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1897Biotechnol. & Biotechnol. eq. 24/2010/2

Article DOi: 10.2478/v10133-010-0041-3 MB

Biotechnol. & Biotechnol. eq. 2010, 24(2), 1897-1903Keywords: fungus, immobilization, hybrid bionanomatrices, alpha-galactosidase

IntroductionThe sol-gel method has definitely proved its exceptional perspective of the possibility to synthesize a significant number of new materials with specific properties. Organic-inorganic hybrids are a relatively new type of composites with interesting mechanical, optical, structural and thermal properties (13, 18, 22). Such hybrids are promising materials for various applications: biocatalysts and as materials for immobilization of different biomolecules (8, 12, 19). As it is known, there are many silica hybrids containing different natural polymers. Silica-gelatin hybrid coatings can be applied to biological substrates such as leather (27). Sol-gel derived silica/chitosan hybrids have the potential to be used as bioactive coating materials for applications in hard tissue engineering (21). Novel nanoporous lignin (lignocellulose)/SiO2 hybrid materials was applied as a sorbents of potential inorganic and organic pollutants (30). the formation of silica/alginate biocomposites by the sol-gel route using aqueous precursors, in the presence of the polymer were also investigated (12). the sol-gel synthesis, structure and application for bacterial immobilization of inorganic-organic hybrids based on different silica precursor and addition of the organic compounds such as sepharose and lactic acid were investigated by Chernev et al. (10, 11).

α-D-Galactosidases (α-D-galactopyranoside galactohydrolase, E.C. 3.2.1.22) catalyze the cleavage of terminal galactosyl residues from a wide range of substrates, including linear and branched oligosaccharides,

polysaccharides and synthetic substrates such as p-nitrophenyl α-D-galactopyranoside. In the food industry microbial α-galactosidases are used for different biotechnological applications, e.g., for the hydrolysis of raffinose in beet sugar syrups or galacto-oligosaccharides in soybean milk (20) and for the improvement of the gelling properties of galactomannans used as food thickeners (9). these enzymes are of interest in medicine, e.g., for the treatment of Fabry’s disease by enzyme replacement therapy (16) or for the blood type conversion (B type cell to o type cell) (31).

α-Galactosidases are produced mainly by filamentous fungi from the genera Aspergillus, Penicillium and Humicola (1, 5, 24, 25).

We previously reported that the fungal mutant strain Humicola lutea 120-5 producer of extracellular α-galactosidase was used for immobilization in sol-gel nanomaterials with algal heteropolysaccharide (6, 15) as well as in nanomatrix, consisting of tetraethylortosilicate (TEOS) and a mixture of polyethyleneglycol (PEG) and polyvinylalcohol (PVA) (28). the use of nonmechanically agitated bioreactors for fungal cell fermentation may contribute to reducing the process costs, because they are considered to have a lower power requirement for comparable values of the oxygen mass transfer coefficient than the mechanically agitated vessels (17). The potential of such a device was previously demonstrated in the submerged cultivation of free as well as immobilized Humicola lutea cells (2, 3, 6) producing different hydrolyzing enzymes.

here we report on the structure and characterization of both nanomatrices, consisting of teoS as an inorganic precursor and lactic acid or sepharose as an organic part. the possibility of using these matrices for immobilization of the fungus Humicola lutea for the production of α-galactosidase during

IMMOBILIZATION IN NANOMATRICES OF HUMICOLA LUTEA MYCELIUM FOR ALPHA-GALACTOSIDASE BIOSYNTHESIS IN LABORATORY AIR-LIFT BIOREACTOR

P. Djambaski1, P. Aleksieva2, D. Spasova2, G. Chernev1, L. Nacheva2

1University of Chemical Technology and Metallurgy, Sofia, Bulgaria2Institute of Microbiology, Bulgarian Academy of Sciences, Sofia, BulgariaCorrespondence to: Georgi Chernev or Lilyana NachevaE-mail: [email protected]; [email protected]

