黑曲霉木聚糖酶基因的克隆、表达与热稳定性改造研究
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摘要
木聚糖广泛存在于植物细胞壁中,是自然界中含量仅次于纤维素的可再生资源。木聚糖的来源不同,分枝程度不同,其主链和支链带有的基团也不同。由于木聚糖结构的复杂性,它的完全降解需要多种水解酶的参与共同作用完成。木聚糖酶(β-1,4-xylanase,EC 3.2.1.18)能够以内切方式作用于木聚糖分子中的β-1,4糖苷键产生不同长度的木寡糖和少量的木糖,因此是木聚糖降解酶中最关键的酶。
本文以1株木聚糖酶产生菌黑曲霉(Aspergillus niger)F19为实验菌株,系统地研究了黑曲霉木聚糖酶基因xynA与xynB的克隆以及在大肠杆菌(Escherichia coli)中的表达、重组木聚糖酶XynB的纯化及其酶学性质研究,并进行了木聚糖酶XynB的热稳定性改造,得到了SS2、ST5、SST7共3个突变体,主要结论如下:
1.xynA与xynB的克隆以及在大肠杆菌中的表达
以黑曲霉(A.niger)F19的基因组DNA为模板,根据GenBank报道的黑曲霉xynA与xynB的全序列设计两对引物,PCR扩增得到木聚糖酶基因xynA和xvnB。将不带原基因信号肽编码序列的xynA和xynB以正确的阅读框架克隆到大肠杆菌(Ecoli)表达载体pET-28a(+)上,并转化E.coli BL21,获得重组工程菌BLX1和BLX2。经过IPTG诱导,XynA和XynB都获得特异性表达,XynA以包涵体和胞内可溶性蛋白2种形式存在,而XynB表达的蛋白全部以胞内可溶性蛋白形式存在。
2.木聚糖酶XynB的纯化
木聚糖酶XynB重组表达蛋白以胞内可溶性蛋白的形式存在,且木聚糖酶基因xynB是和pET-28a(+)融合表达,木聚糖酶XynB的羧基端带有6×His蛋白表达标签,所以我们用Ni-NTA亲和层析柱对木聚糖酶XynB进行了纯化,并使其纯度达到了电泳纯水平,在SDS-PAGE上呈现出清晰的单一条带。
3.木聚糖酶XynB的酶学性质分析
利用纯酶分析XynB的酶学性质,木聚糖酶XynB的比活达到13500±128.4IU/mg,其K_m和V_(max)分别为12.5±0.08 mg/mL,289±10.8μmol/min/mg。它的最适反应pH为5.0,在pH 4.0～7.0之间保持最高活力的75%以上。该重组酶表现出很高的pH稳定性,在pH 3.0～8.0内常温环境下放置6 h,残余酶活保持在68%以上。XynB的最适反应温度为50℃,反应温度小于30℃或大于60℃的情况下重组木聚糖酶表现出较低的活性。该酶热稳定较差,在40℃以下较为稳定,60℃以上重组酶活性下降很快,60℃条件保温30 min后残余酶活为20%。在10 mmol/L浓度下不同金属离子对酶活力的影响研究表明,Zn~(2+)、Co~(2+)离子对重组酶有激活作用,分别使重组酶活性提高了8%和15%,Mn~(2+)使重组酶的相对活性降低了21%,体现出对该酶活性一定的抑制作用,其他金属离子对重组酶活性的影响不大。
4.木聚糖酶XynB的热稳定性改造
由于XynB的比活较高,但是其热稳定性较差,这在一定程度上限制了木聚糖酶XynB在工业水平上的推广应用。为了提高XynB的热稳定性,我们采用了两种定点诱变的策略:一种是将木聚糖酶表面的Ser/Thr替换为Arg,提高XynB表面蛋白的正电荷数及其电解常数以提高蛋白的热稳定性;另外一种是在木聚糖酶的非催化区域引入二硫键,使其结构更加紧密从而提高其热稳定性。将正确的突变体SS2、ST5、SST7基因xynB'构建到表达载体pET-28a(+)上,并转化BL21表达突变蛋白,但是表达的蛋白大部分形成了包涵体,而且菌体破碎液离心后的上清液没有检测出木聚糖酶活性。目前,实验室正在构建XynB野生型和突变型的高效分泌型酵母表达系统以确定定点突变对该重组酶的热稳定性影响,为该酶的大规模工业应用提供基础资料。
Xylan is the most abundant renewable polysaccharide fibers in nature secondary only to cellulose,which is mainly found in the plant cell walls.Different xylans have different origins,branches and there are many substitute groups on the backbone and side chains. For the complexity of the structure of the xylan,the degradation of xylan needs many kinds of hydrolases.Xylanases(EC 3.2.1.18) can catalyze the hydrolysis of internalβ-1,4 bonds of xylan into xylooligosaccharides and D-xylose,therefore,xylanase is the key hydrolases for the degradation of xylan.
