Determination of in-vitro Optimum Conditions of Xylanase Enzyme Activity Produced by Orpinomyces sp. and Neocallimastix sp.

Halit Yucel¹ and Ferudun Kocer^2*
*Correspondence: Ferudun Kocer, Department of Research and Development, (USKIM), Kahramanmaras Sutcu Imam University, Kahramanmara, Turkey, Tel: 5427137102, Email:

Keywords

Orpinomyces sp •  Neocallimastix sp •  Temperature •  Day • pH

Introduction

Lignocellulotic enzymes, which are members of the Carbohydrate Active Enzyme (CAZy) family, are organic compounds synthesized by some microorganisms [1]. Although CAZy enzymes are the most widely used enzyme group in different biotechnological fields, especially in biofuel production, it has been observed that the cost of optimization processes can be high in some cases [2-4].

In order to produce these enzymes by other microorganisms at a cheaper cost, it has led to the research of different living groups and the examination of the enzyme activities of these organisms, and the presence of AGFs among these organisms has increased the research on these microorganisms [5-7].

Anaerobic Gut Fungi (AGF) are eukaryotic microorganisms that spread symbiotically in the rumen and have lignocellulotic enzymes that provide hydrolysis of the plant cell Wall [8,9]. These microorganisms and other fungi have maximal CAZy activity [10].

Xylanase, a member of the CAZy family, is an important enzyme synthesized by these microorganisms, and it is a new enzyme that is isolated from some AGFs and purified for use in biotechnological fields as a result of cloning, and potentially used in different fields such as bread making [11,12]. At the same time, the use of xylanases, which are thermostable, in paper, feed, food and various bio-converting systems increases the demand for purification from different microorganisms due to the increasing interest in these enzymes [13].    

As a result of the comparison of xylanase enzyme activity, which is a member of the CAZy system of AGFs, Orpinomyces sp. and Neocallimastix sp. It has been observed that this enzyme activity is high in species [14]. Therefore, the potential use of xylanase enzyme in new technological areas and the discovery of this enzyme from AGFs Orpinomyces sp. and Neocallimastix sp., it was aimed to determine the optimum conditions for xylanase activity in vitro.

Materials and Methods

Anaerobic Gut Fungi (AGF) (Orpinomyces sp. and Neocallimastix sp.) used in the study were obtained from Kahramanmaraş Sütçü İmam University Biotechnology Laboratory stock culture collection. Orpin anaerobic nutrient medium was used for the development of AGFs in vitro.

Determination of xylanase enzyme activity

Extracellular:For the enzyme study, the samples were centrifuged at 1200 g for 15 minutes and the upper liquid was separated for extracellular (supernatant) enzyme activity and transferred to sterile eppendorf tubes.

Intracellular:In order to determine the intracellular enzyme activity, the sample was centrifuged at 1200 g for 15 minutes and the pellet remaining at the bottom was taken into a separate epondorphan for intracellular enzyme activity, washed twice with distilled water, and then used with liquid nitrogen to break the cell wall. Afterwards, the obtained samples were taken into 25 mM potassium phosphate buffer solution and centrifuged at 10000 g for 3 minutes and the supernatant was taken [15]. Enzyme activity of the obtained samples was done according to Miller method. The slope standard curve from xylose was used to calculate xylanase activity. One unit of xylanase activity (IU) was defined as the amount of enzyme capable of producing 1 µmole of xylose in 1 minute under reaction conditions at 40°C.

Determination of in-vitro optimum parameters

Orpinomyces sp. and Neocallimastix sp. the effects of different time intervals, different temperatures and pH on the raw enzyme were examined. 1-7 days as different time intervals, temperature values between 30-80°C with 5°C increments; For pH, it was adjusted in 0.5 unit increments in the range of 3-9. The activity of xylanase at different time, temperature and pH changes was determined.

Statistical calculation

The data obtained in the study were analyzed statistically with the help of SPSS-23 program. Descriptive statistical analysis of the data and Pearson correlation statistical test were applied. p<0.05 and p<0.01 were considered statistically significant.

Results and Discussion

In this study, Orpinomyces sp. and Neocallimastix sp. descriptive statistical data according to their extracellular and intracellular status at different time, pH and temperature ranges, and the descriptive statistics data were associated with the person correlation coefficients and the enzyme levels were determined at optimum time, pH and temperature (Table 1).

