Aspergillus ficuum菊粉酶的基因克隆、表达及其结构与功能研究
|
摘要
菊粉酶专一性水解β-2,1-D-呋喃果糖苷键，按其作用方式不同分为内切和外切两类。内切菊粉酶（endoinulinase, EC3.2.1.7）从菊粉链内部随机地切断糖苷键，降解产物主要为菊粉型低聚果糖。外切菊粉酶（exoinulinase, EC3.2.1.80）则从菊粉链的果糖末端逐个地水解糖苷键，产物主要为果糖。菊粉酶广泛存在于各种微生物中，在饲料、食品、医药以及能源工业中有着广阔的应用前景。
本实验室从土壤中成功筛选到一株产菊粉酶的菌株Aspergillus ficuum JNSP5-06，此菌株可以产内切菊粉酶(endo I)和外切菊粉酶(exo I)，且产酶性能稳定。为了进一步提高酶活力，探讨菊粉酶作用机制，本研究运用分子生物学的方法，克隆和表达了内、外切菊粉酶的基因，并在此基础上，通过定向诱变技术研究了内切菊粉酶的催化活性中心和菊粉酶分子的C端结构域的作用。
根据Genbank中记录的菊粉酶基因序列信息，利用PCR技术从Aspergillus ficuum JNSP5-06基因组中扩增和克隆了相关基因片段，获得了endo I的DNA和exo I的DNA、cDNA序列，克隆的endo I和exo I的DNA序列在Genbank中的登录号分别为FJ984582和HM587130。序列分析可知：内切菊粉酶endo I的DNA序列全长1658bp，包含了蛋白质编码区1482bp，3′非编码区176bp；内切酶由493个氨基酸组成，理论分子量Mw为53.2kDa，等电点pI为4.33。外切菊粉酶exo I的DNA序列全长1674bp，包含信号肽编码区57bp，蛋白质编码区1557bp，内含子编码区60bp；exoI全长537个氨基酸，其中信号肽和成熟肽分别由19和518个氨基酸残基组成；其理论分子量Mw为57.2kDa，等电点pI为4.89。
将内、外切菊粉酶成熟肽的编码序列分别插入到大肠杆菌表达载体pET-28a(+)中，构建重组质粒pET28a（+）-endo I和pET28a（+）-exo I，转化大肠杆菌BL21(DE3)，筛选获得基因工程重组菌JPE21/endo I和JPE21/exo I。对菌株的IPTG诱导表达条件优化结果发现：JPE21/endo I在OD600为0.308左右时，加入IPTG至终浓度0.4mmol/L，23℃诱导6h，酶活最高达22.36u/mg；JPE21/exo I则在OD600为0.402左右时，加入IPTG至终浓度为0.4mmol/L，26℃诱导7h，其酶活可达59.24u/mg。经SDS-PAGE检测，重组内、外切菊粉酶的分子量分别约为59kDa、63kDa。对其酶学性质的研究结果表明：（1）重组内切菊粉酶的最适pH为5.0，最适反应温度为55℃。Ag+和Cu~(2+)能完全抑制酶的活性，Fe~(2+)、Fe3+和Al3+对酶有较强的抑制作用，而Zn~(2+)、Mn~(2+)、K+对酶有激活作用。在pH5.0、55℃条件下，内切酶水解菊粉的Km和kcat分别为（8.9±1.5）mM和（1.38±0.12）×103min-1。（2）重组外切菊粉酶作用于菊粉和蔗糖的最适pH值分别为4.0和5.0；最适温度分别为60℃和55℃。金属离子对其活力的影响与底物相关，Cu~(2+)完全抑制外切酶对菊粉的作用，而对蔗糖保持10%的活性；Ag+使酶对菊粉和蔗糖的活力分别降低了45.6%和82.9%；Mg~(2+)、Zn~(2+)、Fe~(2+)、Al3+、Ni~(2+)对酶有较强的抑制作用，而Mn~(2+)对酶有激活作用。在pH5.0、55℃条件下，外切酶水解菊粉Km和kcat分别为（7.1±0.2）mM和（6.1±0.0）×104min-1，水解蔗糖的Km和kcat分别为（347.6±25.9）mM和（7.34±0.49）×105min-1。
通过酶活测定以及酶解产物的薄层层析和液相色谱分析，发现重组内切菊粉酶仅专一性水解菊粉，产物主要为低聚二糖、三糖、四糖；外切菊粉酶不仅能水解菊粉，还能降解蔗糖，另外在高浓度的蔗糖中，还有微弱的转果糖基作用。
对内切菊粉酶endo I进行同源建模和序列比较，定点突变证明内切菊粉酶催化位点的关键氨基酸残基为Glu-20及Glu-210，底物结合位点的关键氨基酸残基为Asp-153。
利用PCR和overlap-PCR技术使得内、外切菊粉酶的C端结构域发生缺失和交换突变，结果发现突变酶的活性急剧下降，且内切突变酶对蔗糖产生降解作用，推测C端结构域起到识别底物、稳定酶分子空间结构、保持酶催化活性的作用。
Inulinases, which can be divided into exo-and endoinulinase according to their hydrolysis patterns,hydrolyze β-2,1-glycosidic linkages of the fructan. Endoinulinase (2,1-β-D-fructan fructanohydrolase, EC3.2.1.7) acts only on inulin and hydrolyze internal linkages of inulin to yield fructooligosaccharidesrandomly. Exoinulinase (β-D-fructan fructohydrolase, EC3.2.1.80) can hydrolyze terminal linkages ofinulin to yield fructose successively. Iinulinase broadly exists in various microorganisms and has beenwidely used in feed, food, medicine and energy industries.
A strain of Aspergillus ficuum JNSP5-06which could secrete endoinulinase (endo I) and exoinulinase(exo I) was got from the soil in our laboratory. In order to improve the activity of A. ficuum JNSP5-06inulinase and to elucidate the catalytic mechanism of the enzyme, we cloned and expressed the gene. Thenwe studied the catalysis activity centres and the function of C-terminal in the molecular structure ofinulinase through directional mutation technique.
According to the information of inulinase in Genbank, the DNA and cDNA of endo I and exo I werecloned from Aspergillus ficuum JNSP5-06. And the coding gene sequences of endo I and exo I wereregistered in the GenBank (FJ984582and HM587130). The DNA sequence of endo I is1658bp, whichcontains1482bp of protein coding region and176bp of3’ noncoding region. Endo I consists of493aminoacids. Mw and pI are53.2kDa and4.33, respectively. The DNA sequence of exo I is1674bp, whichconsists of57bp signal peptide coding region,1557bp of protein coding region and60bp of intron codingregion. Mw and pI are57.2kDa and4.89, respectively.
The encoding sequences of endo I and exo I were inserted into pET-28a(+) respectively. Therecombinant pET28a（+）-endo I and pET28a（+）-exo I were introduced into host E. coli BL21(DE3). Afterinduced by IPTG, the recombinant JPE21/endo I and JPE21/exo I were successfully expressed. The optimalconditions for expression of JPE21/endo I were IPTG concentration of0.4mM, temperature of23°C andreaction time of6h. And the optimal conditions for expression of JPE21/exo I were IPTG concentration of0.4mM, temperature of26°C and reaction time of7h. After optimization, the specific activities ofJPE21/endo I and JPE21/exo I could reach to22.36u/mg and59.24u/mg, respectively.
The recombinant protein was checked by SDS-PAGE, and the Mw of endo I and exo I were59kDaand61kDa, respectively. The optimum pH and temperature of endo were5and60℃. Ag+and Cu~(2+)couldinhibit the activity of endo I thoroughly, and Fe~(2+)、Fe3+and Al3+cound inhibit activity intensively whileZn~(2+)、Mn~(2+)and K+could activate endo I. Kmand kcatof endo I with inulin as substrate were (8.9±1.5) mMand（1.38±0.12）×103min-1respectively. Studies on characterization exo I showed optimum pH andtemperature were4and60℃for inulin, whereas the optimum pH and temperature for sucrose were5and55℃. The influence of the metal ions on the exo I activity was related to the substrate. Cu~(2+)causeddecrease in exo I activity against inulin and sucrose by100%and90%, respectively, while Ag+causeddecrease in exo I activity against inulin and sucrose by45.6%and82.9%. Mg~(2+)、Zn~(2+)、Fe~(2+)、Al3+and Ni~(2+)could inhibit activity intensively while Mn~(2+)could activate the activity. Kmand kcatwere (7.1±0.2) mM and(6.1±0.0)×104min-1for inulin, whereas Kmand kcatfor sucrose were (347.6±25.9) mM and (7.34±0.49)×105 min-1.
Analysis of enzymatic hydrolysis products by TLC and liquid chromatography showed that endo Iuniquely hydrolyzed inulin, which produced disaccharide, trisaccharide and tetrose. While exo I not onlyhydrolyzed inulin, but also degraded sucrose. In addition, exo I had the weak ability of transfructosylation.
Through homology modeling, sequence comparing and site-directed mutagenesis of endo I, it wasproved that Glu-20and Glu-210were catalytically important residues, and Asp-153played an importantrole for substrate recognition in the catalysis reaction.
C-terminal domain was processed by deleted and swapped to orthomutation through over-lap PCR orPCR. It was found that enzyme activities were decreased sharply by C-terminal domain deletion andswapping mutants, and in this case endo I had the ability to degraded sucrose. The results showed thatC-terminal domain had the abililty to recognize long-chain inulin, stable enzyme molecule space structureand keep the enzyme catalysis activity.
