Simultaneous production of ligninolytic enzymes by a ... - NOPR

Indian Journal of Biotechnology Vol 14, January 2015, pp 81-86

Simultaneous production of ligninolytic enzymes by a temperature and pH tolerant strain of Aspergillus niger under different cultural conditions Kusum Dhakar1, Rinu Kooliyottil1, Archana Joshi2 and Anita Pandey1* 1

Biotechnological Applications, G B Pant Institute of Himalayan Environment and Development, Kosi-Katarmal Almora 263 643, India 2

Mody Institute of Technology and Science, Lakshmangarh, Sikar 332 311, India

Received 14 March 2013; revised 27 September 2013; accepted 3 November 2013 Ligninolytic activity, represented by laccase, lignin peroxidase (LiP) and manganese peroxidase (MnP), of a temperature and pH tolerant strain of Aspergillus niger isolated from a temperate location in the Indian Himalayan Region (IHR), has been studied under different cultural (physico-chemical & nutritional) conditions. In plate assays, the fungus exhibited ligninolytic activity at wide range of temperature (5-45°C) and pH (3.5-9.5). In quantitative estimations, carried out at 15, 25 and 35°C, production of laccase was favoured by low temperature (15°), while production of LiP and MnP were favoured by higher temperatures (25 & 35°C). At optimum growth temperature (25°C), laccase production was the maximum at 7.5 pH. LiP and MnP production was favoured between 7.5 to 9.5, and 5.5 to 9.5 pH, respectively. Amongst nutritional sources, nitrogen sources were recorded as better enhancers for enzyme production, followed by vitamins and carbon sources. Folic acid (0.01%) was also found to be a good enhancer for production of all the three enzymes. Keywords Aspergillius niger, ligninolytic, Indian Himalayan Region (IHR), temperature/pH tolerant

Introduction Decomposition is broadly defined as the physical, chemical and biological process that transforms complex organic materials into increasingly simpler forms. In forest ecosystems, decomposition of leaf litter is an important factor controlling nutrient cycling and formation of soil. In high altitudes of mountain ecosystems, the degradation process is slow and takes longer period due to the prevalence of low temperature. Thus, occurrence of temperature tolerant microorganisms, psychrotolerants in particular, in such environments plays important role toward sustainability of soil health1-3. Fungi, mainly basidiomycetes, are well known for their contribution in litter decomposition and nutrient release. However, dominance and contribution of ascomycetous fungi has been recognized for various biological processes under low temperature environments in recent literature4-6. Ligninolytic enzymes, a group of highly versatile enzymes, are known for their role in degradation of —————— *Author for correspondence Tel: +91-5962-241041; Fax: +91-5962-241150 [email protected]

the complex and the recalcitrant polymer lignin. Ligninolytic activity is associated with three major enzymes, viz., lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase. Peroxidases possess heme structure and require H2O2 for catalysis, while LiP has the ability to oxidize non-phenolic structures related to lignin. Moreover, MnP facilitates the decomposition by generating phenoxy radicals from phenolic rings. Laccases are multicopper oxido-reductase glycoproteins with the ability to oxidize phenolics as well as the non-phenolic compounds in presence of the mediators, such as, ABTS (2,2'-azino-bis 3-ethylbenzothiazoline-6sulphonic acid) and hydroxybenzotriazole (HBT)7. Besides ecological importance, these enzymes are also known for their biotechnological applications, such as, in food, pharmaceuticals, textiles and synthetic chemistry8. Such applications have led to the increasing demand of these enzymes at industrial scale. The production of these enzymes can be greatly affected by modifying physico-chemical and nutritional conditions9. The aim of the present study was to determine the ligninolytic activity (due to laccase, LiP & MnP) of a temperature tolerant fungus, initially isolated from the

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soil sample collected from a temperate location in Indian Himalayan Region (IHR), under different cultural conditions. Materials and Methods Fungal Culture

Fungal culture was taken from the microbial culture collection that has been developed in the Microbiology Laboratory of G B Pant Institute of Himalayan Environment and Development, Almora (Uttarakhand), India. The fungus was originally isolated from the soil sample collected from a temperate location in IHR. The soil pH at sampling site ranged from 4.5 to 6.5. The location experiences heavy rainfall and snowfall as well, maintaining low temperature up to subzero levels. The fungal culture was maintained on potato dextrose agar (PDA) slants at 4°C, following sub-culture at prescribed intervals. The fungal isolate (Aspergillus niger) under study has been accessioned by Indian Type Culture Collection, Indian Agricultural Research Institute, New Delhi, India (Acc. No. ITCC2546). The description of the site and properties of the fungal isolate have been reported earlier6. Qualitative and Quantitative Estimation of Ligninolytic Activity

