|Year : 2014 | Volume
| Issue : 2 | Page : 160-167
Improved mannanase production from Penicillium humicola and application for hydrolysis property
Mona A El-refai1, Om Kalthoum H Khattab2, Siham A Ismail Professor Assistant 1, Amal M Hashem1, Amany A Abo-Elnasr2, Shaimaa A Nour1
1 Department of Chemistry of Natural and Microbial Products, Division of Pharmaceutical and Drug Industries, National Research Centre, Cairo, Egypt
2 Department of Plant and Microbiology, Faculty of Science, Helwan University, Helwan, Egypt
|Date of Submission||07-Sep-2014|
|Date of Acceptance||14-Oct-2014|
|Date of Web Publication||18-Dec-2014|
Siham A Ismail
Department of Chemistry of Natural and Microbial Products, Division of Pharmaceutical and Drug, Industries, National Research Centre, El-Behowth St, PO Box 12311, Dokki, Cairo
Source of Support: None, Conflict of Interest: None
The aim of this research is to produce β-mannanases from a new microbial source using wastes in their nutrition medium. The enzyme produced can be used for the production of galactomanno-oligosaccharides, which are very useful in the health and medical fields.
Materials and methods
Seven fungal strains and five bacterial strains were tested for the production of β-mannanases. Enzyme activity, protein content, and biomass production were determined in all the cultures produced using standard methods. Optimization studies to maximize enzyme production from the most potent microorganisms, including culture conditions and medium compositions, were also carried out. Preliminary studies for the production of galactomanno-oligosaccharides from locust bean gum using partially purified enzymes were also carried out and followed by thin-layer chromatography techniques and Somogyi methods.
Results and conclusion
The highest mannanase activities were produced by Penicillium humicola (8.8 U/ml) and Penicillium spp. v (7.75 U/ml) in shaking cultures after 10 days using gum locust bean as a carbon source. Among 13 carbon sources examined, coffee residue and ceratonia seeds were the best carbon sources (10.3 and 8.9 U/ml, respectively) for P. humicola, whereas the best nitrogen source was a mixture of peptone, urea, and ammonium sulfate for the same microorganism. The optimum temperature and pH for enzyme reaction was 55 and 5.5°C, respectively. The enzyme was thermostable and retained 80% of its activity after 1 h at 50°C. The highest reducing sugar of 8900 μg/ml was obtained from locust bean gum hydrolytes after 28 h.
Keywords: Bacterial strains, fungal strains, galactomanno-oligosaccharides, industrial wastes, β-mannanases
|How to cite this article:|
El-refai MA, Khattab OH, Ismail SA, Hashem AM, Abo-Elnasr AA, Nour SA. Improved mannanase production from Penicillium humicola and application for hydrolysis property. Egypt Pharmaceut J 2014;13:160-7
|How to cite this URL:|
El-refai MA, Khattab OH, Ismail SA, Hashem AM, Abo-Elnasr AA, Nour SA. Improved mannanase production from Penicillium humicola and application for hydrolysis property. Egypt Pharmaceut J [serial online] 2014 [cited 2020 Apr 1];13:160-7. Available from: http://www.epj.eg.net/text.asp?2014/13/2/160/147102
| Introduction|| |
Lignocellulose is a major component of plant cell walls and is mainly composed of lignin, cellulose, and hemicellulose. Mannans and heteromannans are a part of the hemicellulose fraction in plant cell walls. The structure of hemicellulose is a key component of many types of sugar such as xylans, mannans, heteromannans, galactans, and arabinans . Mannans and heteromannans are distributed widely in hardwoods and softwoods, seeds of leguminous plants, and bean . They can be divided into four types: unsubstituted (1, 4)-linked β-d mannans, galactomannans, glucomannans, and galactoglucomannans . Mannans, whose main component is d-mannose, are important for several industries, including food, feed, and feed stocks. They can be broken down into simple sugars or oligosaccharides by a synergistic action of endomannanases (EC number 184.108.40.206, mannan endo-1, 4-β-mannosidase) and exo-acting β-mannosidase (EC number 220.127.116.11). The production of β-mannanase by microorganisms is more promising because of its low cost, high production rate, and easily controllable condition .
Various microorganisms can produce mannanases such as Aspergillus awamori , Aspergillus oryzae , Penicillium oxalicum , Trichoderma harzianum , Rhodothermus marinus , Bacillus subtilis , and Streptomyces ipomoea .
Many mannan-based carbon sources including locust bean gum, guar gum, and copra meal have been used to cultivate filamentous fungi ,,. Moreover, various inducers were used to improve mannanase production. Chaiongkarn et al.  isolated microorganisms from soil using copra meal as a carbon source for their growth.