本实验室从土壤中成功筛选到一株产菊粉酶的菌株Aspergillus ficuum JNSP5-06，此菌株可以产内切菊粉酶(endo I)和外切菊粉酶(exo I)，且产酶性能稳定。为了进一步提高酶活力，探讨菊粉酶作用机制，本研究运用分子生物学的方法，克隆和表达了内、外切菊粉酶的基因，并在此基础上，通过定向诱变技术研究了内切菊粉酶的催化活性中心和菊粉酶分子的C端结构域的作用。
根据Genbank中记录的菊粉酶基因序列信息，利用PCR技术从Aspergillus ficuum JNSP5-06基因组中扩增和克隆了相关基因片段，获得了endo I的DNA和exo I的DNA、cDNA序列，克隆的endo I和exo I的DNA序列在Genbank中的登录号分别为FJ984582和HM587130。序列分析可知：内切菊粉酶endo I的DNA序列全长1658bp，包含了蛋白质编码区1482bp，3′非编码区176bp；内切酶由493个氨基酸组成，理论分子量Mw为53.2kDa，等电点pI为4.33。外切菊粉酶exo I的DNA序列全长1674bp，包含信号肽编码区57bp，蛋白质编码区1557bp，内含子编码区60bp；exoI全长537个氨基酸，其中信号肽和成熟肽分别由19和518个氨基酸残基组成；其理论分子量Mw为57.2kDa，等电点pI为4.89。
将内、外切菊粉酶成熟肽的编码序列分别插入到大肠杆菌表达载体pET-28a(+)中，构建重组质粒pET28a（+）-endo I和pET28a（+）-exo I，转化大肠杆菌BL21(DE3)，筛选获得基因工程重组菌JPE21/endo I和JPE21/exo I。对菌株的IPTG诱导表达条件优化结果发现：JPE21/endo I在OD600为0.308左右时，加入IPTG至终浓度0.4mmol/L，23℃诱导6h，酶活最高达22.36u/mg；JPE21/exo I则在OD600为0.402左右时，加入IPTG至终浓度为0.4mmol/L，26℃诱导7h，其酶活可达59.24u/mg。经SDS-PAGE检测，重组内、外切菊粉酶的分子量分别约为59kDa、63kDa。对其酶学性质的研究结果表明：（1）重组内切菊粉酶的最适pH为5.0，最适反应温度为55℃。Ag+和Cu~(2+)能完全抑制酶的活性，Fe~(2+)、Fe3+和Al3+对酶有较强的抑制作用，而Zn~(2+)、Mn~(2+)、K+对酶有激活作用。在pH5.0、55℃条件下，内切酶水解菊粉的Km和kcat分别为（8.9±1.5）mM和（1.38±0.12）×103min-1。（2）重组外切菊粉酶作用于菊粉和蔗糖的最适pH值分别为4.0和5.0；最适温度分别为60℃和55℃。金属离子对其活力的影响与底物相关，Cu~(2+)完全抑制外切酶对菊粉的作用，而对蔗糖保持10%的活性；Ag+使酶对菊粉和蔗糖的活力分别降低了45.6%和82.9%；Mg~(2+)、Zn~(2+)、Fe~(2+)、Al3+、Ni~(2+)对酶有较强的抑制作用，而Mn~(2+)对酶有激活作用。在pH5.0、55℃条件下，外切酶水解菊粉Km和kcat分别为（7.1±0.2）mM和（6.1±0.0）×104min-1，水解蔗糖的Km和kcat分别为（347.6±25.9）mM和（7.34±0.49）×105min-1。
通过酶活测定以及酶解产物的薄层层析和液相色谱分析，发现重组内切菊粉酶仅专一性水解菊粉，产物主要为低聚二糖、三糖、四糖；外切菊粉酶不仅能水解菊粉，还能降解蔗糖，另外在高浓度的蔗糖中，还有微弱的转果糖基作用。
对内切菊粉酶endo I进行同源建模和序列比较，定点突变证明内切菊粉酶催化位点的关键氨基酸残基为Glu-20及Glu-210，底物结合位点的关键氨基酸残基为Asp-153。
利用PCR和overlap-PCR技术使得内、外切菊粉酶的C端结构域发生缺失和交换突变，结果发现突变酶的活性急剧下降，且内切突变酶对蔗糖产生降解作用，推测C端结构域起到识别底物、稳定酶分子空间结构、保持酶催化活性的作用。
Inulinases, which can be divided into exo-and endoinulinase according to their hydrolysis patterns,hydrolyze β-2,1-glycosidic linkages of the fructan. Endoinulinase (2,1-β-D-fructan fructanohydrolase, EC3.2.1.7) acts only on inulin and hydrolyze internal linkages of inulin to yield fructooligosaccharidesrandomly. Exoinulinase (β-D-fructan fructohydrolase, EC3.2.1.80) can hydrolyze terminal linkages ofinulin to yield fructose successively. Iinulinase broadly exists in various microorganisms and has beenwidely used in feed, food, medicine and energy industries.
A strain of Aspergillus ficuum JNSP5-06which could secrete endoinulinase (endo I) and exoinulinase(exo I) was got from the soil in our laboratory. In order to improve the activity of A. ficuum JNSP5-06inulinase and to elucidate the catalytic mechanism of the enzyme, we cloned and expressed the gene. Thenwe studied the catalysis activity centres and the function of C-terminal in the molecular structure ofinulinase through directional mutation technique.
According to the information of inulinase in Genbank, the DNA and cDNA of endo I and exo I werecloned from Aspergillus ficuum JNSP5-06. And the coding gene sequences of endo I and exo I wereregistered in the GenBank (FJ984582and HM587130). The DNA sequence of endo I is1658bp, whichcontains1482bp of protein coding region and176bp of3’ noncoding region. Endo I consists of493aminoacids. Mw and pI are53.2kDa and4.33, respectively. The DNA sequence of exo I is1674bp, whichconsists of57bp signal peptide coding region,1557bp of protein coding region and60bp of intron codingregion. Mw and pI are57.2kDa and4.89, respectively.
The encoding sequences of endo I and exo I were inserted into pET-28a(+) respectively. Therecombinant pET28a（+）-endo I and pET28a（+）-exo I were introduced into host E. coli BL21(DE3). Afterinduced by IPTG, the recombinant JPE21/endo I and JPE21/exo I were successfully expressed. The optimalconditions for expression of JPE21/endo I were IPTG concentration of0.4mM, temperature of23°C andreaction time of6h. And the optimal conditions for expression of JPE21/exo I were IPTG concentration of0.4mM, temperature of26°C and reaction time of7h. After optimization, the specific activities ofJPE21/endo I and JPE21/exo I could reach to22.36u/mg and59.24u/mg, respectively.
The recombinant protein was checked by SDS-PAGE, and the Mw of endo I and exo I were59kDaand61kDa, respectively. The optimum pH and temperature of endo were5and60℃. Ag+and Cu~(2+)couldinhibit the activity of endo I thoroughly, and Fe~(2+)、Fe3+and Al3+cound inhibit activity intensively whileZn~(2+)、Mn~(2+)and K+could activate endo I. Kmand kcatof endo I with inulin as substrate were (8.9±1.5) mMand（1.38±0.12）×103min-1respectively. Studies on characterization exo I showed optimum pH andtemperature were4and60℃for inulin, whereas the optimum pH and temperature for sucrose were5and55℃. The influence of the metal ions on the exo I activity was related to the substrate. Cu~(2+)causeddecrease in exo I activity against inulin and sucrose by100%and90%, respectively, while Ag+causeddecrease in exo I activity against inulin and sucrose by45.6%and82.9%. Mg~(2+)、Zn~(2+)、Fe~(2+)、Al3+and Ni~(2+)could inhibit activity intensively while Mn~(2+)could activate the activity. Kmand kcatwere (7.1±0.2) mM and(6.1±0.0)×104min-1for inulin, whereas Kmand kcatfor sucrose were (347.6±25.9) mM and (7.34±0.49)×105 min-1.
Analysis of enzymatic hydrolysis products by TLC and liquid chromatography showed that endo Iuniquely hydrolyzed inulin, which produced disaccharide, trisaccharide and tetrose. While exo I not onlyhydrolyzed inulin, but also degraded sucrose. In addition, exo I had the weak ability of transfructosylation.
Through homology modeling, sequence comparing and site-directed mutagenesis of endo I, it wasproved that Glu-20and Glu-210were catalytically important residues, and Asp-153played an importantrole for substrate recognition in the catalysis reaction.
C-terminal domain was processed by deleted and swapped to orthomutation through over-lap PCR orPCR. It was found that enzyme activities were decreased sharply by C-terminal domain deletion andswapping mutants, and in this case endo I had the ability to degraded sucrose. The results showed thatC-terminal domain had the abililty to recognize long-chain inulin, stable enzyme molecule space structureand keep the enzyme catalysis activity.
引文
1. Roberfroid M B. Inulin-Type Fructans: Functional Food Ingredients[J]. J. Nutr.,2007,137(11):2493S-2502.
2.王建华.微生物菊粉酶基因结构、酶学性质与应用研究进展[J].天然产物研究与开发,2001，13(1)：83-88.
3. Pandey A, Soccol C R, Selvakumar P, et al. Recent developments in microbial inulinases-Itsproduction, properties, and industrial applications[J]. Applied Biochemistry and Biotechnology,1999,81(1):35-52.
4. Vandamme E J, Derycke DG. Microbial InuIinases: Fermentation Process, Properties, andApplications[J]. Advances in applied microbiology.1983,29:139-176.
5. Nakamura T, Hoashi S, Nakatsu S. Culture conditions for inulinase production by Aspergillus[J].Nippon Nogei Kagakku Kaishi,1978,52(3):105-110.
6. Zittan L. Enzymatic hydrolysis of inulin—an alternative way to fructose production[J]. Starch,1981,33:373-377.
7. Nakamura T, Nakatsu S. General properties of extracellular inulase from Penicillium[J]. Nippon NogeiKagakku Kaishi,1977,51:681-689.
8. Kang S-I, Chang Y-J, Oh S-J, et al. Purification and properties of an endo-inulinase from anArthrobacter sp[J]. Biotechnology Letters,1998,20(10):983-986.
