Patent WO2014000748A1 - Novel carriers based on agriculture wastes...

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Novel carriers based on agriculture wastes for

immobilization of biomolecules
WO 2014000748 A1
ABSTRACT
This patent involves treatment of agriculture waste, sawdust, which represents
a major environmental problem worldwide. Sawdust presents a hazard in
manufacturing industries, especially in terms of its flammability as well as they
may collect in piles and add harmful leachates into local water systems,
creating an environmental hazard. The sawdust was treated to immobilize
covalently proteins and especially enzymes. The presence of the new
functionality has been proved by ATR-FTIR. The degree of treatment could be
easily controlled. Four industrial enzymes, invertase, phytase, glucose oxidase and lipase, were successfully immobilized. The immobilized enzyme and for
example invertase was used for 14 cycles with retention of 100D of its relative activity. The treated sawdust could solve an environmental problem, as well as
producing an excellent solution for production of cheap carriers. The immobilized enzymes could be reused for tens of times, which will reduce the cost of the
enzymes and their products.

DESCRIPTION (OCR text may contain errors)

CLAIMS (OCR text may contain errors)

"Novel Carriers based on Agriculture Wastes for Immobilization of

Claims:

Biomolecules"

1- Agriculture wastes based on cellulosic materials were treated with

Technical Field:
Material Sciences (Biopolymers) and Biochemistry (Proteins). Background Art:
Cellulose-waste represents a major environmental problem worldwide. For
example, sawdust as shown in Figure 1, presents a hazard in manufacturing
industries, especially in terms of its flammability. Sawdust may collect in piles
and add harmful leachates into local water systems, creating an environmental

acid-base followed by secondary treatment with oxidizing agent to produce

free active functionalities for immobilization of biomolecules.
2- The agriculture waste in claim 1 was cellulose waste
3- The treatment of HA-SD in claim 2 was using thorough washing with
Distilled water at 50 C followed by acid-base treatment using H2S04 and
NaOH to produce treated cellulose waste.

hazard. This has placed small sawyers and environmental agencies in a

4- The concentration of the acid-base in claim 3 was from 0.1-2 M and

deadlock. [1]

specially 1 M.

Previous art

5- The soaking time of the treated cellulose waste in claim 3 was at 25- 95

Sawdust as the main by-product of sawmills, unless burned in a sawdust burner

C and especially at 80 C.

or used to make heat for other milling operations. Annually, huge amounts are

6- The treated sawdust in claim 4 was further treated with an oxidizing

produced and only small fraction could be used in manufacturing of

agent, periodate (PI) at concentration of 5-100% mole:mole treated

particleboard and the majority is considered as waste. [1]

cellulose waste.

Disclosure of Invention:

7- The secondary treated sawdust with periodate in claim 6, was used to

Immobilization of biomolecules has been widely spread due to their biological

and biomedical applications. For examples, biomolecules such as proteins,
peptides, drugs, saccharides, lipids, nucleic acids and nucleotides. However,
the immobilization process is limited to the cost and efficacy of the carriers. In
this patent, we immobilized four enzymes as a model of other immobilized
biomolecules. We chose enzymes as the enzyme market in 2005 was around
2.65 billion dollars, with an expected annual growth of more than 9%. [2]

immobilize biomolecules such as proteins, peptides, drugs, saccharides,

lipids, nucleic acids and nucleotides.
8- The biomolecules used in claim 8 were proteins and especially enzymes.
9- The novel treated cellulose waste in claim 7 & 8 could bind to
biomolecules through ionic interaction and/or crosslinking via covalent
bonding.

However, expensive enzymes are not favored to be used in industries in the

Free State as they are difficult to be separated from the products (Fig. 2a) and
consequently are lost after the first use. They were alternatively immobilized on solid supports (Fig. 2 b) so that they can
be easily separated from the products by simple filtration or using a fluidized magnetized bed reactor system. [3] In
immobilization technology, the carriers used are very expensive such as Eupergit C and agraroses (Elnashar et al.,
2008).[3] Accordingly, there is still a need to cheaper and robust carriers.
Novelty
Sawdust has been used for this goal after being modified to incorporate new functionality to bind biomolecules via physical
or covalent bonds. We have chosen for this goal four industrial enzymes to be immobilized and the results were