Orpinomyces sp. and Neocallimastix sp.,depending on time, the lowest value TA=1.20 μmol/min/ml, the highest value TA=58.70 μmol/min/ml and the lowest value SA=22.10 μmol/min/ mg, the highest value was found to be SA=505.40 μmol/min/mg. When examined in terms of pH levels, the lowest value TA=0 μmol/min/ml, the highest value TA=49.10 μmol/min/ml and the lowest value SA=0 μmol/min/mg, the highest value SA=398.70 μmol It was found as /min/mg. In the temperature ranges, the lowest value TA=2.5 μmol/min/ml, the highest value TA=57.60 μmol/min/ml and the lowest value SA=44.10 μmol/min/mg, the highest value SA=501, It was found to be 2 μmol/min/mg. According to the analysis of variance findings, TA=267.97 and SA=23597.84 at different times, pH levels TA=182.29 and SA=7552.92, and at temperature levels TA=232.54 and SA=16941.09.

Time-dependent findings of xylanase enzyme activity

Orpinomyces sp. and Neocallimastix sp. on different days, the changes in xylanase enzyme activity extracellular and intracellular in terms of TA and SA values were investigated. In the Orpinomyces sp. and Neocallimastix sp. on different days, the changes in xylanase enzyme activity extracellular and intracellular in terms of TA and SA values were investigated. In the study, time dependent xylanase enzyme activity of Orpinomyces sp. In the study, extracellular TA=47.5 µmol/min/ml intracellular TA=36.9 µmol/min/ml on the 5th day, while extracellular SA=201.4 SA= It was found to be 204.7 μmol/min/mg. Neocallimastix sp. For the 5th day, extracellular TA=58.7 μmol/min/ml intracellular TA=42.7 μmol/min/ml, while extracellular SA=505.4 SA= 502.4 μmol/min/mg (Table 2).

Enzyme production activity of Neocallimastix sp was investigated in different media. In the study, it has been reported that there are differences in enzyme activity according to the medium, and it changes depending on the time [16]. The effect on xylanase production by Aspergillus niger at different temperatures was investigated. In the study, it was stated that 92 hours (approximately 4 days) were required to achieve the optimum temperature of 28°C and maximum xylanase activity [17]. In our study, the optimum xylanase enzyme activity was reached on the 5^th day. This explains the genus-specific differences for xylanase activity. The optimum enzyme activity level was determined on the 5^th day in our study. The xylanase enzyme activity was determined as 290 U/ml under optimum conditions [18]. In our study, the highest value in terms of enzyme activity was Neocallimastix sp. for 502.4 μmol/min/mg in the cell. Orpinomyces sp. the highest value for intracellular SA=204.7 μmol/min/mg was determined.

Findings of xylanase enzyme activity at different pH levels

Extracellular and intracellular optimum pH level of Orpinomyces sp. was determined to be 7. Extracellular TA=37.6 μmol/min/ml and SA=94.3 μmol/min/mg at pH=7, intracellular TA=41.2 μmol/min/ml and SA=124.3 μmol/min/ determined in mg. Neocallimastix sp. intracellular xylanase was determined at the pH level of TA=41.4 μmol/min/ml with the highest value at 6.5 pH, and SA=398.7 μmol/min/mg at a pH level of 7 (Table 3).

In this study, Orpinomyces sp. and Neocallimastix sp. were found at pH values between 6.5 and 7.5. It was determined that the enzyme activity was at the lowest values below the average pH value of 5. The pH range was determined as 6.50 in the study in which the biogas efficiency was examined using Saccharomyces cerevisiae[19]. It is thought that the use of xylanase enzyme, which will be obtained according to the environment, will be beneficial and guide in researches where the enzyme activity changes according to the cell environment.

Findings of xylanase enzyme activity at different temperatures

Orpinomyces sp. and Neocallimastix sp. at different temperatures, it was determined that the xylanase enzyme activity had different intracellular and extracellular temperature values.The highest extracellular and intracellular Total (TA) Activity value of Orpinomyces sp. was 55 μmol/min/ml at 50^oC, and the Specific (SA) Activity value was 309.1 μmol/min/mg at 50^oC. However, the highest values of TA=44.2 μmol/min/ml and SA=304.3 μmol/min/mg were obtained for intracellular xylanase enzyme at 55^oC. It was observed that the xylanase enzyme activity degraded with the increase in temperature and therefore decreased significantly (Table 4).

Neocallimastix sp.the highest extracellular Total (TA) Activity value was 57.6 μmol/min/ml at 50^oC and specific (SA) activity value was 501.2 μmol/min/mg at 50^oC. In intracellular, the highest Total (TA) Activity value was 43.1 μmol/min/ml at 50^oC and Specific (SA) Activity value was 501.2 μmol/min/mg at 50^oC (Table 4). The interactive effects of temperature (20-60^oC) and pH (2-8) were investigated for xylanase production from Aspergillus niger DFR-5. In the study, it was reported that the maximum activity of the enzyme was 4354 IU/ml at 40oC [20]. Xylanase enzyme optimization was achieved at different pH and temperatures with the mutated strain Neocallimastix patriciarum. In the study, it was reported that it provides the highest activity at pH=6 and temperature at 62^oC [21].