9. Kim D H, Choi Y J, Song S K, et al. Production of inulo-oligosaccharides using endo-inulinase from aPseudomonas sp[J]. Biotechnology Letters,1997,19(4):369-371.
10. Park J P, Bae J T, You D J, et al. Production of inulo-oligosaccharides from inulin by a novelendoinulinase from Xanthomonas sp[J]. Biotechnology Letters,1999,21:1043-1046.
11. Viswanathan P, Kulkarul P R. Enhancement of inulinase production by Aspergillus niger vanTeighem[J]. Journal of Applied Bacteriology,1995,78:384-386.
12. Nakamura T, Nagatomo Y, Hamada S, et al. Occurrence of two froms extracellular endoinulinase fromAspergillus niger mutant817[J]. Journal of Fermentation and Bioengineering,1994,78(2):134-139.
13. Bajon A M, Guiraud J P, Galzy P. Isolation of an inulinase derepressed mutant of Pichia polymorphafor the production of fructose[J]. Biotechnology and Bioengineering,1984,26:128-133.
14. Ricca E, Calabrò V, Curcio S, et al. The state of the art in the production of fructose from inulinenzymatic hydrolysis[J]. Critical Reviews in Biotechnology,2007,27:129-145.
15. Kushi R T, Monti R, Contiero J. Production, purification and characterization of an extracellularinulinase from Kluyveromyces marxianus var. bulgaricus[J]. Journal of Industrial Microbiology&Biotechnology,2000,25(2):63-69.
16. Sharma A D, Kainth S, Gill P K. Inulinase production using garlic (Allium sativum) powder as apotential substrate in Streptomyces sp.[J]. Journal of Food Engineering,2006,77(3): p486-491.
17. Gupta A K, Rathore P, Kaur N, et al. Production, Thermal-Stability and Immobilization of Inulinasefrom Fusarium-Oxysporum[J]. Journal of Chemical Technology and Biotechnology,1990,47(3):245-257
18. Laloux O, Cassart J, Delcour J, et al. Cloning and sequencing of the inulinase gene of Kluyveromycesmarxianus var. marxianus ATCC12424[J]. FEBS Letters.1991,289(1):64-68.
19. Rouwenhorst R J, Visser L E, Vanderbaan A A, et al. Production, Distribution, and Kinetic-Propertiesof Inulinase in Continuous Cultures of Kluyveromyces-Marxianus CBS-6556[J]. Applied andEnvironmental Microbiology,1988,54(5):1131-1137.
20. Ettalibi M, Baratti J C. Purification, properties and comparison of invertase, exoinulinases andendoinulinases of Aspergillus ficuum[J]. Applied Microbiology and Biotechnology,1987,26(1):13-20.
21. Ettalibi M, Baratti J C. Molecular and kinetic properties of Aspergillus ficuum inulinases[J].Agricultural and Biological Chemistry,1990,54(1):61-68.
22.魏文铃，余娴文，戴亚，郑晶，谢忠.克鲁维酵母Y-85菊粉酶的纯化和性质[J].微生物学报,1997,37(6):443-448.
23. Uhm T B, Jeon D Y, Myung B S, et al. Purification and properties of [beta]-fructofuranosidase fromAspergillus niger[J]. Biochimica et Biophysica Acta (BBA)-General Subjects,1987,926(2):119-126.
24. Rouwenhorst R J, Hensing M, Verbakel J, et al. Structure and Properties of the Extracellular Inulinaseof Kluyveromyces-Marxianus CBS-6556[J]. Applied and Environmental Microbiology,1990,56(11):3337-3345.
25. Ohta K, Akimoto H, Moriyama S. Fungal Inulinase: Enzymology, Molecular Biology andBiotechnology[J]. Journal of Applied Glycoscience,2004,51:247-254.
26. Xiao R, Tanida M, Takao S. Purification and some properties of endoinulinase from Chrysosporiumpannorum[J]. Journal of Fermentation and Bioengineering,1989,67(4):244-248.
27.张国青,崔福绵,杨秀清.毛壳霉内切菊粉酶的纯化与性质[J].微生物学报,2004,44(6):785-788.
28. Nakamura T, Shitara A, Matsuda S, et al. Production, purification and properties of an endoinulinaseof Penicillium sp. TN-88that liberates inulotriose[J]. Journal of Fermentation and Bioengineering,1997,84(4):313-318.
29. Cho Y J, Yun J W. Purification and characterization of an endoinulinase from Xanthomonas oryzaeNo.5.[J]. Process Biochemistry,2002,37(11):1325-1331.
30. Carniti P, Beltrame P L, Guardione D. Hydrolysis of Inulin-a Kinetic-Study of the ReactionCatalyzed by an Inulinase from Aspergillus ficuum[J]. Biotechnology and Bioengineering,1991,37(6):575-579.
31. Arand M, Golubev A M, Neto J R B, et al. Purification, characterization, gene cloning and preliminaryX-ray data of the exo-inulinase from Aspergillus awamori[J]. Biochemical Journal,2002,362:131-135.
32. Vullo D L, Coto C E, Sineriz F. Characteristics of an Inulinase Produced by Bacillus Subtilis430a, aStrain Isolated from the Rhizosphere of Vernonia herbacea (Vell Rusby)[J]. Applied andEnvironmental Microbiology,1991,57(8):2392-2394.
33. Workman W E, Day D F. Purification and properties of the [beta]-fructofuranosidase fromKluyveromyces fragilis[J]. FEBS Letters,1983,160(1-2):16-20.
34. Silva-Santisteban B O Y, Converti A, Maugeri F. Intrinsic activity of inulinase from Kluyveromycesmarxianus ATCC16045and carbon and nitrogen balances[J]. Food Technology and Biotechnology,2006,44(4):479-483.
35. Gill P K, Manhas R K, Singh J, et al. Purification and characterization of an exoinulinase fromAspergillus fumigatus[J]. Applied Biochemistry and Biotechnology,2004,117(1):19-32.
36. Gill P K, Manhas R K, Singh P. Purification and properties of a heat-stable exoinulinase isoform fromAspergillus fumigatus [J]. Bioresource Technology,2006,97(7):894-902.
37. Xiao R, Tanida M, Takao S. Purification and characteristics of two exoinulinases from Chrysosporiumpannorum[J]. Journal of Fermentation and Bioengineering,1989,67(5):331-334.
38. Moriyama S, Akimoto H, Suetsugu N, et al. Purification and properties of an extracellularexoinulinase from Penicillium sp strain TN-88and sequence analysis of the encoding gene[J].Bioscience Biotechnology and Biochemistry,2002,66(9):1887-1896.
39. Onodera S, Shiomi N. Purification and subsite affinities of exoinulinase from Penicillium-Trzebinskii[J]. Bioscience Biotechnology and Biochemistry,1992,56(9):1443-1447.
40. Sharma A D, Gill P K. Purification and characterization of heat-stable exo-inulinase fromStreptomyces sp.[J]. Journal of Food Engineering,2007,79(4):1172-1178.
41. Sheng J, Chi Z M, Gong F, et al. Purification and characterization of extracellular inulinase from amarine yeast Cryptococcus aureus G7a and inulin hydrolysis by the purified inulinase[J]. AppliedBiochemistry and Biotechnology,2008,144:111-121.
42. Kato K, Araki T, Kitamura T, et al. Purification and properties of a thermostable inulinase (β-D-fructanfructohydrolase) from Bacillus stearothermophilus KP1289[J]. Starch-St rke,1999,51(7):253-258.
43. Azhari R, Szlak A M, Ilan E, et al. Purification and characterization of endo-inulinase andexo-inulinase[J]. Biotechnology and Applied Biochemistry,1989,11(1):105-117.
44. Nagem R A P, Rojas A L, Golubev A M, et al. Crystal structure of exo-inulinase from Aspergillusawamori: the enzyme fold and structural determinants of substrate recognition[J]. Journal ofMolecular Biology,2004,344(2):471-480.
45. Uhm T B, Chae K-S, Lee D-W, et al. Cloning and nucleotide sequence of the endoinulinase-encodinggene, inu2, from Aspergillus ficuum[J]. Biotechnology Letters,1998,20(8):809-812.
46. Kim H S, Lee D W, Ryu E J, et al. Expression of the INU2gene for an endoinulinase of Aspergillusficuum in Saccharomyces cerevisiae[J]. Biotechnology Letters,1999,21(7):621-623.
47. Park S, Jeong H Y, Kim H S, et al. Enhanced production of Aspergillus ficuum endoinulinase inSaccharomyces cerevisiae by using the SUC2-deletion mutation[J]. Enzyme and Technology.2001,29:107-110.
48.王建华，滕达，姚怡等.黑曲霉菊粉内切酶基因和表达该基因的重组毕赤酵母菌株[P].中国专利,1594542,2005
49. Goosen C, Van der Maarel M J, Dijkhuizen L. Exo-inulinase of Aspergillus niger N402: A hydrolyticenzyme with significant transfructosylating activity[J]. Biocatalysis and Biotransformation,2008,26(1-2):49-58.
50. Zhang L, Wang Y, Ohta Y, et al. Expression of the inulinase gene from Aspergillus niger in Pichiapastoris [J]. Process Biochemistry,2003,38(8):1209-1212.
51. Zhang L H, Zhao C, Zhu D, et al. Inhibition of glucose on an exoinulinase from Kluyveromycesmarxianus expressed in Pichia pastoris [J]. Process Biochemistry,2005,40(5):1541-1545.
52. Wen T Q, Liu F, Huo K, et al. Cloning and analysis of the inulinase gene from Kluyveromycescicerisporus CBS4857[J]. World Journal of Microbiology&Biotechnology,2003,19(4):423-426.
53. Tsujimoto Y, Watanabe A, Nakano K, et al. Gene cloning, expression, and crystallization of athermostable exo-inulinase from Geobacillus stearothermophilus KP1289[J]. Applied Microbiologyand Biotechnology,2003,62(2):180-185.