ABSTRACTThe sol-gel synthesis of both hybrid nanomatrices containing tetraethylortosilicate (TEOS) as an inorganic precursor and lactic acid, or sepharose as an organic component was made. Crystal as well as surface morphology of the hybrids were investigated using different methods: X-ray diffraction, infrared spectra, BET-analysis and atomic force microscopy (AFM). The obtained nanomatrices were applicated for immobilization of the α-galactosidase producing fungal strain Humicola lutea 120-5. The semicontinuous cultivation was carried out in laboratory air-lift bioreactor. Maximal level of enzyme activity (1050-1130 U/l) that was reached in the third to fourth fermentation cycle using TEOS+40% lactic acid was higher than that obtained in the samples with TEOS+20% sepharose (660-770 U/l). The correlation between enzyme productivity and fungal development in the pore structure of the carriers was examined using scanning electron microscopy observation.

1898 Biotechnol. & Biotechnol. eq. 24/2010/2

the cultivation of the entrapped cells in laboratory air-lift bioreactor was investigated. The development of the mycelium in the pore structure of the carriers was examined by scanning electron microscopy (SEM).

Materials and MethodsMaterialsThe organic part involved was lactic acid and sepharose (Merck) in quantity 20 and 40 wt%. tetraethylortosilicate (teoS) with 98% purity (Merck) was used as starting inorganic material.

Preparation of hybridsthe inorganic-organic hybrid materials were prepared by substituting part of the inorganic precursor with lactic acid and sepharose. A poly-step sol-gel procedure was carried out at strictly controlled conditions in order to obtain the desired nanostructred materials, permitting the following processes to be carried out: hydrolysis of silica precursors leading to the separation of alkoxide groups and producing of silanols; condensation of monomers and formation of nanoparticles; cross-linking of nanoparticles and gelation. Sol -gel transparent silica hybrid matrix with 20 to 40 wt% of organic compound were synthesized at room temperature. the inorganic-organic hybrid materials were prepared by substituting part of the inorganic precursor with a concentration 20 and 40 wt%. the quantity of sol-gel precursors varied from 16 ml (for the hybrid materials with 20% substitution) to 12 ml (for the hybrids with 40% substitution) depending on the concentration of organic constituent. For preparation of the hybrid materials in the beginning the precursor was mixed with distilled water and five ml of 0.1 N HCl were introduced to pH~1.5 for increasing hydrolysis rate on a magnetic stirrer. After pre-hydrolysis of the initial mixture, the preliminary dissolved in hot water organic compounds in a quantity from 4 to 8 g depending on the concentration in the final gel, was added. The next step included homogenization of the solution for 15 minutes. no alcohol was added as a co-solvent. In all cases the ratio precursor/H

2o

was kept constant and equal to 1. After the phosphate buffer was added to the homogenic solution the gelation time was from 2 to 5 min depending on the concentration of organic component. the addition of organic component led to a faster polymerization.

For studying the structure of the synthesized hybrids the following methods were used:

X-Ray diffraction (XRD) measurements were performed on a Bruker D8 Advance. The diffracted intensity of Cu Kα radiation (λ= 1.54056 Å, 40 kV and 30 mA) was measured with scan rate of 0,02o/min in 2θ range between 4o and 80o.

Infrared spectra (FT-IR) were obtained using a MATSON 7000 Fourier transforming infra-Red spectrometer. Pellets of ca. 2 mg of hybrid samples were mixed with 200 mg of spectroscopic grade KBr.

BET Analysis: N2 adsorption at 77 K was utilized to determine the specific surface areas and porosities of the

prepared hybrids. A gas adsorption manometry apparatus was used for the n2 adsorption experiments. The BET equation was used for the calculation of the specific surface area.

