The authors are indebted to Dr. C. Abetz and Ms. I. Otto (Bayreuth University) for the SEM micrographs of gels.

Acknowledgements

Laboratory of colloid systems and interfacial processes Shchipunov Yu.A. Karpenko T.Yu. Laboratory of enzyme chemistry Shevchenko N.M. Zvyagintseva T.N. Institute of Chemistry Pacific Institute of Bioorganic Chemistry Far East Department Russian Academy of Sciences

Thank you for your attention