54. Moriyama S, Tanaka H, Uwataki M, et al. Molecular cloning and characterization of an exoinulinasegene from Aspergillus niger strain12and its expression in Pichia pastoris[J]. Journal of Bioscienceand Bioengineering,2003,96(4):324-331.
55. Kwon H J, Jeon S J, You D J, et al. Cloning and characterization of an exoinulinase from Bacilluspolymyxa[J]. Biotechnology Letters,2003,25(2):155-159.
56. Akimoto H, Kiyota N, Kushima T, et al. Molecular cloning and sequence analysis of an endoinulinasegene from Penicillium sp strain TN-88[J]. Bioscience Biotechnology and Biochemistry,2000,64(11):2328-2335.
57. Kang S-I, Kim S-I. Molecular cloning and sequence analysis of an endo-inulinase gene fromArthrobacter sp.[J]. Biotechnology Letters,1999,21(7):569-574.
58. Yun J W, Choi Y J, Song C H, et al. Microbial production of inulo-oligosaccharides by anendoinulinase from Pseudomonas sp. expressed in Escherichia coli[J]. Journal of Bioscience andBioengineering,1999,87(3):291-295.
59. Liebl W, Brem D, Gotschlich A. Analysis of the gene for β-fructosidase (invertase, inulinase) of thehyperthermophilic bacterium Thermotoga maritima, and characterisation of the enzyme expressed inEscherichia coli[J]. Applied Microbiology and Biotechnology,1998,50(1):55-64.
60.王婧,张苓花,王运吉.克鲁维酵母菊粉酶基因在毕赤酵母中的表达[J].大连轻工业学院学报,2003,22(2):135-138.
61.张苓花，王运吉，叶淑红，等.菊粉酶酶源菌株筛选及其基因克隆[J].微生物学报，2002，42(3):321-325.
62. Wang J H，Teng D，Yao Y et al. Expression of Aspergillus niger9891endoinulinase in Pichiapastoris[J]. High Technology Letters,2004,10(4):52-56.
63. Singh P, Gill P K. Production of inulinases: recent advances[J]. Food Technology and Biotechnology,2006,44(2):151-162.
64. Reddy A, Maley F. Studies on identifying the catalytic role of Glu-204in the active site of yeastinvertase[J]. Journal of Biological Chemistry,1996,271:13953-13958.
65. Burne R A, Penders J E C. Characterization of the Streptococcus mutans GS-5fruA gene encodingexo-β-D-fructosidase[J]. Infection and Immunity,1992,60:4621-4632.
66. Gurr S J, Unkles S E, Kinghorn J R. The Structure and Organization of Nuclear Genes of FilamentousFungi, In:Gene Structure in Eukaryotic Microbes[M]. IRL Press, Oxford, UK,1987:93-139.
67. Ohta K, Akimoto H, Matsuda S, et al. Molecular cloning and sequence analysis of two endoinulinasegenes from Aspergillus niger[J]. Bioscience Biotechnology and Biochemistry,1998,62(9):1731-1738.
68. Yuan X L, Goosen C, Kools H, et al. Database mining and transcriptional analysis of genes encodinginulin-modifying enzymes of Aspergillus niger[J]. Microbiology,2006,152(10):3061-3073.
69. Yuan X L, Roubos J A, van den Hondel C A, et al. Identification of InuR, a new Zn(II)2Cys6transcriptional activator involved in the regulation of inulinolytic genes in Aspergillus niger[J].Molecular Genetics and Genomics,2008,279:11-26.
70. Park, S., Han Y, Kim H, et al., Trp17and Glu20residues in conserved WMN(D/E)PN motif aAreessential for Aspergillus ficuum endoinulinase (EC3.2.1.7) activity[J]. Biochemistry,2003.68:658-661
71. Kim K Y, Rhee S., Kim S.I., Role of the N-terminal domain of endoinulinase from Arthrobacter spS37in regulation of enzyme catalysis[J]. Journal of Biochemistry,2005.138(1):27-33.
72. Kim, K.Y., Nascimento A S, Golubev, A M. et al., Catalytic mechanism of inulinase from Arthrobactersp. S37[J]. Biochemical and Biophysical Research Communications,2008.371(4):600-605.
73. Chen H Q, Chen X M, Li Y, et al. Purification and characterization of exo-and endo-inulinase fromAspergillus ficuum JNSP5-06[J]. Food Chemistry,2009,115:1206-1212.
74.王静,金征宇,江波等. Aspergillus ficuum菊粉酶的荧光光谱研究[J].食品科学,2009,30(23):337-341
75. Van den Ende W, Michiels A, De Roover J, et al. Cloning and functional analysis of chicory rootfructan1-exohydrolase I (1-FEH): a vacuolar enzyme derived from a cell-wall invertase ancestor?[J].Mass fingerprint of the1-FEH I enzyme[J]. The Plant Journal,2000,24(4):447-456.
76. Onodera S, Murakami T, Ito H，et al. Molecular cloning and nucleotide sequences of cDNA and geneencoding endo-inulinase from Penicillium purpurogenum[J]. Bioscience Biotechnology andBiochemistry.1996,60(11):1780-1785.
77. Kwon H J, Jeon S J, You D J, et al. Cloning and characterization of an exoinulinase from Bacilluspolymyxa[J]. Biotechnology Letters.2003,25(2):155-159.
78. Garber R C. Isolation of DNA from filamemtous fungi and separation into nuclear mitochondrial,ribosomal, and plasmid components [J]. Anal Biochem,1983,135:416–422.
79. Joseph S, David W R著.黄培堂,王恒樑,周晓巍,等译.分子克隆实验指南[M].北京:化学工业出版社,2007,48–49.
80. Zhang X E, Zhou Y H, Zhang Z P. Engineering E. coli alkaline phosphatase yields changes of catalyticactivity, thermal stability and phosphate inhibition. Biocatalysis and Biotransformation,2002,20(6):381-389.
81. Yang X, McGraw R A, Su X. Canine thyrotropin beta-subunit gene:cloning and expression inEscherichia coli, generation of monoclonal antibodies,and transient expression in the Chinese hamsterovary cells. Domestic Animal Endocrinology,2000,18(4):363-378.
82.陆长梅,袁生,赵庆新.用Overlap-PCR法从Trichoderma reesei QM9414基因组DNA中克隆并表达木聚糖酶III.生物工程学报.2004,20(5):764-769.
83.王建华,刘艳艳.一步法制备高果糖浆工程及产酶菌体营养价值研究[J].食品与发酵工业,2000,26(2):1-4.
84. Yokota A, Yamauchi O and Tomita F. Production of inulotriose from inulin by inulin-degradingenzyme from Streptomyces rochei E87[J]. Letters in Applied Microbiology,1995,21:330-333.
85. Onodera S. and Shiomi N. Purification and substrate specificity of endo-type inulinase fromPenicillium purpurogenum[J]. Agricultural and Biological Chemistry,1988,52(10):2569-2576.
86. Cho Y J, Sinha J, Park J P and Yun J W. Production of inulooligosaccharides from chicory extract byendoinulinase from Xanthomonas oryzae No.5[J]. Enzyme and Microbial Technology,2001,28:439-445.
87. Yun J W, Kim D H, Kim B W, and Song S K. Production of inulo-oligosaccharides from inulin byimmobilized endoinulinase from Pseudomonas sp[J]. Journal of Fermentation and Bioengineering,1997,84(4):369-371.
88. Jin Z Y, Wang J, Jiang B, et al. Production of inulooligosaccharides by endoinulinases fromAspergillus ficuum [J]. Food Research International,2005,38:301–308
89. Ohta K, Hamada S and Nakamura T. Production of high concentrations of ethanol from inulin bysimultaneous saccharification and fermentation using Aspergillus niger and Saccharomycescerevisiae[J]. Applied and Environmental Microbiology,1993,59:729-733.
90. Schorr-Galindo S, Fontana A and Guiraud J P. Fructose syrups and ethanol production by selectivefermentation of inulin[J]. Current Microbiology,1995,30:325-330.
91. Margaritis A and Bajpai P. Ethanol production from Jerusalem artichoke tubers(Helianthus tuberosus)using Kluyveromyces marxianus and Saccharomyces rosei[J]. Biotechnology and Bioengineering,1982,24:941-953.
92. Kim, D. M. and H. S. Kim. Continuous production of gluconic acid and sorbitol fromJerusalem-artichoke and glucose using an oxidoreductase of zymomonas-mobilis and inulinase[J].Biotechnology and Bioengineering,1992,39(3):336-342.
93. Shin YC, Kim YH, Lee HS, et al. Production of exopolysaccharide pullulan from inulin by a mixedculture of Aureobasidium pullulans and Kluyveromyces fragilis[J]. Biotechnol Bioeng.1989,33(1):129-133.
94. Oiwa H, Naganuma M, Ohinuma S, Acetone-butanol pro-duction from dahlia inulin by Clostridiumpasteurianum var.1-53[J]. Agric Biol Chem.1987,51(10):2819-2820
95.华承伟,王建华,滕达,等.菊粉化学和微生物菊粉内切酶研究进展[J].中国食品学报,2004,4(4):103-108
96. Wang J, Jin Z, Jiang B, et al. Production and separation of exo-and endoinulinase from Aspergillusficuum[J]. Process Biochemistry,2003,39:5-11
97. Moriyama S, Muguruma M, and Ohta K. Quantitative expression analysis of inulinase gene cluster ofPenicillium sp. strain TN-88[J]. Journal of Bioscience and Bioengineering,2006,101(3):277-279.
98.周晨妍.宇佐美曲霉木聚糖酶基因的克隆、表达及定向诱变研究[D]:[博士学位论文].无锡:江南大学,2008.