Therefore, this study aimed to search for cheaper carbon and nitrogen sources for the production of mannanase and oligosaccharides.
| Materials and methods|| |
Locust bean gum was obtained from Sigma Chemicals (3050 Spruce St. St. Louis, MO 63103, USA) (a galactomannan from the seeds of Ceratonia siliqua, which consists of a straight-chain polymer of mannose with galactose branches on every fourth mannose). Coffee residue was bought from the local market (Giza, Egypt). All other chemicals used were of analytical grade.
In this study, seven fungal strains, namely, Aspergillus niger, Aspergillus repens, Penicillium corylophilum, Penicillium humicola, Penicillium spp., Penicillium spp. v, and Trichoderma viride, and five bacterial strains, namely, Bacillus circulans, Bacillus licheniformis, Bacillus megaterium, B. subtilis, and Geobacillus stearothermophilus were obtained from the culture collection of the National Research Center, Dokki, Cairo, Egypt. All the fungal strains were maintained on potato dextrose agar and incubated at 30°C for 7 days before storage at 4°C with monthly subculturing. All the bacterial strains were maintained on nutrient agar for 48 h at 37°C and then stored at 4°C with monthly subculturing.
0The basal medium for inoculum and enzyme production included: peptone (2 g/l); ammonium sulfate (1.5 g/l); urea (0.3 g/l); MgSO 4·0.7H 2 O (0.5 g/l); K 2 HPO 4 (10 g/l); and locust bean gum (10 g/l) . The pH of the medium was adjusted at 5.3 before autoclaving. Each 250 ml Erlenmeyer flask contained 50 ml of the medium and was autoclaved for 15 min at 121°C. An inoculum culture was obtained by culturing the fungal strains in the above medium at 30°C for 48 h with shaking at 150 rpm. The culture flasks were inoculated by 10% of the inoculum and incubated at 30°C in a shaking incubator at 150 rpm for different periods. Thereafter, the fermented medium was centrifuged to separate the mycelium from the culture filtrate and the filtrate was used as the crude enzyme solution .
The basal medium for inoculum and enzyme production included beef extract (0.5 w/v %); peptone (1.0 w/v %); yeast extract (0.5 w/v %); NaNO 3 (0.5 w/v %); K 2 HPO 4 (0.5 w/v %); MgSO 4·0.7H 2 O (0.02 w/v %); and locust bean gum (2 w/v %). The pH of the medium was adjusted to 7.0 before autoclaving. The crude enzyme solution was obtained as mentioned above .
Estimation of biomass
The separated cells were washed with water and dried at 60°C until a constant weight was obtained.
An assay was performed by incubating 0.5 ml of appropriately diluted culture filtrate with 1 ml of 1% (w/v) locust bean gum (in 50 mmol/l sodium citrate buffer at pH 5.0) for 10 min at 50°C . The reducing sugars produced were determined using the Nelson-Somogyi technique . One unit of enzyme activity was defined as the amount of enzyme that released 1 μmol of mannose/ml/min.
This was carried out according to the method of Lowry et al. .
Determination of total sugars
Carbohydrate contents were determined using the phenol sulfuric acid method of Dubios et al. .
The enzyme activity was tested at different incubation periods (ranged from 4-12 days) with different culture techniques (static and shaking) (at 150 rpm) culture techniques were tested. The effects of addition of various carbohydrates, and organic and inorganic nitrogen were evaluated in relation to enzyme yield. The experiments were conducted in triplicate, and the results are the average of these three independent trials.
The ability of the produced enzyme to hydrolyze galactomannan polymer was tested in reaction mixture consisted of 2 ml enzyme solution (35 U/ml) and 2 ml 2% (w/v) locust bean gum (in 50 mmol/l citrate phosphate buffer pH 5.5) and carried out at 40°C for 28 h. The reactions were stopped by boiling for 10 min after 1, 2, 3, 4, 5, 6, 7, 8, 17, 20, 24, 26, and 28 h of incubation. The amounts of reducing sugar of hydrolysis products were estimated using Somogyi methods. Hence, the hydrolysis product patterns were determined by thin-layer chromatography using propanol : water (8.5 : 1.5 v/v) as the mobile phase. Detection was performed by spraying with phenol-sulfuric acid reagent as follows:
The chromate plates were sprayed with a solution containing 3 g phenol and 5 ml of concentrated sulfuric acid in 95 ml of ethyl alcohol and then heated for 10-15 min at 110°C .