In this present dissertation,xylanase gene xynA and xynB were cloned and expressed in E.coli,and the purification and characterization of recombinant XynB were investigated,using Aspergillus niger F19 as the experimental material.After this,we tried to improve the thermal stability of the recombinant XynB,the results showed that:
1.The cloning,expression of xynA and xynB gene in E.coli
The xynA and xynB gene were isolated from the genomic DNA of A.niger F19, cloned and expressed in E.coll.Two pairs of primers were designed according to the reported full sequences of xynA and xynB gene from A.niger in GenBank.xynA and xynB gene fragments were successfully amplified by PCR reactions under the suitable conditions.The xynA and xynB gene without origin signal peptide sequences were fused into the E.coli expression vector pET-28a(+) with correct open reading frame,then transfered into E.coli BL21,finally the recombinant strain BLX1 and BLX2 were obtained.After induced by IPTG,the xynA gene was successfully expressed in the recombinant strain BLX1;the expression product existed as inclusion bodies and intracellular soluble form,and xynB gene in BLX2 as intracellular soluble form.
2.Purification of XynB
Most of the product of recombinant XynB existed as the intracellular soluble form, and the xynB gene was expressed in pET-28a(+),therefore the C-terminal of the XynB has 6×His protein tag,thus we purified the XynB with the Ni-NTA affinity column.The purity of recombinant XynB was fine and a single band was founded in the SDS-PAGE analysis.
3.The characterization of XynB
The purified XynB had a K_m of 12.5±0.08 mg/mL,a V_(max) of 289±10.8μmol/min/mg and a specific activity of 13500±128.4 IU/mg,using oat spelt xylan as a substrate.The optimal pH of XynB was 5.0,it sustained above 75%of the enzyme activity of XynB among the pH from 4.0 to 7.0 using the purified XynB for assay.The XynB showed high pH stability in room temperature for 6h among the pH from 3.0 to 8.0,more than 68%of the enzyme activity remained.The optimal temperature of XynB was 50℃,the recombinant XynB showed low enzyme activity at the temperature above 60℃or below 30℃.The thermal stability of XynB is low,and the residual activity of the XynB after incubated at 60℃for 30 min is 20%.It was stable at the temperature below 40℃.The effect of 10 mM/L metallic ions on XynB showed that Zn~(2+) and Co~(2+) activated the recombinant xylanase by 8%and 15%,respectively.The Mn~(2+) restrained the XynB by 21%,and other metallic ions showed no effect on it.