99.李胤.阿拉伯木聚糖溶解、降解机制的研究及酸性木聚糖酶基因的克隆、表达[D]:[博士学位论文].无锡:江南大学,2008.
100.宁正祥.食品成分分析[M].北京：中国轻工业出版社,1997.
101.诸葛健,王正祥.工业微生物实验技术手册[M].北京：中国轻工业出版社,1994.
102. Sambrook J, Frisch E F, Maniatis T. Molecular Cloning: A laboratory manual,2nd edition [M]. NewYork: Cold Spring Harbor Laboratory Press,1989.
103. Sengupta S, Jana M L, Sengupta D, et al. A note on the estimation of microbiol glycosidase activitiesby dinitrosalicylic acid reagent[J]. Appl. Microbiol. Biotechnol.2000,53:732-735.
104. Bradford M M. A rapid sensitive method for the quantitation of microgram quantities of proteinutilizing the principle of protein-dye binding[J]. Analytical Biochemistry,1976,72:248-254.
105. Laemmli U K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature (London),1970,227:680-685.
106. R.G. Harrison. Expression of soluble heterologous proteins via fusion with NusA protein. inNovations.2000,11:4-7.
107. Davis G.D, Elisee C, Newham D.M. er al. New fusion protein systems designed to give solubleexpression in Escherichia coli. Biotechnol. Bioeng.1999,65(4):382-8.
108. Wilkinson D L. and Harrison R G. Predicting the solubility of recombinant proteins in Escherichia coli.Bio/Technology.1991,9:443-448.
109. Fuhrmann M, Hausherr A, Ferbitz L, et al. Monitoring dynamic expression of nuclear genes inChlamydomonas reinhardtii by using a synthetic luciferase reporter gene. Plant Mol Biol.2004Aug;55(6):869-81.）
110. Martinez A, Krappskreg P M, Olafsdottir S, et al. Expression of recombinant human phenylalaninehydroxylase as fusion protein in Escherichia coli circumvents proteolytic degradation by host cellproteases [J]. J Biotechnol,1995,306:589–597
111. Luli G W, Strolh W R. Comparison of growth,acetate production,and acetate inhibition of Escherichiacoli strains in batch and fed-batch fermentations [J]. Appl Environ Microbiol,1990,56:1004–1011
112.唐威华，张景六，王宗阳，等. SDS-PAGE法测定His-tag融合蛋白分子量产生偏差的原因[J].植物生理学报,2000,26(1):64-68.
113. Skowronek, M. and J. Fiedurek. Purification and properties of extracellular endoinulinase fromAspergillus niger20OSM[J]. Food Technology and Biotechnology.2006,44(1):53-58.
114. Kim K Y, and Kang S I. Production of a novel endo-inulinase from Arthrobacter sp. S37[J]..Agricultural Chemistry and Biotechnology.1996,.39(2):99-103.
115. Gupta A K, Singh D P, Kaur N, et al. Production, purification and immobilization of inulinase fromKluyveromyces fragilis[J].. J. Chem. Tech. Biotechnol.,1994,59:377-385.
116. Gaye O B, Sukan S, Vassilew M. Production and properties of inulinase from Aspergillus nige[J].r.Biotechnol. lett.,1994,16(3):275-280.
117. Nakamura T, Kurokawa T, Nakatsu S, et al. Crystallization and general properties of an extracellularinulinase from Aspergillus sp[J].. Nippon Nogeikagaku Kaishi,1978,52:159-166.
118. Warrens A N, Jones M D, Lechler R I. Splicing by overlap extension by PCR using asymmetricamplification: An improved technique for the generation of hybrid proteins of immunologicalinterest[J]. Gene,1997,186(1):29-35
119.刘仙,高国粉,杨丽,等.菊粉酶中色氨酸残基的化学修饰及其荧光光谱[J].高等学校化学学报.2007,28(1):103-105.
120.刘彬,王静云,包永明,等.化学修饰法表征Bacillus smithii T7产耐热菊粉酶催化活性中心的必需氨基酸残基[J].催化学报.2009,30(7):673-678.
121. Takahare S, Morohashi T, Sano T, et al. Fructooligosaccharide consumption enhances femoral bonevolume and mineral concentrations in rates[J]. Journal of Nutrition,2000,130(7):1792-1795.
122. Roberfroid M B, Van L J and Gibson G R. The bifidogenic nature of chicory inulin and its hydrolysisproducts[J]. Journal of Nutrition,1998,128(1):11-19.
123.刘协,胡启玉,李小宁.低聚果糖的润肠通便功能研究[J].江苏预防医学,2002,13(1):62-63
124.尤新.功能性低聚糖[J].中国食品添加剂,2002,(2):40-45.
125. Tomoatsu H. Health effects of oligosaccharides[J]. Food Technology,1994,48:61-65.
126.潘国熙.一种新颖保健甜味剂－低聚果糖[J].广西轻工业,1995,(1):7-9.
127. Ohta A, Ohtsuki M, Uehara M, et al. Dietary fructooligosaccharides prevent post-gastrectomyanemia and osteopenia in rats[J]. Journal of Nutrition,1998,128(3):485-490.
128. Sakai K, Ohta A, Shiga K, et al. The cecum and dietary short-chain fructooligo-saccharides areinvolved in preventing postgastrectomy anemia in rates[J]. Journal of Nutrition,2000,130(6):1608-1612.
129. Mckellar R C and Modler H W. Metabolism of fructo-oligosaccharides by Bifidobacterium spp [J].Applied Microbiology and Biotechnology,1989,31:537-541.
130. Kaplan H and Hutkins R W. Fermentation of fructooligosaccharides by lactic acid bacteria andbifidobacteria[J]. Applied and Environmental Microbiology,2000,66:2682-2684.
131.潘国熙.一种新颖保健甜味剂－低聚果糖[J].广西轻工业,1995,(1):7-9.
132. Arnold K, Bordoli L, Kopp J, et al. The SWISS-MODEL Workspace: A web-based environment forprotein structure homology modelling[J]. Bioinformatics.2006,22:195-201.
133. Kiefer F, Arnold K, Künzli M, et al. The SWISS-MODEL Repository and associated resources[J].Nucleic Acids Research.2009,37:387-392.
134. Peitsch M. C. Protein modeling by E-mail[J]. Bio/Technology.1995,13:658-660.
135. Fisher C L, Pei G K. Modification of a PCR-based site-directed mutagenesis method[J].Biotechniques.1997,23:570-574.
136.李松林.曲霉CJ22-326产壳聚糖酶基因的克隆、表达与定点突变[D]:[博士学位论文].无锡:江南大学,2009.
137. Reddy A, Maley F. Identification of an active-site residue in yeast invertase by affinity labeling andsite-directed mutagenesis[J]. J. Biol. Chem.1990,265:10817-10820.
138. Reddy A, Maley F. Studies on identifying the catalytic role of Glu-204in the active site of yeastinvertase[J]. J. Biol. Chem.1996,271:13953-13958.
139. Alberto F, Bignon C, Sulzenbacher G, et al. The threedimensional structure of invertase(beta-fructosidase) from Thermotoga maritima reveals a bimodular arrangement and an evolutionaryrelationship between retaining and inverting glycosidases[J]. J. Biol. Chem.2004,279:18903–189
140. Henrissat B. A classification of glycosyl hydrolases based on amino-acid sequence similarities[J].Biochem. J.1991,280:309-316
141. Cantarel BL, Coutinho PM, Rancurel C, et al. The Carbohydrate-Active EnZymes database (CAZy):an expert resource for Glycogenomics[J]. Nucleic Acids Res.2009,37:D233-238
142. Coutinho PM, Henrissat B. Carbohydrate-active enzymes: an integrated database approach. In: GilbertHJ, Davies GJ, Henrissat B, Svensson B, eds. Recent advances in carbohydrate bioengineering[J].Cambridge, UK: Royal Society of Chemistry.1999,3–12.
143. Naumoff DG. Beta-fructosidase superfamily: homology with some alpha-L-arabinases andbeta-D-xylosidases[J]. Proteins–Structure Function and Genetics.2001,42:66–76.
144. Pons T, Naumoff DG, Martinez-Fleites C, et al. Three acidic residues are at the active site of abeta-propeller architecture in glycoside hydrolase families32,43,62, and68[J]. Proteins: Structure,Function, and Bioinformatics.2004,54:424–432.
145. Holm L, Sander C. Mapping the protein universe[J]. Science.1996,273:595–603.
146. Peumans WJ, Van Damme EJ. Lectins as plant defense proteins[J]. Plant Physiology.1995,109:347–352.
147. Altenbach D, Nuesch E, Meyer AD, et al. The large subunit determines catalytic specificity of barleysucrose: fructan6-fructosyltransferase and fescue sucrose:sucrose1-fructosyltransferase[J]. FEBSLetters.2004,567:214–218.
148. Verhaest M, Lammens W, Le Roy K, et al. X-ray diffraction structure of a cell wall invertase fromArabidopsis thaliana[J]. Acta Crystallographica Section D-Biological Crystallography.2006,62:1555–1563.
149. Le Roy K, Verhaest M, Rabijns A, et al. N-glycosylation affects substrate specificity of chicory fructan1-exohydrolase: evidence for the presence of an inulin binding cleft[J]. New Phytologist.2007b,176:317–324.
2.王建华.微生物菊粉酶基因结构、酶学性质与应用研究进展[J].天然产物研究与开发,2001，13(1)：83-88.
3. Pandey A, Soccol C R, Selvakumar P, et al. Recent developments in microbial inulinases-Itsproduction, properties, and industrial applications[J]. Applied Biochemistry and Biotechnology,1999,81(1):35-52.
4. Vandamme E J, Derycke DG. Microbial InuIinases: Fermentation Process, Properties, andApplications[J]. Advances in applied microbiology.1983,29:139-176.
