Partial Purification and Characterization of a Xylanase from Trichoderma Harzianum.
Summary: Among the fungi, the soft rot fungus Trichoderma has shown to be efficient producers of xylan degrading enzyme. In this paper we report partial purification and biochemical characterization of a xylanase (XYN) from a local isolate of a filamentous fungus Trichoderma harzianum. Xylanase was produced by growing the T. harzianum in Vogel's medium where 1 % xylan was used as carbon source in medium to induce the production of enzyme. During purification 40% ammonium sulphate saturation was found to be optimal for xylanase precipitation. The crude sample was dialysed and was also followed by Sephadex G-200 chromatography steps. The specific activity of xylanase was increased about 3.14-fold compared with the crude preparations. The pH and temperature optima of the partially purified xylanase was 6.0 and 65 oC respectively.
The apparent Km and Vmax values of the crude xylanase using birchwood xylan as a substrate were 1.52 mg mL-1 and 0.647 (mu)mol min-1 mg-1, respectively.
Keywords: Xylanase, Trichoderma harzianum, Purification, Characterization.
Hemicellulases represent about 20-30% of lignocellulosic biomass. In recent years biomass conversion has received much attention because of its practical applications in various agro-industrial processes such as efficient conversion of hemicellulose, biomass to fuels and chemicals, delignification of paper and pulp, digestibility, enhancement of animal feed stock and clarification of juices . Hydrolysis of xylan requires action of different enzymes, two enzymes are responsible for the main change; endo-(Beta)-1-4 xylanase cleaves the backbone to xylo-oligosaacharides and (Beta)-xylosidase hydrolyses them to D-xylose .
Heteroxylan, the major component of hemicellulose, is an important biomass reservoir in the plant cell wall. As hemicellulose is a recyclable material, xylanolytic enzymes from microorganisms have been intensively investigated over the past few decades . Xylanolytic enzymes constitute a commercially important class of enzymes. They have a have a wide range of potential biotechnological applications . Xylanases have been extensively studied and could potentially be employed for the production of hydrolysate from agro-industrial wastes, nutritional improvements of lignocellulosic feeds, processing of food  and increasing animal feed digestibility, biobleaching of paper pulp . Other potential applications include the clarification of fruit juices and wine, the extraction of plant oil, coffee and starch, the production of oligosaccharides and improvement of the nutritional value of feed [7, 8].
Xylanases in conjunction with other enzymes such as cellulases are used for the generation of biological fuels such as ethanol and xylitol from lignocellulosics, for degumming of bast fibers such as flax, hemp, jute, and ramie, and deinking of waste newspapers, which are used in the papermaking process [9, 10].
The use of xylanase in biobleaching process allows the attainment of pulps with high brightness with savings of bleaching chemicals  and is widely used in the bleaching of non-woody pulps . Currently, xylanases and cellulases together with pectinases accounts for 20% of the world enzyme market [13, 14].
A purified xylanase is the pre-requisite for various biochemical studies needed to be done for better understanding of the xylanolytic system [13,15].
A number of fungal species are known for the production of xylanases such as Aspergilus niger, Sporotrichum spp., Chaetmoium thermophilum, Humicola lanuginosa and Trichoderma harzianum [16-18]. Filamentous fungi such as Aspergillus spp. and Trichoderma spp. are of particular interest for xylanase production, because they can secrete higher levels of xylanase than yeast and bacteria [19, 3]. Trichoderma species are reported to produce enzymes involved in the degradation of cellulose, xylan and pectin to fermentable sugars . Among the fungi the soft rot fungi, Trichoderma have been shown to be efficient producer of xylan degrading enzyme activity .
In this paper we report partial purification and biochemical characterization of a xylanase from filamentous fungus Trichoderma harzianum.
Results and Discussion
Purification of Xylanase from Trichoderma harzianum
Trichoderma harzianum was grown with 1% birch wood xylan as a substrate for 120 h in the Vogel's medium  for xylanase purification. The crude culture filtrate was subjected to partial purification using ammonium sulphate precipitation followed by gel filtration on Sephadex G-200 column at 4 oC.
Ammonium Sulphate Precipitation
Total activity and specific activity of the xylanase in the crude enzyme filtrate are given in Table-1. It was found that 40% ammonium sulphate saturation was optimal for xylanase precipitation.
Table-1: A summary of purification of xylanase from T harzianum
Purification###Total activity Specific activity###Purification
Step###(UT mL1)###(U mt')###(Fold)
Gel filtration on###0.023###2.381###3.14
Gel Filtration Chromatography
After ammonium sulphate precipitation the pellet was redissolved in sodium acetate buffer and dialyzed overnight against the same buffer. The dialyzed sample was loaded on to Sephadex G200 column (30 cm) pre-equilibrated with 50 mM sodium acetate buffer, pH 5.0. After Sephadex G-200 chromatography, specific activity of xylanase was increased about 3.14-fold compared with the crude preparations. Summary of purification procedures of xylanase is presented in Table-1.
Properties of Purified Xylanase
A summary of properties of purified xylanase is presented in Table-2.
Table-2: Properties of purified xylanase from T. harzianum.