4.The improvement of thermo-stability on XynB
XynB had a high specific activity but low thermo-stability,so it's difficult to put it into industrial application.To improve the thermal stability of XynB,two site-directed mutagenesis strategies were applied.The first one was to substitute the surface serine and threonine into argnine.The second one was introduce the disulfide bridge into discatalytic domain of XynB.Then the mutated gene constructed to pET-28a(+) and transformed into BL21 for the expression of mutated XynB.However,inclusion bodies formed during the process of expression and no enzyme activity detected on the supernatant of cell disruption liquid.To determine the effect of the site-directed mutagenesis on the thermal stability of XynB,high excretive eukaryotic expression system of wild type and mutated type of XynB is constructing.Still,large-scale industrial application research of this enzyme is in progress.
本文以1株木聚糖酶产生菌黑曲霉(Aspergillus niger)F19为实验菌株,系统地研究了黑曲霉木聚糖酶基因xynA与xynB的克隆以及在大肠杆菌(Escherichia coli)中的表达、重组木聚糖酶XynB的纯化及其酶学性质研究,并进行了木聚糖酶XynB的热稳定性改造,得到了SS2、ST5、SST7共3个突变体,主要结论如下:
1.xynA与xynB的克隆以及在大肠杆菌中的表达
以黑曲霉(A.niger)F19的基因组DNA为模板,根据GenBank报道的黑曲霉xynA与xynB的全序列设计两对引物,PCR扩增得到木聚糖酶基因xynA和xvnB。将不带原基因信号肽编码序列的xynA和xynB以正确的阅读框架克隆到大肠杆菌(Ecoli)表达载体pET-28a(+)上,并转化E.coli BL21,获得重组工程菌BLX1和BLX2。经过IPTG诱导,XynA和XynB都获得特异性表达,XynA以包涵体和胞内可溶性蛋白2种形式存在,而XynB表达的蛋白全部以胞内可溶性蛋白形式存在。
2.木聚糖酶XynB的纯化
木聚糖酶XynB重组表达蛋白以胞内可溶性蛋白的形式存在,且木聚糖酶基因xynB是和pET-28a(+)融合表达,木聚糖酶XynB的羧基端带有6×His蛋白表达标签,所以我们用Ni-NTA亲和层析柱对木聚糖酶XynB进行了纯化,并使其纯度达到了电泳纯水平,在SDS-PAGE上呈现出清晰的单一条带。
3.木聚糖酶XynB的酶学性质分析
利用纯酶分析XynB的酶学性质,木聚糖酶XynB的比活达到13500±128.4IU/mg,其K_m和V_(max)分别为12.5±0.08 mg/mL,289±10.8μmol/min/mg。它的最适反应pH为5.0,在pH 4.0～7.0之间保持最高活力的75%以上。该重组酶表现出很高的pH稳定性,在pH 3.0～8.0内常温环境下放置6 h,残余酶活保持在68%以上。XynB的最适反应温度为50℃,反应温度小于30℃或大于60℃的情况下重组木聚糖酶表现出较低的活性。该酶热稳定较差,在40℃以下较为稳定,60℃以上重组酶活性下降很快,60℃条件保温30 min后残余酶活为20%。在10 mmol/L浓度下不同金属离子对酶活力的影响研究表明,Zn~(2+)、Co~(2+)离子对重组酶有激活作用,分别使重组酶活性提高了8%和15%,Mn~(2+)使重组酶的相对活性降低了21%,体现出对该酶活性一定的抑制作用,其他金属离子对重组酶活性的影响不大。
4.木聚糖酶XynB的热稳定性改造
由于XynB的比活较高,但是其热稳定性较差,这在一定程度上限制了木聚糖酶XynB在工业水平上的推广应用。为了提高XynB的热稳定性,我们采用了两种定点诱变的策略:一种是将木聚糖酶表面的Ser/Thr替换为Arg,提高XynB表面蛋白的正电荷数及其电解常数以提高蛋白的热稳定性;另外一种是在木聚糖酶的非催化区域引入二硫键,使其结构更加紧密从而提高其热稳定性。将正确的突变体SS2、ST5、SST7基因xynB'构建到表达载体pET-28a(+)上,并转化BL21表达突变蛋白,但是表达的蛋白大部分形成了包涵体,而且菌体破碎液离心后的上清液没有检测出木聚糖酶活性。目前,实验室正在构建XynB野生型和突变型的高效分泌型酵母表达系统以确定定点突变对该重组酶的热稳定性影响,为该酶的大规模工业应用提供基础资料。
Xylan is the most abundant renewable polysaccharide fibers in nature secondary only to cellulose,which is mainly found in the plant cell walls.Different xylans have different origins,branches and there are many substitute groups on the backbone and side chains. For the complexity of the structure of the xylan,the degradation of xylan needs many kinds of hydrolases.Xylanases(EC 3.2.1.18) can catalyze the hydrolysis of internalβ-1,4 bonds of xylan into xylooligosaccharides and D-xylose,therefore,xylanase is the key hydrolases for the degradation of xylan.