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outstanding.
The preparation of the modified carriers based on agriculture wastes will not only solve an environmental problem, but also
will be an excellent solution for production of cheap carriers. The estimated lab cost price was approximately $ 1 per lOg
compared to the commercially available matrices such as EupergitCM, which retails at approximately $ 128 per lOg from
Sigma- Aldrich in 2011.
The novelty of this work was treatment of sawdust with acid/base to get rid of some pigments and phenolic compounds
such as lignin. Then treating the residual with periodate to create free aldehyde groups. Finally, immobilizing the enzymes
to the grafted sawdust via the free aldehyde groups (covalent bonds). As a result of this treatment:
1- We partially purified the sawdust from some pigments and phenolic compounds.
2- New functionality has been incorporated to the treated swadust such as the aldehyde groups, which was proved by the
Infrared spectroscopy (IR). The new aldehydic's carbonyl group were formed from the reaction of the periodate with the
vicinal hydroxyl groups has been proved by the Infra Red (IR) technique at a wavelength of 1600 - 1647 cm"1.
3- The degree of formation of free aldehyde groups could be easily controlled by varying the periodate molar ratios from
20 - 100%.
4- Four industrial enzymes, invertase, phytase, glucose oxidase and lipase have been successfully immobilized covalently
to the grafted sawdust.
5- As a result of the strong covalent bond between the enzymes and the gels, the enzymes do not leak and the
immobilized enzymes could be reused on the industrial scale for tens of times, which reduce both the cost of the enzyme
and the enzymatic products. For example, immobilized invertase was reused for 14 times without any loss of activity.
Methodology & Results
The methodology of modifying the raw sawdust for immobilization of enzymes was as follow:
1- Untreated sawdust (HA-SD)
Sawdust supplied by a local wood processing factory, was washed with tap water to remove impurity, and then dried
overnight at 50C. Drying at 50C was chosen because severe drying conditions (high drying temperatures) might cause a
decrease of the number of cellulose-based OH-groups with lower wood moisture content.
2- Chemical treatments of sawdust (HA-SD) with acid and base to produce (HA-SD-treated)
To modify the sportive characteristics of sawdust, the sorbents were treated with 1M H2S04 followed by NaOH with the
same molarity as follows:
Hundred grams of sawdust (SD) was suspended in 1L of IN H2S04 in ratio of 1:10 (g/ml) and the reaction suspension was
stirred for 1 h at 80C. After cooling down at ambient temperature, the reaction suspension was collected by filtration
through cloths. The product was washed with tape water to remove the excess of H2SO4. Subsequently, sorbent was in
re-suspended again in 1L of IN NaOH and the reaction suspension was stirred for 1 h at 80C. After cooling, the reaction
suspension (HA- SD) was filtrated through cloths and then washed with distilled water to remove the traces of NaOH.
3- Chemical modification of treated sawdust (HA-SD-%)
Acid/base treated sawdust (HA-SD) was further treated with sodium periodate (PI) at different mol ratios (1:1, 1:08, 1:06,
1:04, 1:02), HA-SD : PI, to give a percentage of conversion of free vicinal hydroxyl groups to aldehyde groups starting from
20% and ended with 100% (HA-SD-%). 4- Characterization using ATR-FTIR spectroscopy
To prove the treatment of raw sawdust (HA-SD) by the acid base to produce (HA-SD-treated) then followed by the second
treatment to produce free aldehyde groups from (HA-SD-20) to (HA-SD-100), attenuated total reflectance fourier transform
infra red (ATR-FTIR) was used.
As shown in Figure 3, after the first treatement with the acid-base, we get rid of some of the pigments and phenolic
compounds such as legnins, which could be shown from the ATR-IR bands at the range of 1200-1500 nm. As a result,
there is also an increase in the strength of the hydroxyl groups band after the acid-base treatment as shown in Figure 3 at
the range of 3100-3600 nm.
After the second treatment with periodate, as shown in Figure 4, the free OH groups of the treated sawdust (HA-SDtreated), have been used for production of the free aldehyde groups as clearly shown at 3100-3600 nm, where the
strength of the hydroxyl group bands decreases by increasing the percentage of the second treatment with periodate.
Therefore, the secondary treated HA-SD-% showed more aldehyde groups at 1600 nm than that of the firstly trearted
sawdust, HA-SD-treated.
5- Immobilization of enzymes
Four industrial enzymes, invertase, phytase, glucose oxidase and lipase, were immobilized onto secondary treated
sawdust from 20-100% periodate (mole/mole) and the results were shown in Figures 5-8. The results revealed that the
enzymes' loading capacity varies by changing the concentration of the periodate. That could be regarded as the more
periodate used, the more free aldehyde groups were formed and more enzymes could be possibly immobilized. However,