Atomic Force Microscopy (AFM) images were performed using a Digital Instruments multimode AFM equipped with a Nanoscope IIIa controller (Digital Instruments, Santa Barbara, CA). The results were obtained in tapping mode AFM. A vertical engage 4842 JV-scanner and Si probes were applied in all experiments. The driving frequency in tapping mode was chosen at the resonant frequency of the free-oscillating cantilever in the immediate vicinity of the sample surface. height and phase images were recorded simultaneously.

The average roughness (Ra) of the hybrids surface was calculated directly from the AFM image.

Microorganism, mediumthe fungal mutant strain Humicola lutea 120-5 (national Bank for Industrial Microorganisms and Cell Cultures, Bulgaria, No 391) was used in the present study. Soya meal extract (5) was used as a nutrient medium for growth as well as α-galactosidase production by both free and immobilized mycelium.

Immobilization procedure and culture conditionsto obtain one sample the cell immobilization was carried out using 5 ml spore suspension with a density 1010 spores per ml from one test tube. the obtained matrices were dried for 72 h and the thin blocks (3-4 mm) were used for the fermentation process. the sol-gel particles containing the entrapped spores as well as the same quantity of free spores were used as an inoculum in the batch and repeated batch α-galactosidase biosynthesis in an air-lift bioreactor. The cultivation was carried out in a 0.5 l laboratory bioreactor (0.3 l working volume) with a ratio of liquid height/diameter of 4.0 (2). culture temperature was maintained by water flow from a recirculating bath through the water jacket of the reactor. Each batch culture consisted of 250 ml nutrient medium and sol-gel blocks obtained from 3 test tubes. in the case of free cell fermentation, 15 ml spore suspension (or 6%) was used as an inoculum. Air was supplied at 1.5 l min-1. At the end of each cycle (144 h), culture broth was drained and the particles were washed within the reactor with sterile water. The next batch culture was started with the addition of fresh medium. During the experiments with immobilized cells, the deviation of replicates was less than 4%, whereas free cell fermentation demonstrated a deviation of 6%. The culture filtrates were assayed for the α-galactosidase activity, expressed as units per litre.

α-Galactosidase determinationThe α-galactosidase activity was assayed (14) using 0.003 M p-nitrophenyl-α-D-galactopyranoside as substrate in a citrate-phosphate buffer, pH 5.5. The reaction mixture was incubated at 50°C for 15 min; the reaction was stopped by the addition of 0.1 M sodium carbonate. One unit (U) of α-galactosidase activity was defined as the amount of enzyme which liberates 1 μmol of p-nitrophenol per min under the described conditions.


For the scanning electron microscopy (SEM) observations the samples of H. lutea free and entrapped cells were fixed for 2 h with 2% (w/v) glutaraldehyde (7) at room temperature. After washing with distilled water samples were dehydrated in ethanol series ranging from 30 to 100% (v/v). After air-drying, specimens were coated with 120-130 Å gold in argon atmosphere using an edwards apparatus (model S 150A). the SEM observations were made on a Philips SEM 515 at 20 kV accelerating voltage with a 5-6 nm electron beam.

Results and DiscussionCrystal morphology of the hybridsthe X-ray diffraction patterns of hybrids containing sepharose and lactic acid are shown in Fig. 1. The results from the XRD-analysis showed that all the studied hybrids were amorphous. it was observed that the intensity of diffraction peaks decreased with the increase of organic component content according to Yano (32).

Fig. 1. XRD patterns of hybrids containing sepharose and lactic acid

the Ft-iR spectra (Fig. 2) of synthesized hybrids show characteristic peaks at around 1080 cm-1, 790 cm-1 and 460 cm-1 attributed to Sio2 network. They are assigned to νas, νs and δ of Si-O-Si vibrations, but at the same time the band at 1080 cm-1 can be related to the presence of Si-o-c, c-o-c and Si-c bonds (29). the presence of bands at 950 cm-1 is attributed to Si-oh groups (23). the characteristic bands at around 3460 cm-1 and at 1630 cm-1 assigned to H-O-H vibration can also be detected. At the same time the bands around 3460 cm-1 can be associated with ν (OH) ring stretching vibrations. Absence of bands in the region between 645 cm-1 and 720 cm-1 indicate that the used silica precursor is fully hydrolyzed.