In this present dissertation,xylanase gene xynA and xynB were cloned and expressed in E.coli,and the purification and characterization of recombinant XynB were investigated,using Aspergillus niger F19 as the experimental material.After this,we tried to improve the thermal stability of the recombinant XynB,the results showed that:
1.The cloning,expression of xynA and xynB gene in E.coli
The xynA and xynB gene were isolated from the genomic DNA of A.niger F19, cloned and expressed in E.coll.Two pairs of primers were designed according to the reported full sequences of xynA and xynB gene from A.niger in GenBank.xynA and xynB gene fragments were successfully amplified by PCR reactions under the suitable conditions.The xynA and xynB gene without origin signal peptide sequences were fused into the E.coli expression vector pET-28a(+) with correct open reading frame,then transfered into E.coli BL21,finally the recombinant strain BLX1 and BLX2 were obtained.After induced by IPTG,the xynA gene was successfully expressed in the recombinant strain BLX1;the expression product existed as inclusion bodies and intracellular soluble form,and xynB gene in BLX2 as intracellular soluble form.
2.Purification of XynB
Most of the product of recombinant XynB existed as the intracellular soluble form, and the xynB gene was expressed in pET-28a(+),therefore the C-terminal of the XynB has 6×His protein tag,thus we purified the XynB with the Ni-NTA affinity column.The purity of recombinant XynB was fine and a single band was founded in the SDS-PAGE analysis.
3.The characterization of XynB
The purified XynB had a K_m of 12.5±0.08 mg/mL,a V_(max) of 289±10.8μmol/min/mg and a specific activity of 13500±128.4 IU/mg,using oat spelt xylan as a substrate.The optimal pH of XynB was 5.0,it sustained above 75%of the enzyme activity of XynB among the pH from 4.0 to 7.0 using the purified XynB for assay.The XynB showed high pH stability in room temperature for 6h among the pH from 3.0 to 8.0,more than 68%of the enzyme activity remained.The optimal temperature of XynB was 50℃,the recombinant XynB showed low enzyme activity at the temperature above 60℃or below 30℃.The thermal stability of XynB is low,and the residual activity of the XynB after incubated at 60℃for 30 min is 20%.It was stable at the temperature below 40℃.The effect of 10 mM/L metallic ions on XynB showed that Zn~(2+) and Co~(2+) activated the recombinant xylanase by 8%and 15%,respectively.The Mn~(2+) restrained the XynB by 21%,and other metallic ions showed no effect on it.
4.The improvement of thermo-stability on XynB
XynB had a high specific activity but low thermo-stability,so it's difficult to put it into industrial application.To improve the thermal stability of XynB,two site-directed mutagenesis strategies were applied.The first one was to substitute the surface serine and threonine into argnine.The second one was introduce the disulfide bridge into discatalytic domain of XynB.Then the mutated gene constructed to pET-28a(+) and transformed into BL21 for the expression of mutated XynB.However,inclusion bodies formed during the process of expression and no enzyme activity detected on the supernatant of cell disruption liquid.To determine the effect of the site-directed mutagenesis on the thermal stability of XynB,high excretive eukaryotic expression system of wild type and mutated type of XynB is constructing.Still,large-scale industrial application research of this enzyme is in progress.