5. Nakamura T, Hoashi S, Nakatsu S. Culture conditions for inulinase production by Aspergillus[J].Nippon Nogei Kagakku Kaishi,1978,52(3):105-110.
6. Zittan L. Enzymatic hydrolysis of inulin—an alternative way to fructose production[J]. Starch,1981,33:373-377.
7. Nakamura T, Nakatsu S. General properties of extracellular inulase from Penicillium[J]. Nippon NogeiKagakku Kaishi,1977,51:681-689.
8. Kang S-I, Chang Y-J, Oh S-J, et al. Purification and properties of an endo-inulinase from anArthrobacter sp[J]. Biotechnology Letters,1998,20(10):983-986.
9. Kim D H, Choi Y J, Song S K, et al. Production of inulo-oligosaccharides using endo-inulinase from aPseudomonas sp[J]. Biotechnology Letters,1997,19(4):369-371.
10. Park J P, Bae J T, You D J, et al. Production of inulo-oligosaccharides from inulin by a novelendoinulinase from Xanthomonas sp[J]. Biotechnology Letters,1999,21:1043-1046.
11. Viswanathan P, Kulkarul P R. Enhancement of inulinase production by Aspergillus niger vanTeighem[J]. Journal of Applied Bacteriology,1995,78:384-386.
12. Nakamura T, Nagatomo Y, Hamada S, et al. Occurrence of two froms extracellular endoinulinase fromAspergillus niger mutant817[J]. Journal of Fermentation and Bioengineering,1994,78(2):134-139.
13. Bajon A M, Guiraud J P, Galzy P. Isolation of an inulinase derepressed mutant of Pichia polymorphafor the production of fructose[J]. Biotechnology and Bioengineering,1984,26:128-133.
14. Ricca E, Calabrò V, Curcio S, et al. The state of the art in the production of fructose from inulinenzymatic hydrolysis[J]. Critical Reviews in Biotechnology,2007,27:129-145.
15. Kushi R T, Monti R, Contiero J. Production, purification and characterization of an extracellularinulinase from Kluyveromyces marxianus var. bulgaricus[J]. Journal of Industrial Microbiology&Biotechnology,2000,25(2):63-69.
16. Sharma A D, Kainth S, Gill P K. Inulinase production using garlic (Allium sativum) powder as apotential substrate in Streptomyces sp.[J]. Journal of Food Engineering,2006,77(3): p486-491.
17. Gupta A K, Rathore P, Kaur N, et al. Production, Thermal-Stability and Immobilization of Inulinasefrom Fusarium-Oxysporum[J]. Journal of Chemical Technology and Biotechnology,1990,47(3):245-257
18. Laloux O, Cassart J, Delcour J, et al. Cloning and sequencing of the inulinase gene of Kluyveromycesmarxianus var. marxianus ATCC12424[J]. FEBS Letters.1991,289(1):64-68.
19. Rouwenhorst R J, Visser L E, Vanderbaan A A, et al. Production, Distribution, and Kinetic-Propertiesof Inulinase in Continuous Cultures of Kluyveromyces-Marxianus CBS-6556[J]. Applied andEnvironmental Microbiology,1988,54(5):1131-1137.
20. Ettalibi M, Baratti J C. Purification, properties and comparison of invertase, exoinulinases andendoinulinases of Aspergillus ficuum[J]. Applied Microbiology and Biotechnology,1987,26(1):13-20.
21. Ettalibi M, Baratti J C. Molecular and kinetic properties of Aspergillus ficuum inulinases[J].Agricultural and Biological Chemistry,1990,54(1):61-68.
22.魏文铃，余娴文，戴亚，郑晶，谢忠.克鲁维酵母Y-85菊粉酶的纯化和性质[J].微生物学报,1997,37(6):443-448.
23. Uhm T B, Jeon D Y, Myung B S, et al. Purification and properties of [beta]-fructofuranosidase fromAspergillus niger[J]. Biochimica et Biophysica Acta (BBA)-General Subjects,1987,926(2):119-126.
24. Rouwenhorst R J, Hensing M, Verbakel J, et al. Structure and Properties of the Extracellular Inulinaseof Kluyveromyces-Marxianus CBS-6556[J]. Applied and Environmental Microbiology,1990,56(11):3337-3345.
25. Ohta K, Akimoto H, Moriyama S. Fungal Inulinase: Enzymology, Molecular Biology andBiotechnology[J]. Journal of Applied Glycoscience,2004,51:247-254.
26. Xiao R, Tanida M, Takao S. Purification and some properties of endoinulinase from Chrysosporiumpannorum[J]. Journal of Fermentation and Bioengineering,1989,67(4):244-248.
27.张国青,崔福绵,杨秀清.毛壳霉内切菊粉酶的纯化与性质[J].微生物学报,2004,44(6):785-788.
28. Nakamura T, Shitara A, Matsuda S, et al. Production, purification and properties of an endoinulinaseof Penicillium sp. TN-88that liberates inulotriose[J]. Journal of Fermentation and Bioengineering,1997,84(4):313-318.
29. Cho Y J, Yun J W. Purification and characterization of an endoinulinase from Xanthomonas oryzaeNo.5.[J]. Process Biochemistry,2002,37(11):1325-1331.
30. Carniti P, Beltrame P L, Guardione D. Hydrolysis of Inulin-a Kinetic-Study of the ReactionCatalyzed by an Inulinase from Aspergillus ficuum[J]. Biotechnology and Bioengineering,1991,37(6):575-579.
31. Arand M, Golubev A M, Neto J R B, et al. Purification, characterization, gene cloning and preliminaryX-ray data of the exo-inulinase from Aspergillus awamori[J]. Biochemical Journal,2002,362:131-135.
32. Vullo D L, Coto C E, Sineriz F. Characteristics of an Inulinase Produced by Bacillus Subtilis430a, aStrain Isolated from the Rhizosphere of Vernonia herbacea (Vell Rusby)[J]. Applied andEnvironmental Microbiology,1991,57(8):2392-2394.
33. Workman W E, Day D F. Purification and properties of the [beta]-fructofuranosidase fromKluyveromyces fragilis[J]. FEBS Letters,1983,160(1-2):16-20.
34. Silva-Santisteban B O Y, Converti A, Maugeri F. Intrinsic activity of inulinase from Kluyveromycesmarxianus ATCC16045and carbon and nitrogen balances[J]. Food Technology and Biotechnology,2006,44(4):479-483.
35. Gill P K, Manhas R K, Singh J, et al. Purification and characterization of an exoinulinase fromAspergillus fumigatus[J]. Applied Biochemistry and Biotechnology,2004,117(1):19-32.
36. Gill P K, Manhas R K, Singh P. Purification and properties of a heat-stable exoinulinase isoform fromAspergillus fumigatus [J]. Bioresource Technology,2006,97(7):894-902.
37. Xiao R, Tanida M, Takao S. Purification and characteristics of two exoinulinases from Chrysosporiumpannorum[J]. Journal of Fermentation and Bioengineering,1989,67(5):331-334.
38. Moriyama S, Akimoto H, Suetsugu N, et al. Purification and properties of an extracellularexoinulinase from Penicillium sp strain TN-88and sequence analysis of the encoding gene[J].Bioscience Biotechnology and Biochemistry,2002,66(9):1887-1896.
39. Onodera S, Shiomi N. Purification and subsite affinities of exoinulinase from Penicillium-Trzebinskii[J]. Bioscience Biotechnology and Biochemistry,1992,56(9):1443-1447.
40. Sharma A D, Gill P K. Purification and characterization of heat-stable exo-inulinase fromStreptomyces sp.[J]. Journal of Food Engineering,2007,79(4):1172-1178.
41. Sheng J, Chi Z M, Gong F, et al. Purification and characterization of extracellular inulinase from amarine yeast Cryptococcus aureus G7a and inulin hydrolysis by the purified inulinase[J]. AppliedBiochemistry and Biotechnology,2008,144:111-121.
42. Kato K, Araki T, Kitamura T, et al. Purification and properties of a thermostable inulinase (β-D-fructanfructohydrolase) from Bacillus stearothermophilus KP1289[J]. Starch-St rke,1999,51(7):253-258.
43. Azhari R, Szlak A M, Ilan E, et al. Purification and characterization of endo-inulinase andexo-inulinase[J]. Biotechnology and Applied Biochemistry,1989,11(1):105-117.
44. Nagem R A P, Rojas A L, Golubev A M, et al. Crystal structure of exo-inulinase from Aspergillusawamori: the enzyme fold and structural determinants of substrate recognition[J]. Journal ofMolecular Biology,2004,344(2):471-480.
45. Uhm T B, Chae K-S, Lee D-W, et al. Cloning and nucleotide sequence of the endoinulinase-encodinggene, inu2, from Aspergillus ficuum[J]. Biotechnology Letters,1998,20(8):809-812.
46. Kim H S, Lee D W, Ryu E J, et al. Expression of the INU2gene for an endoinulinase of Aspergillusficuum in Saccharomyces cerevisiae[J]. Biotechnology Letters,1999,21(7):621-623.
47. Park S, Jeong H Y, Kim H S, et al. Enhanced production of Aspergillus ficuum endoinulinase inSaccharomyces cerevisiae by using the SUC2-deletion mutation[J]. Enzyme and Technology.2001,29:107-110.
48.王建华，滕达，姚怡等.黑曲霉菊粉内切酶基因和表达该基因的重组毕赤酵母菌株[P].中国专利,1594542,2005
49. Goosen C, Van der Maarel M J, Dijkhuizen L. Exo-inulinase of Aspergillus niger N402: A hydrolyticenzyme with significant transfructosylating activity[J]. Biocatalysis and Biotransformation,2008,26(1-2):49-58.
50. Zhang L, Wang Y, Ohta Y, et al. Expression of the inulinase gene from Aspergillus niger in Pichiapastoris [J]. Process Biochemistry,2003,38(8):1209-1212.