Fig. 2. Ft-iR spectra of hybrids containing sepharose and lactic acid

Surface morphology of the hybridsThe results of BET analysis proved that the pore diameter size is about 2 nm, and the specific surface area is in the range from 156 to 400 m2/g depending on the hybrid chemical composition (Fig. 3).

0

50

100

150

200

250

300

350

Surf

ace

area

, m2 /g

TEOS+20 wt. %Sepharose

TEOS+40 wt. %Sepharose

TEOS+20 wt. %Lactic acid

TEOS+40 wt. %Lactic acid

Hybrid materials

Fig. 3. Bet analysis of hybrids containing sepharose and lactic acid

Nanostructure with well-defined nanounits and their aggregates, formed by self-organizing processes, was observed by AFM studies. The size of nanoparticles were from 10 to 18 nm and the dimensions of their self-assembled aggregates were about 34-55 nm (Fig. 4). In the same figure the height distribution profiles of surfaces roughness were shown. The histograms of the surface height distribution profiles, obtained from AFM topography images, show that all of the hybrids had surface with irregularities of quite small height. the sample synthesized with 20% sepharose (Fig. 4a) had a quite homogeneous and much more flat surface with RMS roughness value of 2.9 nm. The sample synthesized with 40% sepharose (Fig. 4b) had a still homogeneous and flat surface with a bit higher RMS roughness of 3.6 nm. In the studied


Fig. 4. AFM images (three and two dimensional) and height distribution profi le of hybrids containing: (a) 20 wt.% sepharose; (b) 40 wt.% sepharose; (c) 20 wt.% lactic acid; (d) 40 wt.% lactic acid


samples with 20% lactic acid the maximum profile peak heights determined from the histograms of samples is 16.3 nm (Fig. 4c). The structure becomes heterogeneous. Voids and aggregates are observed in sample with 40% lactic acid. The highest surface roughness and heights, corresponding to the separated aggregates, are found only for the sample with 40% sepharose (Fig. 4d).

Bioreactor performance for α-galactosidase biosynthesis by the immobilized fungusα-Galactosidase yield by H. lutea mycelium immobilized in both nanomatrices (teoS + lactic acid and teoS + sepharose) and free cells (as a control) during semicontinuous cultivation in an air-lift laboratory bioreactor was shown in Fig. 5. The enzyme titre rapidly increased during the first three or four reincubations, which may have been caused by the accelerated growth of the cells entrapped in the nanomaterials. The maximal level of enzyme activity (1050-1130 U/l) that was reached in the third to fourth cycles when using teoS + lactic acid- immobilized cells was higher than that obtained in the experiments with TEOS + sepharose- entrapped mycelium where the maximal enzyme titre (660-770 U/l) did not exceeded the α-galactosidase produced by free cells (750 U/l or 100%) after 120 h batch cultivation in an air-lift bioreactor. The half-life time was 5 runs or 30 days (the duration of one batch was 144 h) in both nanomatrices, but the use of teoS + lactic acid resulted in 140-150% intensified enzyme productivity as compared with teoS + sepharose- entrapped mycelium (102%) and free cells. The half-life is defined as the number of cycles in which half of the maximal enzyme production was reached during the semicontinuous experiments.