引文
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44 Kung L, Treacher R J, Nauman G A, Smagala A M, Endres K M, Cohen M A. The effect of treating forages with fibrolytic enzymes on its nutritive value and lactation performance of dairy cows. J Dairy Sci, 2000, 83: 115-122
45 Lapidot A. Mechly A. Shoham Y, Journal of Biotechnology, 1996, 51: 259-264
46 Liu M Q, Weng X Y, Sun J Y. Expression of recombinant Aspergillus niger xylanase A in Pichia pastoris and its action on xylan. Prot Expres Purif, 2006,48: 292-299
47 Mohammad M A, Satoshi K, Wang Q, Kei Y R, Mitiko G, Kiyoshi H. Capacity of thermomonospora albs XylA to impart thermostability in familiar F/10 chimeric xylanase. Enryme and Microbiology Technology, 2001, 28: 8-15
48 Neeta K, Abhay S, Maraa R. Molecular and biotechnological aspects of xylanase. FEMS Microbiology Reviews, 1999, 23: 411-456
49 Nuyens F, Van W H, Zyl D, et al. Heterologous expression of the Bacillus pumilus endo-beta-xylanase gene in the yeast Saccharomyces cerevisiae. Appl Microbiol Biotechnol, 2001, 56(3-4): 431-434
50 Paes G, O'Donohue M J. Engineering increased thermostability in the thermo- statble GH-11 x- ylanase from Thermobacillus xylanilyticus. J Biotechnol, 2006, 125: 338-350.
51 Palackal N, Brennan Y, Callen W N, Dupree P, Frey G, Goubet F, Hazlewood G P, Healey S, Kang Y E, Kretz K A, Lee E, Tan X, TomLinson G L, Verruto J, Wong V W K, Mathur E J, Short J M, Robertson D E, Steer B A. An evolutionary route to xylanase process fitness. Protein Sci, 2004,13: 494-503
52 Panbangred W, Fukusaki E, Epifanio E C, et al. Expression of a xylanases gene of Bacillus subtilis. Appl Microbiol Biotechnol, 1985,22: 233-239
53 Petersen M, Jonson P H, Petersen S B. Amino acid neighbours and detailed conformational analysis of cysteines in proteins, Protein Engineering. 1999, 12: 535-548
55 Polizeli, MLTM, Rizzatti, A C S, Monti R. Xylanases from fungi properties and industrial applications. Appl Microbiol Biotechnol, 2005, 67: 577-591
56 Rakhee K, Narayan B B. Application of thermoalkalophilic xylanase from Arthrobacter sp. MTCC 5214 in bioleaching of kraft pulp. Biores Technol, 2007, 98: 897-903
57 Ruanglek V, Sriprang R, Ratanaphan N, Tirawongsaroj P, Chantasigh D, Tanapongpipat S, Pootanakit K, Eurwilaichitr L. Cloning, expression, characterization, and high cell-density production of recombinant endo-1,4-xylanase from Aspergillus niger in Pichia pastoris. Enzyme and Microbial Technology, 2007,41: 19-25
58 Sambrook J, Russell D W. Molecular Cloning: A Laboratory Manual, third ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001
59 Savitha S, Sadhasivam S, Swaminathan K. Application of Aspergillus fumigatus xylanase for quality improvement of waste paper pulp. Bull Environ Contam Toxicol, 2007,78:217-221
60 Schlacher A, Holzmann K, Hayn M, Steiner W, Schwab H. Cloning and characterization of the gene for the thermostable xylanase XynA from Thermomyces lanuginosus. J Biotechnol 1996, 49: 211-218
61 Shibuya H, Kaneko S, Hayashi K. Enhancement of the thermostability and hydrolytic activity of xylanase by random gene shuffling. Biochem J, 2000, 349: 651-656
