Biological sciences / 3. Mycology and phycology

Nekleva Y.S., Krasinko V.O.

National University of Food Technologies, Ukraine

Syrchin S.O.

DK Zabolotny Institute of Microbiology and Virology, National Academy of Science of Ukraine, Ukraine

The increasing of xylanase and endoglucanase activity in Fennellia flavipes 2608 micromycete grown on the pretreated substrate

Introduction

The development of effective technology of enzymatic hydrolysis of plant biomass with subsequent processing of mono- and disaccharides (glucose, xylose and cellobiose) into industrially valuable products (biogas, bioethanol, organic acids) might become one of the promising ways of plant biomass processing [1].

In natural lignocellulosic materials cellulose fibers are encapsulated with hemicelluloses microfibrils and lignin. Pretreatment of lignocellulosic materials allows to solubilize lignin and to make cellulose more accessible to enzymes. The effectiveness of microwave radiation used for the pretreatment of rice straw and improving of cellulases and xylanases yield was previously shown [2].

Cellulase complex is a system of enzymes whichact on the cellulosic substratein a complex way and exhibit the synergistic effects. The complex consist of 1,4-β-D-glucanglucanhydrolase orendoglucanase (EC 3.2.1.4),1,4-β-D-glucancellobiohydrolase (exoglucanase or cellobiohydrolase EC 3.2.1.91), β-glucosidase (β-D-glucosideglucohydrolase EC 3.2.1.21) [3].

Cellulases have a great biotechnological potential and accounts for about 30% of world enzymes production.

Xylanases (EC 3.2.1.8 ) are enzymes which decompose a linear polysaccharide β -1,4-xylan to xylose, thus destroying hemicellulose - a major component of plant cell walls.

Xylanases are used in production of animal feed. Treatment of grain feed silageusing xylanases improves the nutritional value and digestion in ruminants. It has been proven that the use of feed enzymes systems based on xylanase increases the digestibility of nutrients and reduces quality variability of the key grain ingredients [4].

 Endoglucanase is able to non-selectively hydrolyse glycosidic β-1,4 bonds in cellulose, lichenin and β-glucans, which are distant from the ends of the polymer chain to form a polymer chain fragments and cello-oligosaccharides. This enzyme first attacks the cellulose molecule.

Endoglucanase is also widely used in the textile industry. Using the biotreatment high-quality processing of products based on cotton, linen and viscose fiber is achieved [5].

Thus, the search for the effective producers of cellulolytic’s complex enzymes, including xylanases, and developing of effective technological methods of their preparation is an important task of modern biotechnology.

Materials and methods

The object of study is Fennellia flavipes 2608, which is the museum culture of the physiology and taxonomy department of  DK Zabolotny Institute of Microbiology and Virology. Mushroom submerged cultivation was carried out at temperature of 28°C during the 4 days on the Capek's medium, which contained pretreated straw in concentration (g/L):

·                   Straw - 5,0;

·                   NaNO3 – 2,0;

·                   KH2PO4  - 1,0;

·                   MgSO4 · 7H2O – 0,5;

·                   FeSO4 · 7H2O – 0,01;

·                   KCl – 0,5;

​​8 different combinations of pretreatment with chemical (NaOH - 2% and 5%) and physical (microwave irradiation power - 450 W and 900 W, irradiation time – 5min and 10 min.) methods were used (Table 1). To determine the activity cultural fluid was sampled once a day.

 

Table 1

Options of straw pretreatment

Option #

Concentration of NaOH, %

Irradiation power, W

Irradiation time, min

1

2

450

5

2

2

900

5

3

2

450

10

4

2

900

10

5

5

450

5

6

5

900

5

7

5

450

10

8

5

900

10

 

The amount of enzyme that hydrolyzes xylose to form 1 µM of reducing sugars in 1 min. at 50°C and pH 4.6 (standard conditions) was taken as the xylanase activity unit [6].

Measuring of endoglucanase activity can be performed either viscometrically orby determination of CMC activity using photoelectric colorimeter. Methodof determining of CMC activity is more convenient because it requires less measuring time, is more accurate and sensitive.

The amount of glucose in µM, which was formed in the reaction under standard conditions for 30 min at 50°C and pH 4.6 was taken as the activity unit [7].

 

Results and discussion

The xylanase activity was synchronous in almost all variants of the experiment except option without pretreatment. Peak of xylanase activity in pretreated straw fell on the first day of cultivation and was 21.2 U / ml under followingpretreatment options: concentration of  NaOH - 5%, irradiation power-900 W, irradiation time - 10 min, and  concentration of NaOH - 5%,  irradiation power - 450 W,  irradiation time -10 min. In the version without pretreatment highest peak of activity (18.5 U / ml) was observed on the third day (Figure 1).

 

 

Figure 1.Xylanase activity of F. flavipes 2608 grown on the pretreated substrate

 

During the study of endoglucanase activity an option without pretreatment showed quite good results on the third day (Figure 2), but pretreatment option number 6 (concentration of sodium hydroxide - 5%, irradiation power - 900 W, irradiation time - 5 minutes) had the best activity values on the fourth day of the experiment.

Figure 2. CMC activity of F. flavipes 2608 grown on the pretreated substrate

 

Conclusions

Thus, pretreatment of the substrate allows not only to increase the xylanasesyield  by 53% and endoglucanaseyield by 55%, but also to decrease significantly the time of primary producers cultivation during the xylanases obtaining, which is economically efficient. Nutrient medium with pretreated straw is promising in future studies as they provide improved enzyme activity and, in some cases, reduce the duration of cultivation.

 

Literature:

1.     Dashtban, М. Fungal Bioconversion of Lignocellulosic Residues; Opportunities & Perspectives / M.Dashtban, H.Schraft, W.Sin // International Journal of Biological Sciences. - 2009. - V.5. - P.578-595.

2.   Singh A, Tuteja S, Singh N, Bishnoi NR. Enhanced saccharification of rice straw and hull by microwave-alkali pretreatment and lignocellulolytic enzyme production.
Bioresour Technol. 2011 Vol.102 N2:P.1773-1782

3.   Kumar D., Murthy G. S. Stochastic molecular model of enzymatic hydrolysis of cellulose for ethanol production// Biotechnology for Biofuels 2013. – V.6. –N 63, . – 20 p.

4.   Гришутин С. Г. Свойства ксиланаз, β-глюканази ксилоглюканаз Aspergillus japonicus:дис…кандидат ихим. Наук/ Гришутин Сергей. – М., 2004р. – 177с.

5.   Чешкова А.В. Использование биопроцессов при отделке тканей из смеси хлопка и химических волокон // Химические волокна. – 2007. – № 4. – 52-54 с.

6.   ГОСТ 31488-2012. Препараты ферментные. Методы определения ферментативной активности ксиланазы.

7.   Zhang Y.H.P., et al. Cellulase Assays // in: Mielenz (ed.), Biofuels: Methods and Protocols, Methods in Molecular Biology, 2009. – V. 581. – Р. 213 – 231