红色亚栖热菌海藻糖合酶结构与功能的关系及定向进化的研究
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摘要
海藻糖是一种应用广泛的非还原性双糖，在医学、化妆品行业、制药学、食品加工业以及农业等领域都有十分诱人的应用前景。而利用酶法制备海藻糖是现今生产海藻糖的主要手段。在可制备海藻糖的各种酶类中，海藻糖合酶可以利用麦芽糖为底物通过一步反应生产海藻糖，整个过程简单可控，原料成本低廉，因此引起了人们越来越多的关注。然而目前天然的海藻糖合酶其结构与功能之间的关系还未被人们所了解，并且存在着催化效率较低，反应产生的副产物葡萄糖较多等问题，所以急需对该酶的结构功能进行研究，并且建立适合于海藻糖合酶定向进化的方法，对该酶进行改造。
我们前期已经克隆得到来源于红色亚栖热菌的海藻糖合酶基因，并成功地将其在大肠杆菌中进行异源表达。该酶具有良好的嗜热性以及稳定性，因此具有工业应用潜力。但是如果将该酶直接用于工业生产，那么它的活性还不是很高，有进一步提升的空间。同时对该酶的结构、作用机理以及热稳定性机制也还不清楚，有必要进行研究，为进一步提升酶的催化效率或热稳定性提供理论依据。
在本研究中，首先构建了红色亚栖热菌N端缺失突变子与C端缺失突变子两种缺失突变蛋白。通过对二者活性及二级结构分析发现，仅含C端结构域的蛋白与全长蛋白的二级结构相似，均为α/β型，这也是α淀粉酶家族的典型结构。、缺失C端而仅含有N端结构域的蛋白二级结构发生了变化，说明红色亚栖热菌海藻糖合酶的C端区域对维持蛋白结构稳定性有重要作用。
通过构建一系列长度递减的C端片段缺失蛋白，我们发现C端缺失44个氨基酸后，蛋白依然具有活性，而且最适反应温度依然保持在50℃，但在60℃保温1h后残余酶活低于全长蛋白，说明红色亚栖热菌海藻糖合酶的C端能够影响蛋白的热稳定性。而C端缺失44个氨基酸的截短蛋白对底物选择性也发生了改变，它对麦芽糖的亲和力更高，说明红色亚栖热菌海藻糖合酶C端结构域可能对酶与底物的结合有影响。而当C端缺失68个或者更多氨基酸残基时，异源表达的蛋白无法正确折叠为可溶性蛋白，即使经过蛋白质复性后依然没有活性，说明从C末端第68个氨基酸开始的C端片段能够影响蛋白结构与功能。
其次，构建了红色亚栖热菌海藻糖合酶与嗜热栖热菌海藻糖合酶C端与N端互换的杂合蛋白TSTtMr与TSMrTt。通过对两种杂合蛋白的表达纯化，测定酶学动力学常数以及温度耐受性等酶学性质，发现拥有相同N端结构域的蛋白，其酶学动力学常数也大致相似，而且有相同的最适反应温度与类似的温度耐受性，说明海藻糖合酶的N端结构域与催化活性密切相关，同时影响酶的嗜热性和热稳定性。而杂合蛋白TSTtMr与麦芽糖的亲和能力较红色亚栖热菌海藻糖合酶有所提高，而能够反映催化效率的kcat/Km值是红色亚栖热菌海藻糖合酶的2倍，说明该杂合蛋白催化效率更高。
第三，为了研究影响红色亚栖热菌海藻糖合酶活性与热稳定性的具体区域或位点，本研究对该酶的三维结构进行模拟，并对预测得到的关键位点进行定点突变研究。通过Swiss-Model同源建模，发现红色亚栖热菌海藻糖合酶N端第3位-第543位氨基酸残基属于α淀粉酶家族成员，拥有类似于β/α的结构，该模拟结构中含有一个“口袋”区域，其中含有4个α淀粉酶家族保守区及关键氨基酸残基。通过定点突变，能够确定位于保守区中的H104、D200以及第三保守区对海藻糖合酶的活性起至关重要的作用，当将这些关键位点突变后，蛋白彻底失去活性。此外，位于口袋结构中的Y135、R388、R392位点也影响蛋白的功能，将它们突变为丙氨酸后，蛋白发生了活性丧失或下降。R392A的突变还会影响蛋白的嗜热性，使其在50℃失活，而在30℃具有活性，但与野生型蛋白相比，催化能力大大降低。说明R392位点或附近相关区域能够影响蛋白活性及嗜热性。
第四，对红色亚栖热菌海藻糖合酶的定向进化进行了研究。确立了利用甲苯透性化细胞制备粗酶，经过DNS反应后在570nm处测定吸光度，以检验红色亚栖热菌海藻糖合酶活性高低的高通量筛选方法。运用易错PCR以及分段DNA改组法对红色亚栖热菌海藻糖合酶进行定向进化的初步研究，经过一轮易错PCR和一轮分段DNA改组筛选得到一株粗酶活力为野生型红色亚栖热菌海藻糖合酶1.6倍的突变株，该突变子共发生了6个氨基酸位点的突变。将突变蛋白纯化后，研究该酶的催化动力学常数，发现其对麦芽糖为底物时的Km值约是野生型的一半，表示突变子对底物的亲和能力提高了一倍，而突变子的kcat/Km值是野生型的2倍，说明该突变子的催化效率则是野生型的2倍。
Trehalsoe is a non-reducing disaccharide widely used in pharmaceutical industry,cosmetic industry, food, agriculture, and many other fields. Because of its ability ofprotection, trehalose attracts lots of interest. Nowadays, trehalose is mainlymanufactured through enzymatic pathways. Among the enzymes which couldproduce trehalose, trehalose synthase (TreS) could convert maltose into trehalose inone step reaction, and the raw material is cheap, which makes it get more and moreconcerning. However, the relationship between the structure and function of TreS hasnot been investigated deeply. Moreover, the efficiency of some native TreS is not veryhigh. So it is important to study about the relationship between the structure andfunction and construct the method for directed evolution of TreS.