Modified Kirk and Farrell medium10 was used for the production of lignin degrading enzymes. The medium contained (g/L): malt extract 2.0, glucose 2.0, NH4NO3 2.0, Na2HPO4 0.26, KH2PO4 0.26, MgSO4.7H2O 0.5, CuSO4.5H2O 0.01, CaCl2.2H2O 0.006, FeSO4.7H2O 0.005, ZnSO4.7H2O 0.0005, Na2MoO4 0.00002, MnSO4.H2O 0.00009 and H3BO3 0.00007. Plate assay was done by supplementing 0.35 g/L ABTS. The inoculated plates were observed for development of green colour zone around the colony, following 7 d of incubation. For quantitative estimation, 50 mL medium (pH=5.0±0.5) was prepared in 250 mL Erlenmeyer flasks and 5 mm disc of 7-d-old fungal culture grown on PDA was used for inoculation. Laccase activity was determined by using ABTS (ε420=36000 M-1cm-1)11 at 420 nm in citrate-phosphate buffer (pH=3.0). LiP activity was assayed by veratryl alcohol (ε310=9300 M-1 cm-1)12. MnP activity was determined by reading the formation of stable tartrate complex through the oxidation of Mn+2 to Mn+3 (ε238= 6500 M-1 cm-1)12 in tartrate buffer (pH=4.5). Enzyme activity was defined as 1 µM of substrate oxidized per minute under the given conditions.

Effect of Physico-chemical Conditions on Production of Ligninolytic Enzymes

The production of ligninolytic enzymes was investigated at 3 temperatures (15, 25 & 35°C). At 15°C, the readings were recorded at weekly intervals up to 5 wk. At 25 and 35°C, the readings were recorded at every 3rd d up to 15 d. For determination of effect of pH on production of ligninolytic enzymes, the experiments were conducted at 25°C in 4 sets, maintaining pH of the media at 3.5, 5.5, 7.5 and 9.5. Enzyme activity was estimated at 12th d of incubation, in each case. Effect of Nutritional Ligninolytic Enzymes

Supplements

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Production

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Effect of carbon sources, replacing glucose by arabinose, fructose, galactose, lactose, maltose, trehalose, pectin, starch, cellulose and chitin; nitrogen sources, replacing ammonium nitrate by ammonium sulfate, ammonium acetate, ammonium ferrous sulfate, potassium nitrate, urea, casein, yeast extract, peptone and allylthiourea (0.2% each, separately); and five vitamin supplements [pyridoxine, B1, biotin, nicotinic acid & folic acid (0.01% each, separately)] were studied on the production of lignin degrading enzymes. Vitamins were added in the medium at 5th d of incubation. The readings were taken following 12 d incubation at 25°C. All the experiments were conducted in triplicate and the mean values with standard deviation were shown as the error bars. Results and Discussion Plate assays revealed the production of ligninolytic enzymes at wide range of temperature (4-35°C) by A. niger. In quantitative estimations conducted at 3 temperatures, the production of laccase was favoured by low temperature. The maximum (9.2±1.8 U/L) production of laccase was estimated at 15°C on 21st d of incubation, following decline to 4.2±1.1 U/L (35th d). At 25 and 35°C, the production of laccase was the maximum (8.0±1.0 & 2.4±0.7 U/L, respectively) at 12th and 6th d, respectively. Contrary to these results, production of LiP and MnP was favoured by higher temperatures (25 & 35°C). The maximum LiP was produced (165.3±15.7 U/L) at 25°C at 9th d of incubation, then declined to 91.1±10.6 U/L (15th d). A similar trend of LiP activity was recorded at 35°C. At 15°C, the maximum LiP activity (137.0±7.8 U/L) was recorded at 21st d, declined to 96.6±9.5 U/L at 35th d of incubation. The production of MnP was also favoured by higher