| Results and discussion|| |
The results in [Table 1] show that among the fungal cultures investigated, P. humicola and Penicillium spp. v were the most potent microorganisms and produced the highest mannanase activity (8.8 and 7.75 U/ml, respectively) after 10 days, so they selected for further studies. [Table 1] also indicated that the mannanase activity of other cultures was ranged from 0.06 to 0.79 U/ml. No definite correlation was found between mycelial dry weight and mannanase activity of the culture filtrate. The protein content of the culture filtrate was not a criterion in the productivity of mannanase activity. All the bacterial tested were not promising (data not shown).
|Table 1: Survey of some fungal strains for the production of extracellular b-mannanase in shaking cultures|
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Time course of mannanase production
The time course of mannanase production by the static culture technique (in an incubator) or shaking culture (on a rotary shaker at 150 rpm) was studied for P. humicola and Penicillium spp. v. The results presented in [Table 2] show that mannanase was maximally produced after 10 days of fermentation by the shaking technique (8.8 U/ml for P. humicola and 7.75 U/ml for Penicillium spp. v). Blibech et al.  found that the maximum mannanase yield (8 U/ml) by Penicillium accitanis was observed on the sixth day of incubation. Generally, the highest level of β-mannanase produced from fungal strains was obtained after 3-11 days as reported by other authors ,.
|Table 2: Biosynthesis of b-mannanase using shaking and static culture techniques at different incubation periods|
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Effect of different carbon sources
Agroindustrial byproducts are available in large amounts and they have been used for the production of several enzymes [23,24]. Several types of agroindustrial byproducts were evaluated as substrates for mannanase production in comparison with locust bean gum (control) [Table 3]. P. humicola and Penicillium spp. v were grown, with significant differences in the raw materials. In case of P. humicola sesame waste was enhanced the mycelial growth (2.34 g) but the enzyme activity was very low (0.51U/ml), whereas rice bran, hyphaene (dom), wheat bran, wheat husk, and defatted palm kernel wastes reduced the activity to about 12-23% of the control. It seems that defatted palm kernel waste improved the growth of Penicillium spp. v (3.53 g/flask), but the enzyme activity was low as 3.21 U/ml [Table 3]. From all the agricultural waste used, ceratonia seeds promoted the biosynthesis of β-mannanase for Penicillium spp. v and yielded 8.39 U/ml. It is clear that both coffee residue and ceratonia seeds were the best inducers for mannanase synthesis by P. humicola (10.3 and 8.87 U/ml, respectively). Coffee residue increased the activity by 1.2-fold compared with locust bean gum. Chantorn et al.  found that Robusta coffee residue appeared to be a more efficient carbon source for Penicillium oxalium KUB-SN2-1 mannanase. The large variation in the mannanase activity by using different wastes in the nutrition medium may be due to the different in the nature of cellulose, or hemicellulose and/or the presence of some other components in these materials (activators or inhibitors) and variation in substrate accessibility. On comparison with other fungal producer and wastes [Table 4], it was observed that our results were higher than those reported by other mannanase producers.
|Table 3: Effect of different carbon sources on the production of Penicillium humicola and Penicillium spp. v extracellular b-mannanase|
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|Table 4: Comparison of mannanase activities obtained by several fungi reported by other authors with our results|
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Therefore, coffee residue and ceratonia seeds were used for P. humicola and ceratonia seeds waste for Penicillium spp. v as a sole carbon sources in the subsequent experiments (replacing locust bean gum).
Effect of nitrogen source
The mechanisms that govern the formation of extracellular enzymes are influenced by the availability of precursors for protein synthesis. Furthermore, the nitrogen source can significantly affect the pH of the medium during the course of fermentation . The effect of various inorganic and organic nitrogen sources on mannanase synthesis was studied. This was done by eliminating (NH 4 ) 2 SO 4 , peptone, and urea from the original culture medium and using (on equivalent N-basis) ammonium sulfate; baker's yeast; peptone; sodium nitrate; urea; or yeast extract. The results in [Table 5] indicated that for P. humicola, the control media or peptone or yeast extract appeared to be the best nitrogen sources that enabled the production of the highest mannanase activity using coffee residue as a sole carbon source (10.3, 10.0, and 9.5 U/ml, respectively) and yielding 8.87, 8.8, and 8.5 U/ml, respectively, when using ceratonia seeds as a carbon source [Table 5]. Also, the effect of different nitrogen sources using Penicillium spp. v showed that the mixture of ammonium sulfate, peptone, and urea (control) produced the highest mannanase activity (8.4 U/ml), followed by the use of only yeast extract as a nitrogen source (7.4 U/ml), whereas inhibition occurred with the other nitrogen sources [Table 6]. Our results are in agreement with those obtained previously by Hashem et al. . Some investigators reported that fungi produce more enzymes on the addition of complex organic nitrogen sources ,. Naganagouda et al.  found that the best nitrogen source was the combined form of yeast extract and ammonium nitrate for Aspergillus spp.; however, Youssef et al.  reported that ammonium chloride was the best preferable nitrogen source for A. niger.