62 Sriprang R, Asano K, Gobsuk J, Tanapongpipat S, Champreda V, Eurwilaichitr L. Improvement of thermostability of fungal xylanase by using site-directed mutagenesis. J Biotechnol, 2006,126: 454-462
63 Stephens D E, Rumbold K, Permaul K, Prior B A, Singh S. Directed evolution of the thermostable xylanase from Thermomyces lanuginosus. J Biotechnol, 2007, 127: 348-354
64 Suchita N, Ramesh C K. Bleaching of wheat straw-rich soda pulp with xylanase from a thermoalkalophilic Streptomyces cyaneus SN32. Biores Technol, 2006, 97: 2291-2295
65 Techapun C, Poosaran N, Watanabe M, Sasaki K. Thermostable and alkaline-tolerant microbial cellulose-free xylanases produced from agricultural wastes and the properties required for use in pulp bleaching bioprocesses: a review. Process Biochem, 2003,38:1327-1340
66 Turunen O, Etuaho K, Fenel F, Vehmaanpera J, Wu X, Rouvinen J, Leisola M. A combination of weakly stabilizing mutations with a disulfide bridge in the alpha-helix region of Trichoderma reesei endo-1,4-beta-xylanase II increases the thermal stability through synergism. J Biotechnol, 2001, 88: 37-46
67 Turunen O, Vuorio M, Fenel F, Leisola M. Engineering of multiple arginines into the Ser/Thr surface of trichoderma reesei endo-1,4-beta-xylanase II increases the thermotolerance and shifts the pH optimum towards alkaline pH. Protein Eng, 2002, 15:141-145
68 Viikari L, Ranua M, Kantelinen A, Linko M, Sundquist J. Bleaching with enzymes in the Pulp and Paper Industry . Proc 3rd Int Conf, Stockholm, 1986: 67- 69
69 Visser J, CiBeldman M A, Kusters V, Someren A C, Voragen J. Progress in biotechnology 7 Xylans and Xylanases. ELSEVIER SCIENCE PUBLISHERS BV, the Netherlands, (1992)
70 Wakarchuk W W, Sung W L, Campbell R L. Thermostabilization of the bacillus cireulans xylanase bv the introduction of disulfide bonds. Protein Engineering, 1994, 7(11): 1379-1386
71 Whitehead R T, Hespell B R, FEMS Microbiology Letters, 1990, 66: 61-66
72 Wang Y R, Zhang H L, He Y Z, Luo H Y, Yao B. Characterization, gene cloning, and expression of a novel xylanase XYNB from Streptomyces olivaceoviridis Al. Aquaculture, doi: 10.1016/j.aquaculture.2007.03.005
73 Wong K K Y, Tan L U L, Saddler J N. Multiplicity of β-1, 4-xylanase in microorganisms: functions and applications, Microbiol Rev, 1988, 52: 305-317
74 Yang H M, Yao B, Meng K, Wang Y R, Bai Y G, Wu N F. Introduction of a disulfide bridge enhances the thermostability of a Streptomyces olivaceoviridis xylanase mutant. J Ind Microbiol Biotechnol, 2007, 34,213-218
75 Zhao J, Li X Z, Qu Y B. Application of enzymes in producing bleached pulp from wheat straw. Biores Technol 2006, 97: 1470-1476
76 Zhou C Y, Bai J Y, Deng S S, Wang J, Zhu J, Wu M C, Wang W. Cloning of a xylanase gene from Aspergillus usamii and its expression in Escherichia coli. doi:10.1016/j.biortech.2007.01.035
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40 Jurasek L, Paice M. Pulp, paper and biotechnology. Chemtechnology, 1986, 16: 360- 365
41 Ken K Y Wong, Larry U L Tan, John N Saddler.: Multiplicity of p-1,4-xylanase in microorganisms: Functions and Applications. Microbiological Reviews, 1988, 52: 305-317
