| Xylanases(endo-1,4-β-D-xylanohydrolase;EC 3.2.1.8),the family of xylan-degrading enzymes,reported predominantly from bacteria,fungi,actinomycetes,and yeast,have been widely used in the animal feed,pulp and paper,textile,and food industries.Results from previous studies conducted in our laboratories have revealed that Streptomyces griseorubens LH-3 exhibited high xylanase activity.In this study,we have investigated the production of xylanase by fermentation and its purification by anion-exchange and Gel chromatographies.The purified enzyme has been characterized and its action on unbleached kraft pulp has been evaluated.The potential of crude xylanase in biobleaching and biobeating of unbleached kraft pulp has also been investigated.In order to achieve selective production of xylanase,hemicellulose extracted from black liquor of bagasse kraft pulping was used as the carbon source in this study.The optimal fermentation conditions for producing xylanase by strain LH-3 were determined based on the analysis of single factor synthesis and orthogonal test,and the results are as follows:optimal fermentation medium contains hemicellulose(30.0 g/L),yeast extract(3.0 g/L),K2HPO4(0.6 g/L),FeS04·7H20(0.01 g/L),MgS04-7H20(0.6 g/L),and NaCl(0.6 g/L);optimal fermentation conditions constitute a seed volume of 12%,rotating speed of 160 r/min,a culture temperature of 34℃,the initial pH of 9.0,a fermentation time of 96 h,and a liquid volume of 20%.Under these conditions,the activity of xylanase isolated from S.griseorubens LH-3 can be as high as 105.4 IU/mL.Evaluation of the enzymatic properties revealed that the crude xylanase exhibits thermal stability in the temperature range from 55℃ to 70 ℃,is active across a wide pH range(4.0～9.0),and is cellulose-free.Taken together,these observations indicate that the xylanase from S.griseorubens LH-3 can be a potential bleaching agent for pulp.A combination of ammonium sulphate precipitation,Mono Q ion exchange chromatography,and Gel chromatography was used to separate xylanase from the S.griseorubens LH-3.The apparent molecular mass of the enzyme was 45 kDa subunit as determined in SDS gel electrophoresis.Evaluation of enzyme activity revealed that the pH for the optimal activity of pure xylanase is 5.0,the enzyme is stable in the pH range from 5.0 to 8.0,the temperature for optimal activity is 60℃,and the thermal stability is up to 50℃.Metal ions such as Fe2+and Mg2+ enhanced the enzyme activity.While some ions,such as Ca2+,Co2+,Zn2+,and Cu2+,partially inhibited the activity of pure xylanase,other ions,such as Hg2+ and Al3+,strongly inhibited its activity.With xylan obtained from birchwood as the substrate,the Km of the reaction catalyzed by xylanase was 1.5 mg/mL and the highest reaction rate(Vmax)was 2.0 mg/mL·min.Only oligomers of xylose were observed in the HPLC analysis of the degraded products(xylose is not observed)and indicated that isolated the enzyme was an endo-xylanase.A sample of the pulp treated with the xylanase was subjected to scanning electron microscope,ultraviolet,FT-IR,and HPLC analyses.The results revealed that treatment with xylanase can degrade part of the xylan and promote lignin dissolution,in particular,the effect of enzyme treatment on unbleached bagasse kraft pulp was better than that on unbleached eucalyptus kraft pulp.In order to evaluate the effect of S.griseorubens LH-3 xylanase on biobleaching of kraft pulp,the quantity of chromophores released from pulp on treatment with crude xylanase was measured.Highest levels of released chromophores were observed when a dosage of 20 IU/g(dry pulp)of crude xylanase was used to treat unbleached eucalyptus kraft pulp.Similarly,highest release of chromophores was observed when 30 IU/g(dry pulp)of crude xylanase was used to treat unbleached bagasse kraft pulp.When compared to the brightness of the untreated sample,biobleaching by xylanase increased the brightness of eucalyptus kraft pulp by 12.9%and that of bagasse kraft pulp by 17.9%.Subsequently,the amount of hydrogen peroxide used for bleaching the enzyme-treated and-untreated pulps to a predetermined level of ISO brightness were compared.Bleaching enzyme-treated eucalyptus kraft pulp with hydrogen peroxide to a predetermined level of ISO brightness reduced hydrogen peroxide consumption by 16.7%when compared to the amount of hydrogen peroxide required to bleach untreated eucalyptus kraft pulp to the same brightness level;furthermore,the chemical treatment of enzyme-treated kraft pulp increased yield and viscosity of pulp by 1.47%and 1.53%,respectively.Similarly,the consumption of hydrogen peroxide was reduced by 20.0%for bleaching enzyme-treated bagasse kraft pulp and the viscosity of the pulp was increased by 5.96%.These results show that biobleaching of kraft pulp with crude xylanase from S.griseorubens LH-3 not only increases brightness and reduces Kappa number significantly,but also reduces the chemical consumption effectively.Moreover,the yield and viscosity of pulp is also increased.The effect of saving energy in the refining of enzyme treatment on unbleached kraft pulps before and during beating was also investigated.When unbleached bagasse kraft pulp treated with crude enzyme was processed by beating,the highest growth rate in beating degree was observed when the pretreatment dosage of xylanase was 30 IU/g(dry pulp);the observed increase in the beating degree was 18.9%.Similarly,the highest rate of increase in beating degree of pretreated eucalyptus kraft pulp was at a pretreatment dosage of 20 IU/g(dry pulp)and the observed increase in beating degree was 16.2%.These results show that both untreated and xylanase treated pulp can be processed by beating to obtain a pulp with identical physical properties;however,pretreatment of the pulp with xylanase results in 16.0%and 12.5%lesser consumption of energy during the beating of bagasse and eucalyptus kraft pulps,respectively.When the crude xylanase enzyme was added to the unbleached bagasse kraft pulp during the process of beating,the highest growth rate of beating degree was achieved at a dosage of 40 IU/g(dry pulp)and the increase in beating degree was 32.3%.Similarly,highest increase in the growth rate of beating degree for unbleached eucalyptus kraft pulp was observed at a dosage of 30 IU/g(dry pulp)and beating degree increased by 25.5%.Pulps of identical physical properties,i.e.,similar beating degrees,can be obtained in the presence or absence of the crude xylanase enzyme through the beating process.However,inclusion of the xylanase enzyme during the beating process reduces energy consumption by 20.0%and 18.8%for unbleached bagasse and eucalyptus kraft pulps,respectively.These results show that biobeating of kraft pulp with crude xylanse obtained from S.griseorubens LH-3 not only increases the beating degree significantly,but also effectively reduces energy consumption.In particular,these studies reveal that the biobeating effect is more significant when xylanase is added to the pulp during the process of beating than that when the beating is pursued after pulp is initially treated with xylanase.In conclusion,the above results show that this crude preparation of the xylanase enzyme exhibits promise for potential industrial applications. |
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