Orpinomyces sp. and Neocallimastix sp., it was determined that there was no significant difference between the breeds and the environment (p<0.01). However, it was statistically determined that there was a negative relationship between the breeds in terms of SA values. When different days (time) were considered, it was determined that there was a statistically significant positive correlation between TA=0.732 and SA=0.546 (p<0.01). It was determined that there was a statistically positive and significant relationship between TA=0.622 and SA= 0.520 at different pH levels. This situation differs in terms of temperature levels. It was determined that there was a statistically negative significant relationship between genders and SA=-0.354 (p<0.05). In addition, a statistically positive and significant relationship was found within the SA values. TA values were not found to be statistically significant (Table 5).

Orpinomyces sp.in study was conducted on thermostability improvement of xylanase obtained from Xylanase from Orpinomyces shows optimum activity at neutral or acidic pH. In the study, xylanase activities using different mutant species were maximum at 60oC and pH range of 5-7. It was stated that the temperature ranges of xylanase activities of different mutants changed [22-24]. The synergistic effect of pH and temperature on xylanase enzyme was investigated by using Aspergillus niger species. In the study, the optimum temperature values for the enzyme were found between 35oC and 60oC, and the optimum pH range was between 4 and 5.5 [25-27].

Conclusion

Orpinomyces sp. and Neocallimastix sp., with different applications, optimum endoxylanase and exoxylanase TA and SA values were determined. Since the concepts of pH, temperature and time are important for enzyme activity in industrial and in-vitro laboratory applications, it has been interpreted statistically by making it with both sexes. Although both sexes showed similar characteristics in the study, a statistically negative significant relationship was found (p<0.01). In industrial applications, it is important to apply the data obtained from these parameters in order to obtain the optimum product.

References

1. Bajaj P and Mahajan R. Cellulase and xylanase synergism in industrial biotechnology.  Appl Microbiol Biotechnol 103 (2019): 8711-8724.

   [Crossref][Googlescholar][Indexed]

2. Barrett K, Jensen K, Meyer AS and Frisvad JC, et al. Fungal secretome profile categorization of CAZymes by function and family corresponds to fungal phylogeny and taxonomy: Example Aspergillus and Penicillium. Sci Rep 10 (2020): 1-12.

   [Crossref][Googlescholar][Indexed]

3. Chadha BS, Kaur B, Basotra N and Tsang A, et al. Thermostable xylanases from thermophilic fungi and bacteria: current perspective. Bioresour Technol 277 (2019): 195-203.

   [Crossref][Googlescholar][Indexed]

4. Dagar SS, Kumar S, Mudgil P and Puniya AK, et al. Comparative evaluation of lignocellulolytic activities of filamentous cultures of monocentric and polycentric anaerobic fungi. Anaerobe 50 (2018): 76-79.

   [Crossref][Googlescholar][Indexed]

5. Dehhaghi M, Panahi HKS, Jouzani GS and Nallusamy S, et al. Anaerobic Rumen Fungi for Biofuel Production. In Fungi In Fuel Biotechnol  (2020): 149-175.

   [Googlescholar]

6. Farinas CS, Loyo MM, Junior AB and Tardioli PW, et al. Finding stable cellulase and xylanase: evaluation of the synergistic effect of pH and temperature. N Biotechnol 27 (2010): 810-815.

   [Crossref][Googlescholar][Indexed]

7. Flad V, Young D, Seppala S and Hooker C, et al. The Biotechnological Potential of Anaerobic Gut Fungi. Genet Mol Biol 2 (2020): 413-437.

   [Indexed]

8. Flint HJ, McPherson CA and Martin J. Expression of two xylanase genes from the rumen cellulolytic bacterium Ruminococcus flavefaciens 17 cloned in pUC13. J Gen Microbiol 137 (1991): 123-129.

   [Crossref][Googlescholar][Indexed]

9. Haitjema CH, Solomon KV, Henske JK and Theodorou MK, et al. Anaerobic gut fungi: advances in isolation, culture, and cellulolytic enzyme discovery for biofuel production.  Biotechnol Bioeng 111 (2014): 1471-1482.

   [Crossref][Googlescholar][Indexed]

10. Hanafy RA, Elshahed MS and Youssef NH. Feramyces austinii, gen. nov., sp. nov., an anaerobic gut fungus from rumen and fecal samples of wild Barbary sheep and fallow deer. Mycologia 110 (2018): 513-525.