42 Kindel P K , Liao S Y , Liske M R. Arabinoxylans from rye and wheat seed that interact with ice. Carbohydr Res, 1989, 187(2):173-85
43 Kulkarni N, Shendye A, Rao M. Molecular and biotechnological aspects of xylanases. FEMS Microbiol Rev, 1999,23:411-456
44 Kung L, Treacher R J, Nauman G A, Smagala A M, Endres K M, Cohen M A. The effect of treating forages with fibrolytic enzymes on its nutritive value and lactation performance of dairy cows. J Dairy Sci, 2000, 83: 115-122
45 Lapidot A. Mechly A. Shoham Y, Journal of Biotechnology, 1996, 51: 259-264
46 Liu M Q, Weng X Y, Sun J Y. Expression of recombinant Aspergillus niger xylanase A in Pichia pastoris and its action on xylan. Prot Expres Purif, 2006,48: 292-299
47 Mohammad M A, Satoshi K, Wang Q, Kei Y R, Mitiko G, Kiyoshi H. Capacity of thermomonospora albs XylA to impart thermostability in familiar F/10 chimeric xylanase. Enryme and Microbiology Technology, 2001, 28: 8-15
48 Neeta K, Abhay S, Maraa R. Molecular and biotechnological aspects of xylanase. FEMS Microbiology Reviews, 1999, 23: 411-456
49 Nuyens F, Van W H, Zyl D, et al. Heterologous expression of the Bacillus pumilus endo-beta-xylanase gene in the yeast Saccharomyces cerevisiae. Appl Microbiol Biotechnol, 2001, 56(3-4): 431-434
50 Paes G, O'Donohue M J. Engineering increased thermostability in the thermo- statble GH-11 x- ylanase from Thermobacillus xylanilyticus. J Biotechnol, 2006, 125: 338-350.
51 Palackal N, Brennan Y, Callen W N, Dupree P, Frey G, Goubet F, Hazlewood G P, Healey S, Kang Y E, Kretz K A, Lee E, Tan X, TomLinson G L, Verruto J, Wong V W K, Mathur E J, Short J M, Robertson D E, Steer B A. An evolutionary route to xylanase process fitness. Protein Sci, 2004,13: 494-503
52 Panbangred W, Fukusaki E, Epifanio E C, et al. Expression of a xylanases gene of Bacillus subtilis. Appl Microbiol Biotechnol, 1985,22: 233-239
53 Petersen M, Jonson P H, Petersen S B. Amino acid neighbours and detailed conformational analysis of cysteines in proteins, Protein Engineering. 1999, 12: 535-548
55 Polizeli, MLTM, Rizzatti, A C S, Monti R. Xylanases from fungi properties and industrial applications. Appl Microbiol Biotechnol, 2005, 67: 577-591
56 Rakhee K, Narayan B B. Application of thermoalkalophilic xylanase from Arthrobacter sp. MTCC 5214 in bioleaching of kraft pulp. Biores Technol, 2007, 98: 897-903
57 Ruanglek V, Sriprang R, Ratanaphan N, Tirawongsaroj P, Chantasigh D, Tanapongpipat S, Pootanakit K, Eurwilaichitr L. Cloning, expression, characterization, and high cell-density production of recombinant endo-1,4-xylanase from Aspergillus niger in Pichia pastoris. Enzyme and Microbial Technology, 2007,41: 19-25
58 Sambrook J, Russell D W. Molecular Cloning: A Laboratory Manual, third ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001
59 Savitha S, Sadhasivam S, Swaminathan K. Application of Aspergillus fumigatus xylanase for quality improvement of waste paper pulp. Bull Environ Contam Toxicol, 2007,78:217-221