###Km###1.52 mg mL-1###
###Vmax###0.647 (mu)mol min-1 mg -1
Effect of pH and Temperature on Xylanase Activity pH and Temperature of the enzyme are very important factors when we intend to study the industrial importance of the enzyme. T. harzianum purified xylanase activity was observed at pH 6. pH optimum of T. harzianum xylanase compares well with those reported for other fungal xylanases. Thus, our results for optimum pH values are similar as reported for A. oryzae NRRL 3485 , Fusarium graminearum , and Trichoderma sp. .
Difference in pH and temperature tolerance for xylanase secreted might be due to the effect of different enzymes mixtures secreted, and /or the post-translational modifications in xylanase secretion process, such as glycosylation, that improves stability in more extreme pH and temperature conditions .
In our present study, the T. harzianum xylanase preparation exhibited unusually (for Trichoderma) high pH and temperature optima of pH 6 and 65 degC, as compared to another strain of T. harzianum , thereby rendering it of potential industrial importance.
Kinetic Parameters Km and Vmax
The apparent Km and Vmax values of the xylanase using birchwood xylan as a substrate were 1.52 mg mL-1 and 0.647 (mu)mol min-1 mg-1, respectively (Fig. 3). Km value for xylanase from Penicillium capsulatum using birchwood xylan as a substrate was 4.0 mg mL-1 . Km and Vmax of xylanase from Aspergillus nidulans with oat-spelt xylan as a substrate were found to be 0.97 mg mL-1 and 1,091 (mu)mol min-1 mg-1respectively . Earlier Km and Vmax values of the xylanase from Trichoderma harzianum strain T4 were reported to be 1.61 mg mL-1 and 10.03 IU mL-1, respectively using soluble birchwood xylan as a substrate . Lower Km values demonstrate greater affinity for the substrate thus showing higher efficiency of the enzyme to convert the substrate into product.
All the chemicals used were of analytical grade unless otherwise stated.
Trichoderma harzianum, a local isolate, was used in this study and was maintained at 4 oC after growing for 7 days in MYG medium (0.2% malt extract, 0.2 % yeast extract, 2% glucose and 2 % agar) at 28 oC .
Microorganism and Culture Conditions
For the production of xylanase in liquid state fermentation the fungus was grown in 500 mL Erlenmeyer flask containing 100 mL of the Vogel's medium . One percent glucose was used as carbon source for inoculum preparations whereas 1% xylan was used as carbon source in cultivation medium. pH of the medium was adjusted to 5.5 with 1 M NaOH/1 M HCl before autoclaving. Inoculum preparations were completed by 24 h of cultivation at 28 oC on an orbital shaker (150 rpm). A 10 mL of liquid culture from the inoculum was transferred to 1000 mL Erlenmeyer flasks containing 250 mL Vogel's medium containing the substrate for 5 days at 28 oC with shaking at 150 rpm . Liquid states cultures were harvested by centrifugation at 10, 000 rpm, for 20 min at 4 oC . The resulting supernatant was used as crude enzyme preparation.
Xylanase activity was assayed using 1% (w/v) of birchwood xylan as a substrate. Reaction mixture contained 1 mL of appropriately diluted enzyme and 1% xylan in citrate phosphate buffer. The mixture was incubated at 50 oC for 30 min. After predetermined periods the releasing sugars were estimated with 3,5-dinitrosalysilic acid (DNS) using xylose as standard . One unit of xylanase activity was defined as the amount of enzyme that released 1 (mu)mol reducing sugars equivalent xylose min-1.
All the purification steps were performed at 4 oC unless otherwise stated. Xylanse was purified by ammonium sulphate precipitation followed by gel filtration chromatography on Sephadex G200 column. For the purification of xylanase the crude extract of 5 days culture grown on 1% xylan was subjected to ammonium sulphate precipitation at different concentrations of (NH4)2 SO4 saturation (0 to 80%). After precipitation the pellet was re- dissolved in sodium acetate buffer and dialyzed overnight against the same buffer. The dialyzed sample was loaded on to Sephadex G200 column (30 cm) pre-equilibrated with 50 mM sodium acetate buffer, pH 5.0. The xylanase was eluted at a linear flow rate of 30 cm h-1. Different fractions of the xylanase were pooled, concentrated and subjected to enzyme activity. Protein concentration was determined by Bradford method  using bovine serum albumin as standard. Proteins in the column effluents were monitored by measuring A280.
Effect of Temperature and pH on Xylanase Activity
For determination of optimum temperature for the purified xylanase activity, the reactions were carried out at 40 to 75 oC with an interval of 5 oC at pH 6. Enzyme assays were carried out as described above. The pH optima of the purified xylanase activity was estimated using DNS assay at various pH values between 4.0 to 7.0.
Kinetic parameters were determined by incubating the enzyme under the optimal conditions of temperature and pH. The Km and Vmax of the enzyme were determined by measuring the enzyme activity at varying concentrations of xylan (0.5 to 20 mg mL-1) and plotting the values in Lineweaver Burk plot.
The research was supported by a grant from Higher Education Commission, Pakistan.
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|Author:||Abbas, Aamir; Ahmad, Sibtain; Mushtaq, Zahid; Jamil, Amer|
|Publication:||Journal of the Chemical Society of Pakistan|
|Date:||Dec 31, 2012|
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