In our previous study, the treS gene had been cloned from a thermophilicMeiothermus ruber strain CBS-01and expressed in Escherichia coli to characterizeits properties. Because of its thermophilicity and thermostability, TreS from M. ruber(TSM) was fit to produce trehalose in industry. However, the activity of TSM was nothigh enough to be applied in industry. It was necessary to improve the efficiency ofTSM for application and investigate the thermoadapatation mechanism of theenzyme.
Firstly, the N-and C-terminal domains of TSM were constructed and expressedin E. coli. The C-termianl domain and wild-type TSM shared the similar secondarystructure, both of which were α/β type，a typical structure of α-amylase super family.While the N-terminal domain without C-terminal region underwent the change insecondary structure. It implied that the C-terminal domain was important for themaintenance of the structure of protein.
Moreover, a series of proteins with different deletion at C terminus of TSM wereconstructed to find the region which affected the function of TSM. In a result, thetruncated protein ΔC44, which was deleted44amino acid residues from C terminus,changed the affinity to the substrates. It preferred maltose to trehalose as its substrate. Meanwhile, the truncated protein ΔC68and more amino acid residues missed from Cterminus could not fold correctly in solution form. The result showed that the44amino acid residues from C terminus may affect the linkage between the protein andsubstrate, while the region from the residue68from C terminus played a key role inthe protein folding.
Secondly, the N-terminal and C-terminal domains of TSM and trehalosesynthase from Thermus thermophilus (TST) were switched to ascertain which of themplayed the important role in the characteristics and function of TreS. Two fusionproteins TSTtMr (N-terminal domain of TST fused with C-terminal domain of TSM)and TSMrTt (N-terminal domain of TSM and C-terminal domain of TST) wereconstructed. The enzymes with the same N-terminal domains shared the similarkinetics parameters and optimum temperature, indicating that the N-terminal domainsthemselves also play a major role in determining the thermostability and activity ofenzymes. Additionally, the fusion protein TSTtMr displayed the higher kcat/Kmvaluethan that of TSM, indicating that it could convert maltose to trehalose moreefficiently from a kinetic point of view.