60 Schlacher A, Holzmann K, Hayn M, Steiner W, Schwab H. Cloning and characterization of the gene for the thermostable xylanase XynA from Thermomyces lanuginosus. J Biotechnol 1996, 49: 211-218
61 Shibuya H, Kaneko S, Hayashi K. Enhancement of the thermostability and hydrolytic activity of xylanase by random gene shuffling. Biochem J, 2000, 349: 651-656
62 Sriprang R, Asano K, Gobsuk J, Tanapongpipat S, Champreda V, Eurwilaichitr L. Improvement of thermostability of fungal xylanase by using site-directed mutagenesis. J Biotechnol, 2006,126: 454-462
63 Stephens D E, Rumbold K, Permaul K, Prior B A, Singh S. Directed evolution of the thermostable xylanase from Thermomyces lanuginosus. J Biotechnol, 2007, 127: 348-354
64 Suchita N, Ramesh C K. Bleaching of wheat straw-rich soda pulp with xylanase from a thermoalkalophilic Streptomyces cyaneus SN32. Biores Technol, 2006, 97: 2291-2295
65 Techapun C, Poosaran N, Watanabe M, Sasaki K. Thermostable and alkaline-tolerant microbial cellulose-free xylanases produced from agricultural wastes and the properties required for use in pulp bleaching bioprocesses: a review. Process Biochem, 2003,38:1327-1340
66 Turunen O, Etuaho K, Fenel F, Vehmaanpera J, Wu X, Rouvinen J, Leisola M. A combination of weakly stabilizing mutations with a disulfide bridge in the alpha-helix region of Trichoderma reesei endo-1,4-beta-xylanase II increases the thermal stability through synergism. J Biotechnol, 2001, 88: 37-46
67 Turunen O, Vuorio M, Fenel F, Leisola M. Engineering of multiple arginines into the Ser/Thr surface of trichoderma reesei endo-1,4-beta-xylanase II increases the thermotolerance and shifts the pH optimum towards alkaline pH. Protein Eng, 2002, 15:141-145
68 Viikari L, Ranua M, Kantelinen A, Linko M, Sundquist J. Bleaching with enzymes in the Pulp and Paper Industry . Proc 3rd Int Conf, Stockholm, 1986: 67- 69
69 Visser J, CiBeldman M A, Kusters V, Someren A C, Voragen J. Progress in biotechnology 7 Xylans and Xylanases. ELSEVIER SCIENCE PUBLISHERS BV, the Netherlands, (1992)
70 Wakarchuk W W, Sung W L, Campbell R L. Thermostabilization of the bacillus cireulans xylanase bv the introduction of disulfide bonds. Protein Engineering, 1994, 7(11): 1379-1386
71 Whitehead R T, Hespell B R, FEMS Microbiology Letters, 1990, 66: 61-66
72 Wang Y R, Zhang H L, He Y Z, Luo H Y, Yao B. Characterization, gene cloning, and expression of a novel xylanase XYNB from Streptomyces olivaceoviridis Al. Aquaculture, doi: 10.1016/j.aquaculture.2007.03.005
73 Wong K K Y, Tan L U L, Saddler J N. Multiplicity of β-1, 4-xylanase in microorganisms: functions and applications, Microbiol Rev, 1988, 52: 305-317
74 Yang H M, Yao B, Meng K, Wang Y R, Bai Y G, Wu N F. Introduction of a disulfide bridge enhances the thermostability of a Streptomyces olivaceoviridis xylanase mutant. J Ind Microbiol Biotechnol, 2007, 34,213-218
75 Zhao J, Li X Z, Qu Y B. Application of enzymes in producing bleached pulp from wheat straw. Biores Technol 2006, 97: 1470-1476
76 Zhou C Y, Bai J Y, Deng S S, Wang J, Zhu J, Wu M C, Wang W. Cloning of a xylanase gene from Aspergillus usamii and its expression in Escherichia coli. doi:10.1016/j.biortech.2007.01.035