Fig. 5. α-Galactosidase yield by H. lutea cells immobilized in teoS + lactic acid and teoS + sepharose as compared to the free mycelium during semicontinuous cultivation in air-lift laboratory bioreactor

According to Aleksieva et al. (6) the maximal α-galactosidase yield using the teoS + algal polysaccharide- immobilized fungus H. lutea was 1200 U/l or 160% as compared to the control and half-life of enzyme biosynthesis was 7 batches (42 days). Fig. 5 demonstrates also a rapid decrease of the enzyme value (490 U/l or 65%) in the first reincubation with

free mycelium. After three runs the α-galactosidase level was 180 U/l or 24%. these results are in agreement with the rapid loss of the enzyme titre by free H. lutea cells during repeated batch shake flask cultivation (28).

A

B Fig. 6. Scanning electron micrographs of free H. lutea cells after three reincubation. (A) deformed wrinkled hyphae; damage of cell division and lysis (B) fragmentation of the filaments and formation of single and chain of lemon-shaped spores. Bars= 10 μm

Scanning electron microscopy observations showed the growth of the fungus Humicola lutea in free (Fig. 6) and immobilized form (Fig. 7 and Fig. 8). electron micrographs of free cell culture (Fig. 6A, Fig. 6B) after three runs in the air-lift bioreactor demonstrated lysed hyphae and spore formation which is in accordance with significant decreasing of the enzyme titre (Fig. 5). in contrast, the immobilization of the fungal cells in nanomatrice containing teoS + lactic acid (Fig. 7A) resulted in development of long vegetable hyphae which grew in the pore structure of the carrier (Fig. 7B). After three or four batches in the bioreactor when the α-galactosidase activity reached its maximum (Fig. 5), a more condensed mycelial mass (Fig. 7C, Fig. 7D) was observed. Fig. 8A demonstrates hybrid nanomaterial prepared by teoS and sepharose including the fungal spores after 144 h of cultivation. Viable and branched filaments (Fig. 8B) were observed in the same matrix in the fourth reincubation, when maximal enzyme value was registered. A similar relationship between the fungal development and α-galactosidase yield was also established when humicola lutea cells were immobilized


A

C

B

D Fig. 7. Scanning electron micrographs of teoS + lactic acid-immobilized H. lutea. (A) matrix with entrapped H. lutea spores after 144 h of cultivation. Bar= 100 μm (B) the fungal development in the matrice after the first run (C) a dense mycelial network after three cycles when maximal enzyme level was reached (D) mycelial growth after four reincubations. Bars= 10 μm

Fig. 8. Scanning electron micrographs of teoS + sepharose-immobilized H. lutea. (A) matrix with entrapped H. lutea spores after 144 h of cultivation. Bar= 100 μm (B) thin branched hyphae filing the pores of the matrice after four batches. Bar= 10 μm

A B


in hybrid nanomatrice containing TEOS and polyvinylalcohol (PVA) mixed with polyethyleneglycol (PEG) (28).

the results presented in Fig. 5, Fig 7 and Fig 8 clearly demonstrated the advantage of the sol-gel-immobilized over the free cells.

Conclusionsinorganic-organic silica hybrids containing lactic acid and sepharose as an organic component were obtained using the sol-gel method. The FT-IR spectra of investigated hybrids displayed that interactions between the organic and inorganic parts are only by Van der Waals, hydrogen or electrostatic forces. the presence of a heterogeneous structure with well-defined nano units is clearly observed with the help of AFM. The RMS analysis shows that with the increase of organic compounds content the surface of hybrids becomes rougher. the nanomatrices play the role of a barrier which protects the cells against premature aging. For that reason the immobilized mycelia preserve their viability and synthetic capability for a long time (five or six batches) while the free cells lyze quickly and their enzyme activity decreases already after the first two or three runs, as it was previously discussed by Aleksieva et al. (4).

Acknowledgements This work was supported by Grant NT 2-01 and NT 2-02 of the National Fund for Scientific Research, Republic of Bulgaria.

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IMMOBILIZATION IN NANOMATRICES OF HUMICOLA · PDF filethe sol-gel synthesis, ... applications, e.g., for the hydrolysis of raffinose in beet sugar ... condensation of monomers and