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most Figures showed that the optimum enzyme loading concentration is at 60% periodate, after- which the enzyme
activities started to decline. That could be regarded to enzymes multiple point attachment or saturation of the carrier's
surface with the enzymes. As an example of immobilization of enzymes using the covalent bonding, invertase enzyme was
immobilized onto 60% periodate-treated sawdust and being used for 14 cycles without loosing any of its activity as shown
in Figure 9. Figures legends:
Fig.l. Untreated sawdust (HA-SD).
Fig. 2. Schematic diagram of free and immobilized enzyme reactions, (a), Reaction of free enzyme with substrate and
formation of product, which has to be separated via dialysis; (b), Reaction of immobilized enzyme with substrate and
formation of product, which can be separated via filtration or using a fluidized magnetized bed reactor system.
Fig. 3/9. ATR-FTIR of untreated (HA-SD) and acid/base treated sawdust (HA-SD- treated).
Fig. 4/9. ATR-FTIR of untreated (HA-SD), acid/base treated sawdust (HA-SD- treated) and secondary treated sawdust
HA-SD-%.
Fig. 5/9. Immobilization of invertase onto secondary treated sawdust using 20-100% periodate (HA-SD-%).
Fig. 6/9. Immobilization of phytase onto secondary treated sawdust using 20-100% periodate (HA-SD-%).
Fig. 7/9. Immobilization of glucose-oxidase onto secondary treated sawdust using 20- 100% periodate (HA-SD-%).
Fig. 8/9. Immobilization of lipase onto secondary treated sawdust using 20-100% periodate (HA-SD-%).
Fig. 9/9. Reusability of immobilized invertase onto secondary treated sawdust using 60% periodate.
References
1- http://en.wikipedia.org/wiki/Sawdust
2- Ayala M. Torres E. (2004). Enzymatic activation of alkanes: constraints and prospective. Applied Catalysis A: General.
272, 1.
3- Elnashar, M.M. (2010): Review article: "Immobilized Molecules Using Biomaterials and Nanobiotechnology". Journal of
Biomaterials and Nanobiotechnology 1, 61-76.
4- Elnashar, M.M., Yassin, A.M. and Kahil, T. (2008): Novel thermally and mechanically stable hydrogel for enzyme
immobilization of penicillin G acylase via covalent technique. Journal of Applied Polymer Sciences 109, 4105-4111.

NON-PATENT CITATIONS
Reference

1
2

APPLIED CATALYSIS A: GENERAL vol. 272, 2004, page 1

*

DOAA A. R. MAHMOUD: "Immobilization of invertase by a new economical method using wood sawdust waste", AUSTRALIAN JOURNAL OF
BASIC AND APPLIED SCIENCES, vol. 1, no. 4, 1 January 2007 (2007-01-01) , pages 364-372, XP055054886,

ELNASHAR, M.M.: 'Immobilized Molecules Using Biomaterials and Nanobiotechnology' JOURNAL OF BIOMATERIALS AND
NANOBIOTECHNOLOGY vol. 1, 2010, pages 61 - 76

ELNASHAR, M.M.; YASSIN, A.M.; KAHIL, T.: 'Novel thermally and mechanically stable hydrogel for enzyme immobilization of penicillin G acylase
via covalent technique' JOURNAL OF APPLIED POLYMER SCIENCES vol. 109, 2008, pages 4105 - 4111

KOTWAL S M ET AL: "Immobilized invertase", BIOTECHNOLOGY ADVANCES, ELSEVIER PUBLISHING, BARKING, GB, vol. 27, no. 4, 1 July
2009 (2009-07-01), pages 311-322, XP026127880, ISSN: 0734-9750, DOI: 10.1016/J.BIOTECHADV.2009.01.009 [retrieved on 2009-01-31]

P. GEMEINER ET AL: "Aldehydic derivatives of bead cellulose--relationships between matrix structure and function in immobilization of enzymes
catalyzing hydrolysis of high molecular substrates", BIOTECHNOLOGY AND BIOENGINEERING, vol. XXIV, 1 January 1982 (1982-01-01), pages
2573-2582, XP055054884,

VARAVINIT S ET AL: "Covalent immobilization of a glucoamylase to bagasse dialdehyde cellulose", WORLD JOURNAL OF MICROBIOLOGY
AND BIOTECHNOLOGY, vol. 17, no. 7, October 2001 (2001-10), pages 721-725, XP002693056, ISSN: 0959-3993

* Cited by examiner

CLASSIFICATIONS
International Classification

C12N11/12

Cooperative Classification

C12N11/12

LEGAL EVENTS
Date

Code

Feb 26, 2014

121

Event

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