    [Crossref][Googlescholar][Indexed]

11. Henske JK, Gilmore SP, Haitjema CH and Solomon KV, et al. Biomass‐degrading enzymes are catabolite repressed in anaerobic gut fungi.  AIChE J 64 (2018): 4263-4270.

    [Crossref][Googlescholar][Indexed]

12. Hooker CA, Lee KZ and Solomon KV. Leveraging anaerobic fungi for biotechnology.  Curr Opin Biotechnol 59 (2019): 103-110.

    [Crossref][Googlescholar][Indexed]

13. Huang Y, Zhang NJ and Zhao Z. Immobilization of mutated xylanase from Neocallimastix patriciarum in E. coli and application for kraft pulp biobleaching.  Braz J Biol 83 (2021): 243629.

    [Crossref][Googlescholar][Indexed]

14. Karaman A, Yucel H, Ekinci K and Comertpay S, et al. Comparing Cellulotic Enzyme Activities of Neocallimastix sp. in Orpin’s and Menke’s Media. Mantar Dergisi 13 (2022): 55-61.

    [Googlescholar]

15. Leggieri PA, Kerdman-Andrade C, Lankiewicz TS and Valentine MT, et al. Non-destructive quantification of anaerobic gut fungi and methanogens in co-culture reveals increased fungal growth rate and changes in metabolic flux relative to mono-culture. Microb Cell Fact 20 (2021): 1-16.

    [Crossref][Googlescholar][Indexed]

16. Malik SM, Ahanger FA, Sahay S and Jain K, et al. Industrial Applications And Production of Cold Active Xylanase: A. Int J Res Anal Rev 5 (2018): 1-10.

    [Googlescholar]

17. Miller GL. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31 (1959): 426-428.

    [Crossref][Googlescholar]

18. Orpin CG. The characterization of the rumen bacterium Eadie's oval, Magnoovum gen. nov. eadii sp. nov. Arch Microbiol 111 (1976): 155-159.

    [Crossref][Googlescholar][Indexed]

19. Pal A and Khanum F. Purification of xylanase from Aspergillus niger DFR-5: Individual and interactive effect of temperature and pH on its stability. Process Biochem 46 (2011): 879-887.

    [Crossref][Googlescholar][Indexed]

20. Ranganathan A, Smith OP, Youssef NH and Struchtemeyer CG, et al. Utilizing anaerobic fungi for two-stage sugar extraction and biofuel production from lignocellulosic biomass.  Front Microbiol 8 (2017): 635.

    [Crossref][Googlescholar][Indexed]

21. Singh B, Mal G, Gautam SK and Mukesh M, et al. Anaerobic Gut Fungi-A Biotechnological Perspective. Adv Anim Biotechnol  (2019): 31-38.

    [Crossref][Googlescholar]

22. Swift CL, Brown JL, Seppala S and O’Malley MA, et al.  Co-cultivation of the anaerobic fungus Anaeromyces robustus with Methanobacterium bryantii enhances transcription of carbohydrate active enzymes. J Ind Microbiol Biotechnol 46 (2019): 1427-1433.

    [Crossref][Googlescholar][Indexed]

23. Trevizano LM, Ventorim RZ, de Rezende ST and Junior FPS, et al. Thermostability improvement of Orpinomyces sp. xylanase by directed evolution. J Mol Catal B Enzym 81 (2012): 12-18.

    [Crossref][Googlescholar][Indexed]

24. Vallejo-Hernández LH, Elghandour MM, Greiner R and Anele UY, et al. Environmental impact of yeast and exogenous xylanase on mitigating carbon dioxide and enteric methane production in ruminants. J Clean Prod 189 (2018): 40-46.

    [Crossref][Googlescholar][Indexed]

25. Wen S, Wu G and Wu H. Biochemical characterization of a GH10 xylanase from the anaerobic rumen fungus Anaeromyces robustus and application in bread making. Biotech 11 (2021): 1-12.

    [Crossref][Googlescholar][Indexed]

26. Wilken SE, Monk JM, Leggieri PA and Lawson CE, et al. Experimentally validated reconstruction and analysis of a genome-scale metabolic model of an anaerobic Neocallimastigomycota fungus. Msystems 6 (2021): 2-21.

    [Crossref][Googlescholar][Indexed]

27. Yuan QP, Wang JD, Zhang H and Qian ZM, et al. Effect of temperature shift on production of xylanase by Aspergillus niger. Process Biochem 40 (2005): 3255-3257.

    [Crossref][Googlescholar][Indexed]