DHAKAR et al: LIGNINOLYTIC ENZYMES BY TEMPERATURE AND pH TOLERANT A. NIGER

temperatures, being maximum (2326.9±226.7 U/L) at 35°C at 12th d of incubation and then declined to 2022.4±217.2 U/L at 15th d. The production of MnP was higher at 25° in comparison to 15°C. Results on the production of all the 3 enzymes are presented in Table 1. The fungus under study (A. niger) has been reported to tolerate a wide range of temperature with its optimum growth in mesophilic range6. Importance of temperature in production of bioactive molecules by various fungi, initially isolated from low temperature environments, has been reported in recent years13-14. In the present study, the production of different ligninolytic enzymes varied with respect to temperature. While the production of laccase was favoured by psychrophilc range, production of LiP and MnP was better under mesophilic temperature range. Temperature for optimum production of laccase has been reported in between 25 to 30°C15. The production of the ligninolytic enzymes also varied with respect to pH, most pronounced in case of laccase. The minimum production of laccase (0.8±0.3 U/L) was recorded at pH 3.5, which increased with the increase of pH up to pH 7.5 (8.9±0.6 U/L) and then it declined to pH 9.5 (4.1±0.5 U/L). The production of LiP was recorded almost stable at pH 7.5 and 9.5, showing the activities at 211.6±14.5 and 214.9±15.2 U/L, respectively. The minimum production (110.0±28.2 U/L) of LiP was recorded at pH 3.5. The maximum production (1737.4±74.5U/L) of MnP was recorded at pH 5.5, showing stability at pH 9.5 (1618.3±96.3 U/L), while

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the minimum MnP production (1439.1±164.5 U/L) was recorded at pH 3.5 (Table 2). The pH of the medium is known to alter the structure of catalytic site of enzymes affecting the activity. In Rhizoctonia praticola, pH 7.5 has been reported optimum for the production of laccase16. In general, acidic pH is known to be more suitable for the production of the enzymes17. In the present study, the production of ligninolytic enzymes has been recorded at a much wider range of pH. The effect of carbon sources on production of ligninolytic enzymes is presented in Fig. 1. Among 10 carbon sources used at 0.2 % concentration, arabinose, trehalose and cellulose were found to be the best enhancers of laccase production (10.6±1.5, 8.9±1.6 & 10.0±2.1 U/L, respectively). Other carbon sources, namely, fructose, galactose, lactose, maltose, pectin, starch and chitin resulted in inhibitory effect on laccase production. Fructose, maltose, trehalose, pectin and chitin were found to be the effective enhancers (100.5±7.1, 97.3±9.2, 91.2±10.6, 92.1±12.9 & 91.7±11.7 U/L, respectively) of LiP; while arabinose, galactose, lactose, starch and cellulose exhibited inhibitory effects on LiP production. All the Table 2—Effect of pH on production of ligninolytic enzymes pH

Laccase (U/L)

LiP (U/L)

MnP (U/L)

3.5 5.5 7.5 9.5

0.8±0.3 7.6±1.4 8.9±0.7 4.1±0.5

110.0±28.2 149.3±13.2 211.6±14.5 214.9±15.2

1439.1±164.5 1737.4±74.5 1642.8±177.0 1618.3±96.3

Values are mean±SD (n= 3)

Table 1—Effect of temperature on production of ligninolytic enzymes Incubation (d)

Laccase (U/L)

LiP (U/L)

MnP (U/L)

A 7 14 21 28 35

15°C 0.9±0.3 3.3±0.5 9.2±1.8 5.9±0.9 4.2±1.1

15°C 86.5±17.7 126.7±8.5 137.0±7.8 104.5±14.5 96.6±9.5

15°C 1667.1±133.1 1782.4±105.2 2131.7±288.5 2028.6±262.4 1974.8±197.3

B 3 6 9 12 15

25°C 1.4±0.4 1.6±0.6 3.9±0.7 8.0±1.0 4.3±1.0

Values are mean ± SD (n= 3)

35°C 1.3±0.3 2.4±0.7 2.1±0.2 1.7±0.5 1.9±0.3

25°C 52.8±6.9 85.8±7.6 165.3±15.7 92.1±9.3 91.1±10.6

35°C 79.7±4.1 94.3±6.1 168.4±5.7 89.7±10.0 80.6±5.7

25°C 1783.9±87.5 1751.9±124.0 2193.4±261.4 1909.6±145.5 1933.3±203.1

35°C 1779.4±104.3 2028.8±192.1 2205.1±165.8 2326.9±226.7 2022.4±217.2

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Fig. 1 (A-C)—Effect of carbon sources on production of ligninolytic enzymes: A, Laccase; B, LiP; & C, MnP. [G, Glucose; F, Fructose; Gal, Galactose; Lac, Lactose; M, Maltose; Tre, Trehalose; P, Pectin; St, Starch; Cel, Cellulose; & Chi, Chitin]

carbon sources enhanced the production of MnP; maximum being in case of chitin (2307.9±284.1 U/L). Addition of nitrogen sources was more effective in enhancing the production of ligninolytic enzymes (Fig. 2). Out of 10 sources, ammonium ferrous sulfate, urea, yeast extract and peptone were found to enhance the laccase production (10.3±1.8, 10.5±1.2, 9.5±1.5 & 9.9±1.6 U/L, respectively). However, ammonium sulfate, ammonium acetate, potassium nitrate, casein and allylthiourea exhibited inhibitory effects on laccase production. Seven of the nitrogen sources (ammonium acetate, ammonium ferrous sulfate, urea, casein, yeast extract, peptone and allylthiourea) resulted in enhancement of LiP production, which was recorded as 109.2±13.8, 360.2±74.1, 174.3±12.1, 116.4±12.5, 94.3±14.5, 191.5±27.7, 170.1±12.4 and 145.2±11.8 U/L,