51. Zhang L H, Zhao C, Zhu D, et al. Inhibition of glucose on an exoinulinase from Kluyveromycesmarxianus expressed in Pichia pastoris [J]. Process Biochemistry,2005,40(5):1541-1545.
52. Wen T Q, Liu F, Huo K, et al. Cloning and analysis of the inulinase gene from Kluyveromycescicerisporus CBS4857[J]. World Journal of Microbiology&Biotechnology,2003,19(4):423-426.
53. Tsujimoto Y, Watanabe A, Nakano K, et al. Gene cloning, expression, and crystallization of athermostable exo-inulinase from Geobacillus stearothermophilus KP1289[J]. Applied Microbiologyand Biotechnology,2003,62(2):180-185.
54. Moriyama S, Tanaka H, Uwataki M, et al. Molecular cloning and characterization of an exoinulinasegene from Aspergillus niger strain12and its expression in Pichia pastoris[J]. Journal of Bioscienceand Bioengineering,2003,96(4):324-331.
55. Kwon H J, Jeon S J, You D J, et al. Cloning and characterization of an exoinulinase from Bacilluspolymyxa[J]. Biotechnology Letters,2003,25(2):155-159.
56. Akimoto H, Kiyota N, Kushima T, et al. Molecular cloning and sequence analysis of an endoinulinasegene from Penicillium sp strain TN-88[J]. Bioscience Biotechnology and Biochemistry,2000,64(11):2328-2335.
57. Kang S-I, Kim S-I. Molecular cloning and sequence analysis of an endo-inulinase gene fromArthrobacter sp.[J]. Biotechnology Letters,1999,21(7):569-574.
58. Yun J W, Choi Y J, Song C H, et al. Microbial production of inulo-oligosaccharides by anendoinulinase from Pseudomonas sp. expressed in Escherichia coli[J]. Journal of Bioscience andBioengineering,1999,87(3):291-295.
59. Liebl W, Brem D, Gotschlich A. Analysis of the gene for β-fructosidase (invertase, inulinase) of thehyperthermophilic bacterium Thermotoga maritima, and characterisation of the enzyme expressed inEscherichia coli[J]. Applied Microbiology and Biotechnology,1998,50(1):55-64.
60.王婧,张苓花,王运吉.克鲁维酵母菊粉酶基因在毕赤酵母中的表达[J].大连轻工业学院学报,2003,22(2):135-138.
61.张苓花，王运吉，叶淑红，等.菊粉酶酶源菌株筛选及其基因克隆[J].微生物学报，2002，42(3):321-325.
62. Wang J H，Teng D，Yao Y et al. Expression of Aspergillus niger9891endoinulinase in Pichiapastoris[J]. High Technology Letters,2004,10(4):52-56.
63. Singh P, Gill P K. Production of inulinases: recent advances[J]. Food Technology and Biotechnology,2006,44(2):151-162.
64. Reddy A, Maley F. Studies on identifying the catalytic role of Glu-204in the active site of yeastinvertase[J]. Journal of Biological Chemistry,1996,271:13953-13958.
65. Burne R A, Penders J E C. Characterization of the Streptococcus mutans GS-5fruA gene encodingexo-β-D-fructosidase[J]. Infection and Immunity,1992,60:4621-4632.
66. Gurr S J, Unkles S E, Kinghorn J R. The Structure and Organization of Nuclear Genes of FilamentousFungi, In:Gene Structure in Eukaryotic Microbes[M]. IRL Press, Oxford, UK,1987:93-139.
67. Ohta K, Akimoto H, Matsuda S, et al. Molecular cloning and sequence analysis of two endoinulinasegenes from Aspergillus niger[J]. Bioscience Biotechnology and Biochemistry,1998,62(9):1731-1738.
68. Yuan X L, Goosen C, Kools H, et al. Database mining and transcriptional analysis of genes encodinginulin-modifying enzymes of Aspergillus niger[J]. Microbiology,2006,152(10):3061-3073.
69. Yuan X L, Roubos J A, van den Hondel C A, et al. Identification of InuR, a new Zn(II)2Cys6transcriptional activator involved in the regulation of inulinolytic genes in Aspergillus niger[J].Molecular Genetics and Genomics,2008,279:11-26.
70. Park, S., Han Y, Kim H, et al., Trp17and Glu20residues in conserved WMN(D/E)PN motif aAreessential for Aspergillus ficuum endoinulinase (EC3.2.1.7) activity[J]. Biochemistry,2003.68:658-661
71. Kim K Y, Rhee S., Kim S.I., Role of the N-terminal domain of endoinulinase from Arthrobacter spS37in regulation of enzyme catalysis[J]. Journal of Biochemistry,2005.138(1):27-33.
72. Kim, K.Y., Nascimento A S, Golubev, A M. et al., Catalytic mechanism of inulinase from Arthrobactersp. S37[J]. Biochemical and Biophysical Research Communications,2008.371(4):600-605.
73. Chen H Q, Chen X M, Li Y, et al. Purification and characterization of exo-and endo-inulinase fromAspergillus ficuum JNSP5-06[J]. Food Chemistry,2009,115:1206-1212.
74.王静,金征宇,江波等. Aspergillus ficuum菊粉酶的荧光光谱研究[J].食品科学,2009,30(23):337-341
75. Van den Ende W, Michiels A, De Roover J, et al. Cloning and functional analysis of chicory rootfructan1-exohydrolase I (1-FEH): a vacuolar enzyme derived from a cell-wall invertase ancestor?[J].Mass fingerprint of the1-FEH I enzyme[J]. The Plant Journal,2000,24(4):447-456.
76. Onodera S, Murakami T, Ito H，et al. Molecular cloning and nucleotide sequences of cDNA and geneencoding endo-inulinase from Penicillium purpurogenum[J]. Bioscience Biotechnology andBiochemistry.1996,60(11):1780-1785.
77. Kwon H J, Jeon S J, You D J, et al. Cloning and characterization of an exoinulinase from Bacilluspolymyxa[J]. Biotechnology Letters.2003,25(2):155-159.
78. Garber R C. Isolation of DNA from filamemtous fungi and separation into nuclear mitochondrial,ribosomal, and plasmid components [J]. Anal Biochem,1983,135:416–422.
79. Joseph S, David W R著.黄培堂,王恒樑,周晓巍,等译.分子克隆实验指南[M].北京:化学工业出版社,2007,48–49.
80. Zhang X E, Zhou Y H, Zhang Z P. Engineering E. coli alkaline phosphatase yields changes of catalyticactivity, thermal stability and phosphate inhibition. Biocatalysis and Biotransformation,2002,20(6):381-389.
81. Yang X, McGraw R A, Su X. Canine thyrotropin beta-subunit gene:cloning and expression inEscherichia coli, generation of monoclonal antibodies,and transient expression in the Chinese hamsterovary cells. Domestic Animal Endocrinology,2000,18(4):363-378.
82.陆长梅,袁生,赵庆新.用Overlap-PCR法从Trichoderma reesei QM9414基因组DNA中克隆并表达木聚糖酶III.生物工程学报.2004,20(5):764-769.
83.王建华,刘艳艳.一步法制备高果糖浆工程及产酶菌体营养价值研究[J].食品与发酵工业,2000,26(2):1-4.
84. Yokota A, Yamauchi O and Tomita F. Production of inulotriose from inulin by inulin-degradingenzyme from Streptomyces rochei E87[J]. Letters in Applied Microbiology,1995,21:330-333.
85. Onodera S. and Shiomi N. Purification and substrate specificity of endo-type inulinase fromPenicillium purpurogenum[J]. Agricultural and Biological Chemistry,1988,52(10):2569-2576.
86. Cho Y J, Sinha J, Park J P and Yun J W. Production of inulooligosaccharides from chicory extract byendoinulinase from Xanthomonas oryzae No.5[J]. Enzyme and Microbial Technology,2001,28:439-445.
87. Yun J W, Kim D H, Kim B W, and Song S K. Production of inulo-oligosaccharides from inulin byimmobilized endoinulinase from Pseudomonas sp[J]. Journal of Fermentation and Bioengineering,1997,84(4):369-371.
88. Jin Z Y, Wang J, Jiang B, et al. Production of inulooligosaccharides by endoinulinases fromAspergillus ficuum [J]. Food Research International,2005,38:301–308
89. Ohta K, Hamada S and Nakamura T. Production of high concentrations of ethanol from inulin bysimultaneous saccharification and fermentation using Aspergillus niger and Saccharomycescerevisiae[J]. Applied and Environmental Microbiology,1993,59:729-733.
90. Schorr-Galindo S, Fontana A and Guiraud J P. Fructose syrups and ethanol production by selectivefermentation of inulin[J]. Current Microbiology,1995,30:325-330.
91. Margaritis A and Bajpai P. Ethanol production from Jerusalem artichoke tubers(Helianthus tuberosus)using Kluyveromyces marxianus and Saccharomyces rosei[J]. Biotechnology and Bioengineering,1982,24:941-953.
92. Kim, D. M. and H. S. Kim. Continuous production of gluconic acid and sorbitol fromJerusalem-artichoke and glucose using an oxidoreductase of zymomonas-mobilis and inulinase[J].Biotechnology and Bioengineering,1992,39(3):336-342.
93. Shin YC, Kim YH, Lee HS, et al. Production of exopolysaccharide pullulan from inulin by a mixedculture of Aureobasidium pullulans and Kluyveromyces fragilis[J]. Biotechnol Bioeng.1989,33(1):129-133.