|Table 5: Effect of different nitrogen sources with coffee waste and ceratonia waste on the production of Penicillium humicola extracellular b-mannanase|
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|Table 6: Effect of different nitrogen sources with ceratonia waste on the production of Penicillium spp. v extracellular b-mannanase|
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β-Mannanase obtained from P. humicola (10.8 U/ml) was higher than that obtained from Penicillium spp. v; therefore, P. humicola was chosen for further work and the properties of its crude enzyme were studied.
Some properties of the crude Penicillium humicola β-mannanase
Effect of pH value of the reaction mixture
P. humicola mannanase showed optimum activity at pH 5.5 [Figure 1]. In general, optimum pH for the activity of most fungal mannanases was in the acidic range ,,,.
Effect of temperature of the reaction mixture
P. humicola mannanase had an optimum temperature for activity at 50°C. It was still active at a higher temperature and retained 89 and 83% of its activity at 55-60°C, respectively [Figure 2]. There are several commercial advantages in carrying out enzymatic reactions at a higher temperature . Enzymatic digestion at a high temperature (60-65°C) may reduce microbial contamination of the material being processed. In addition, higher temperatures increase the rate of substrate digestion and increase the solubility of the polymeric substrates such as carbohydrates, rendering them more amenable to enzymatic attack .
The pH stability of the tested enzyme was investigated after preincubation of the crude enzyme in buffer with different pHs (4.5-6) for different periods (15-60 min). The crude enzyme showed good stability within a broad pH range of 4.5-6 for 60 min. The enzyme retained about 81 and 82% of its original activity after 60 min of incubation at pH 5 and 5.5, respectively. Also, it retained 80% of its activity after 60 min of exposure at pH 6.0 [Figure 3].
The enzyme solution was incubated in the absence of its substrate at 40, 45, 50, and 55°C in a water bath and the residual activities at different periods up to 1 h were determined under the optimum conditions (pH 5.5, 50°C). The enzyme retained 98.4% of its activity after 60 min at 40°C and 95% of its activity at 45°C. After heating the enzyme at 50°C up to 15 min, most of the activity was retained (96%) and still retained 79.8% of its activity after 60 min [Figure 4]. The enzyme was still active after 15 and 30 min of incubation at 55°C and yielded 86 and 56.4% activity, respectively. The most adverse effect on the crude enzyme was after 1 h (36.9%) at 55 and 60°C.
The time course of hydrolysis of locust bean gum by crude extracellular P. humicola β-mannanase was studied. The amount of reducing sugars released is shown in [Table 7]. The highest reducing sugar (8900 μmol/ml) was achieved after 28 h, followed by 26 h (8870 μmol/ml). In addition, various sizes of oligosaccharides detected by thin-layer chromatography were obtained from the hydrolysis locust bean gum as shown in [Figure 5]a and b. Similarly, mannanase from P. oxalicum KUB-SNS-1 hydrolyzed copra mannan into mannotriose and mannabiose . Hence, some fungal mannanases hydrolyze mannotetraose to mannotriose and mannabiose through a transglycosylation reaction . The authors showed that P. humicola mannanase efficiently hydrolyzed locust bean gum into galactomanno-oligosaccharides. Mannanase produced from P. humicola had interesting properties and could be applied in a prebiotic preparation. However, the degradation products and the most suitable conditions to obtain higher quantities of oligosaccharides still need further investigation.
|Table 7: Hydrolysis of locust bean gum by Penicillium humicola extracellular b-mannanase at different time intervals|
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| Conclusion|| |
This study focuses on the optimization of culture parameters for the maximal production of crude extracellular mannanase from P. humicola using agricultural waste as a carbon source. The modified medium with an initial pH of 5.5 at 30°C produced the highest mannanase activity of 10.3 U/ml after 10 days. Finally, the enzyme tested was found to have the ability to hydrolyze galactomannan and produced galactomanno-oligosaccharides, which can be used in food and feed industries.
| Acknowledgements|| |
The authors are grateful to National Research Center for supporting this research study.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]