Fig. 2 (A-C)—Effect of nitrogen sources on produciton of ligninolytic enzymes: A, Laccase; B, LiP; & C, MnP. [AN, Ammonium nitrate; AS, Ammonium sulfate; AA, Ammonium acetate; AFS, Ammonium ferrous sulfate; KN, Potassium nitrate; U, Urea; Cs, Casein; Ye, Yeast extract; Pep, Peptone; & ATU, Allythiourea]

respectively. On the contrary, ammonium sulfate and potassium nitrate were found to be inhibitory for LiP production. In case of MnP, allylthiourea was found to be the best enhancer that resulted in approx 3-fold enhancement (6499.9±701.8 U/L). Ammonium ferrous sulfate, casein, yeast extract, peptone and allylthiourea were the other enhancers (5307.6±780.2, 2576.9±378.8, 2532.1±640.2, 2436.14±390.3 & 6499.6±701.8 U/L, respectively) of MnP. However, MnP production was inhibited by 4 nitrogen sources, namely, ammonium sulfate, ammonium acetate, potassium nitrate and urea. Addition of different carbon and nitrogen sources for regulating the production of ligninolytic enzymes has been well reported18-20. In the present study, among various nitrogen sources, organic nitrogen sources, in

DHAKAR et al: LIGNINOLYTIC ENZYMES BY TEMPERATURE AND pH TOLERANT A. NIGER

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enhancement of laccase production by Cyathus bulleri21, and effect of physico-chemical and nutritional parameters as well as other supplements on production of ligninolytic enzymes from Trametes sp. have also been reported earlier19. Literature on ascomycetous fungi in lignin degradation, specifically from low temperature environments, is limited. Various species from the ascomycetous genera, mainly Aspergillus, Paecilomyces and Penicillium, have been of interest because of their presence and activities in various geographical locations, including low temperature environments under mountain ecosystem4-6,22. The psychrotolerant strain of A. niger, used in the present study, was originally isolated from the mixed forest of Cedrus-Taxus in IHR, where litter degradation is slow due to the prevalence of low temperature. The present study highlights the importance of temperature and pH in production of the ligninolytic enzymes. The fungus showed its ability to produce ligninolytic enzymes at a wide and different range of temperature and pH. Because of the stability at low temperature, laccase is likely to be more applicable for degradation in the low temperature environments. Research on microorganisms dominating the low temperature environments, such as, Arctic, Antarctic, Andes and IHR, is getting increasing attention of the scientific community. The focus in these studies is mainly on the phylogeny, adaptation mechanisms, biogeography, bioremediation, biotechnological applications and ecological resilience23-28.

Fig. 3 (A-C)—Effect of vitamins on production of ligninolytic enzymes: A, Laccase; B, LiP; & C, MnP. [C, Control; P, Pyridoxine; B1, Thiamine; B, Biotin; N, Nicotinic acid; & F, Folic acid]

particular, was found to be more effective in comparison to the addition of carbon sources. Vitamin supplements also affected production of ligninolytic enzymes. Pyridoxine, biotin and folic acid resulted in enhanced production (10.3±1.6, 10.1±1.5 & 11.4±1.2 U/L, respectively) of laccase. On the contrary, addition of B1 and nicotinic acid was found to be inhibitory for the production of laccase. While all the vitamins showed strong positive effect on production of LiP and MnP, maximum enhanced production (662.3± 108.7 & 3525.6±483.9 U/L, respectively) was recorded in case of folic acid (Fig. 3). Effect of biotin and pyridoxine in

Acknowledgment Authors are thankful to the Director, G B Pant Institute of Himalayan Environment and Development, Almora for encouragement and providing the facilities. The Department of Science and Technology, and the Union Ministry of Environment, Forests and Climate Change, Government of India, New Delhi, are acknowledged for financial support. Senior author (DK) is also thankful to Indian Council of Medical Research, New Delhi for the award of fellowship. References 1

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