94. Oiwa H, Naganuma M, Ohinuma S, Acetone-butanol pro-duction from dahlia inulin by Clostridiumpasteurianum var.1-53[J]. Agric Biol Chem.1987,51(10):2819-2820
95.华承伟,王建华,滕达,等.菊粉化学和微生物菊粉内切酶研究进展[J].中国食品学报,2004,4(4):103-108
96. Wang J, Jin Z, Jiang B, et al. Production and separation of exo-and endoinulinase from Aspergillusficuum[J]. Process Biochemistry,2003,39:5-11
97. Moriyama S, Muguruma M, and Ohta K. Quantitative expression analysis of inulinase gene cluster ofPenicillium sp. strain TN-88[J]. Journal of Bioscience and Bioengineering,2006,101(3):277-279.
98.周晨妍.宇佐美曲霉木聚糖酶基因的克隆、表达及定向诱变研究[D]:[博士学位论文].无锡:江南大学,2008.
99.李胤.阿拉伯木聚糖溶解、降解机制的研究及酸性木聚糖酶基因的克隆、表达[D]:[博士学位论文].无锡:江南大学,2008.
100.宁正祥.食品成分分析[M].北京：中国轻工业出版社,1997.
101.诸葛健,王正祥.工业微生物实验技术手册[M].北京：中国轻工业出版社,1994.
102. Sambrook J, Frisch E F, Maniatis T. Molecular Cloning: A laboratory manual,2nd edition [M]. NewYork: Cold Spring Harbor Laboratory Press,1989.
103. Sengupta S, Jana M L, Sengupta D, et al. A note on the estimation of microbiol glycosidase activitiesby dinitrosalicylic acid reagent[J]. Appl. Microbiol. Biotechnol.2000,53:732-735.
104. Bradford M M. A rapid sensitive method for the quantitation of microgram quantities of proteinutilizing the principle of protein-dye binding[J]. Analytical Biochemistry,1976,72:248-254.
105. Laemmli U K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature (London),1970,227:680-685.
106. R.G. Harrison. Expression of soluble heterologous proteins via fusion with NusA protein. inNovations.2000,11:4-7.
107. Davis G.D, Elisee C, Newham D.M. er al. New fusion protein systems designed to give solubleexpression in Escherichia coli. Biotechnol. Bioeng.1999,65(4):382-8.
108. Wilkinson D L. and Harrison R G. Predicting the solubility of recombinant proteins in Escherichia coli.Bio/Technology.1991,9:443-448.
109. Fuhrmann M, Hausherr A, Ferbitz L, et al. Monitoring dynamic expression of nuclear genes inChlamydomonas reinhardtii by using a synthetic luciferase reporter gene. Plant Mol Biol.2004Aug;55(6):869-81.）
110. Martinez A, Krappskreg P M, Olafsdottir S, et al. Expression of recombinant human phenylalaninehydroxylase as fusion protein in Escherichia coli circumvents proteolytic degradation by host cellproteases [J]. J Biotechnol,1995,306:589–597
111. Luli G W, Strolh W R. Comparison of growth,acetate production,and acetate inhibition of Escherichiacoli strains in batch and fed-batch fermentations [J]. Appl Environ Microbiol,1990,56:1004–1011
112.唐威华，张景六，王宗阳，等. SDS-PAGE法测定His-tag融合蛋白分子量产生偏差的原因[J].植物生理学报,2000,26(1):64-68.
113. Skowronek, M. and J. Fiedurek. Purification and properties of extracellular endoinulinase fromAspergillus niger20OSM[J]. Food Technology and Biotechnology.2006,44(1):53-58.
114. Kim K Y, and Kang S I. Production of a novel endo-inulinase from Arthrobacter sp. S37[J]..Agricultural Chemistry and Biotechnology.1996,.39(2):99-103.
115. Gupta A K, Singh D P, Kaur N, et al. Production, purification and immobilization of inulinase fromKluyveromyces fragilis[J].. J. Chem. Tech. Biotechnol.,1994,59:377-385.
116. Gaye O B, Sukan S, Vassilew M. Production and properties of inulinase from Aspergillus nige[J].r.Biotechnol. lett.,1994,16(3):275-280.
117. Nakamura T, Kurokawa T, Nakatsu S, et al. Crystallization and general properties of an extracellularinulinase from Aspergillus sp[J].. Nippon Nogeikagaku Kaishi,1978,52:159-166.
118. Warrens A N, Jones M D, Lechler R I. Splicing by overlap extension by PCR using asymmetricamplification: An improved technique for the generation of hybrid proteins of immunologicalinterest[J]. Gene,1997,186(1):29-35
119.刘仙,高国粉,杨丽,等.菊粉酶中色氨酸残基的化学修饰及其荧光光谱[J].高等学校化学学报.2007,28(1):103-105.
120.刘彬,王静云,包永明,等.化学修饰法表征Bacillus smithii T7产耐热菊粉酶催化活性中心的必需氨基酸残基[J].催化学报.2009,30(7):673-678.
121. Takahare S, Morohashi T, Sano T, et al. Fructooligosaccharide consumption enhances femoral bonevolume and mineral concentrations in rates[J]. Journal of Nutrition,2000,130(7):1792-1795.
122. Roberfroid M B, Van L J and Gibson G R. The bifidogenic nature of chicory inulin and its hydrolysisproducts[J]. Journal of Nutrition,1998,128(1):11-19.
123.刘协,胡启玉,李小宁.低聚果糖的润肠通便功能研究[J].江苏预防医学,2002,13(1):62-63
124.尤新.功能性低聚糖[J].中国食品添加剂,2002,(2):40-45.
125. Tomoatsu H. Health effects of oligosaccharides[J]. Food Technology,1994,48:61-65.
126.潘国熙.一种新颖保健甜味剂－低聚果糖[J].广西轻工业,1995,(1):7-9.
127. Ohta A, Ohtsuki M, Uehara M, et al. Dietary fructooligosaccharides prevent post-gastrectomyanemia and osteopenia in rats[J]. Journal of Nutrition,1998,128(3):485-490.
128. Sakai K, Ohta A, Shiga K, et al. The cecum and dietary short-chain fructooligo-saccharides areinvolved in preventing postgastrectomy anemia in rates[J]. Journal of Nutrition,2000,130(6):1608-1612.
129. Mckellar R C and Modler H W. Metabolism of fructo-oligosaccharides by Bifidobacterium spp [J].Applied Microbiology and Biotechnology,1989,31:537-541.
130. Kaplan H and Hutkins R W. Fermentation of fructooligosaccharides by lactic acid bacteria andbifidobacteria[J]. Applied and Environmental Microbiology,2000,66:2682-2684.
131.潘国熙.一种新颖保健甜味剂－低聚果糖[J].广西轻工业,1995,(1):7-9.
132. Arnold K, Bordoli L, Kopp J, et al. The SWISS-MODEL Workspace: A web-based environment forprotein structure homology modelling[J]. Bioinformatics.2006,22:195-201.
133. Kiefer F, Arnold K, Künzli M, et al. The SWISS-MODEL Repository and associated resources[J].Nucleic Acids Research.2009,37:387-392.
134. Peitsch M. C. Protein modeling by E-mail[J]. Bio/Technology.1995,13:658-660.
135. Fisher C L, Pei G K. Modification of a PCR-based site-directed mutagenesis method[J].Biotechniques.1997,23:570-574.
136.李松林.曲霉CJ22-326产壳聚糖酶基因的克隆、表达与定点突变[D]:[博士学位论文].无锡:江南大学,2009.
137. Reddy A, Maley F. Identification of an active-site residue in yeast invertase by affinity labeling andsite-directed mutagenesis[J]. J. Biol. Chem.1990,265:10817-10820.
138. Reddy A, Maley F. Studies on identifying the catalytic role of Glu-204in the active site of yeastinvertase[J]. J. Biol. Chem.1996,271:13953-13958.
139. Alberto F, Bignon C, Sulzenbacher G, et al. The threedimensional structure of invertase(beta-fructosidase) from Thermotoga maritima reveals a bimodular arrangement and an evolutionaryrelationship between retaining and inverting glycosidases[J]. J. Biol. Chem.2004,279:18903–189
140. Henrissat B. A classification of glycosyl hydrolases based on amino-acid sequence similarities[J].Biochem. J.1991,280:309-316
141. Cantarel BL, Coutinho PM, Rancurel C, et al. The Carbohydrate-Active EnZymes database (CAZy):an expert resource for Glycogenomics[J]. Nucleic Acids Res.2009,37:D233-238
142. Coutinho PM, Henrissat B. Carbohydrate-active enzymes: an integrated database approach. In: GilbertHJ, Davies GJ, Henrissat B, Svensson B, eds. Recent advances in carbohydrate bioengineering[J].Cambridge, UK: Royal Society of Chemistry.1999,3–12.
143. Naumoff DG. Beta-fructosidase superfamily: homology with some alpha-L-arabinases andbeta-D-xylosidases[J]. Proteins–Structure Function and Genetics.2001,42:66–76.
144. Pons T, Naumoff DG, Martinez-Fleites C, et al. Three acidic residues are at the active site of abeta-propeller architecture in glycoside hydrolase families32,43,62, and68[J]. Proteins: Structure,Function, and Bioinformatics.2004,54:424–432.
145. Holm L, Sander C. Mapping the protein universe[J]. Science.1996,273:595–603.
146. Peumans WJ, Van Damme EJ. Lectins as plant defense proteins[J]. Plant Physiology.1995,109:347–352.
147. Altenbach D, Nuesch E, Meyer AD, et al. The large subunit determines catalytic specificity of barleysucrose: fructan6-fructosyltransferase and fescue sucrose:sucrose1-fructosyltransferase[J]. FEBSLetters.2004,567:214–218.
148. Verhaest M, Lammens W, Le Roy K, et al. X-ray diffraction structure of a cell wall invertase fromArabidopsis thaliana[J]. Acta Crystallographica Section D-Biological Crystallography.2006,62:1555–1563.
149. Le Roy K, Verhaest M, Rabijns A, et al. N-glycosylation affects substrate specificity of chicory fructan1-exohydrolase: evidence for the presence of an inulin binding cleft[J]. New Phytologist.2007b,176:317–324.