Thirdly, the three-dimensional structure of the N-terminal domain (3-543residues) of TSM was predicted at the tertiary level as a GH13domain of α-amylasesuper family. There was a putative catalytic cleft in the N-terminal domain. Fourconserved region and key sites of α-amylase super family were located in the cleft.Through site-directed mutagenesis, H104, D200, and the third conserved region werefound to play important roles in the activity of TSM. Besides, Y135, R388, and R392,which were also located in the cleft, could affect the function of TSM as well. Themutagenesis at these sites led to the complete loss or sharp decrease in the activity.The mutant R392A was inactive at50℃and kept a little activity at30℃, suggestingthat R392or the region near R392could influence the activity and thermophilicity ofTSM.
Fourthly, the method for the directed evolution of TSM was constructed. Aproper process to prepare the crude enzyme was achieved with cell suspension treatedby2%toluene to get permeabilized cells. After DNS analysis, the absorbance at570nm was used to compare the activity of the mutants. Moreover, error-prone PCR and DNA shuffling for the directed evolution of TSM were performed to construct thelibrary of mutants. After screening the library, a mutant with6site substitution wasselected. The activity of the mutant was1.6fold of that of wild-type TSM. The Kmvalue of the mutant was a half of TSM, implying the affinity of mutant was2-foldhigher. And the catalytic efficiency was2-fold of wild-type TSM.
我们前期已经克隆得到来源于红色亚栖热菌的海藻糖合酶基因，并成功地将其在大肠杆菌中进行异源表达。该酶具有良好的嗜热性以及稳定性，因此具有工业应用潜力。但是如果将该酶直接用于工业生产，那么它的活性还不是很高，有进一步提升的空间。同时对该酶的结构、作用机理以及热稳定性机制也还不清楚，有必要进行研究，为进一步提升酶的催化效率或热稳定性提供理论依据。
在本研究中，首先构建了红色亚栖热菌N端缺失突变子与C端缺失突变子两种缺失突变蛋白。通过对二者活性及二级结构分析发现，仅含C端结构域的蛋白与全长蛋白的二级结构相似，均为α/β型，这也是α淀粉酶家族的典型结构。、缺失C端而仅含有N端结构域的蛋白二级结构发生了变化，说明红色亚栖热菌海藻糖合酶的C端区域对维持蛋白结构稳定性有重要作用。
通过构建一系列长度递减的C端片段缺失蛋白，我们发现C端缺失44个氨基酸后，蛋白依然具有活性，而且最适反应温度依然保持在50℃，但在60℃保温1h后残余酶活低于全长蛋白，说明红色亚栖热菌海藻糖合酶的C端能够影响蛋白的热稳定性。而C端缺失44个氨基酸的截短蛋白对底物选择性也发生了改变，它对麦芽糖的亲和力更高，说明红色亚栖热菌海藻糖合酶C端结构域可能对酶与底物的结合有影响。而当C端缺失68个或者更多氨基酸残基时，异源表达的蛋白无法正确折叠为可溶性蛋白，即使经过蛋白质复性后依然没有活性，说明从C末端第68个氨基酸开始的C端片段能够影响蛋白结构与功能。
其次，构建了红色亚栖热菌海藻糖合酶与嗜热栖热菌海藻糖合酶C端与N端互换的杂合蛋白TSTtMr与TSMrTt。通过对两种杂合蛋白的表达纯化，测定酶学动力学常数以及温度耐受性等酶学性质，发现拥有相同N端结构域的蛋白，其酶学动力学常数也大致相似，而且有相同的最适反应温度与类似的温度耐受性，说明海藻糖合酶的N端结构域与催化活性密切相关，同时影响酶的嗜热性和热稳定性。而杂合蛋白TSTtMr与麦芽糖的亲和能力较红色亚栖热菌海藻糖合酶有所提高，而能够反映催化效率的kcat/Km值是红色亚栖热菌海藻糖合酶的2倍，说明该杂合蛋白催化效率更高。
第三，为了研究影响红色亚栖热菌海藻糖合酶活性与热稳定性的具体区域或位点，本研究对该酶的三维结构进行模拟，并对预测得到的关键位点进行定点突变研究。通过Swiss-Model同源建模，发现红色亚栖热菌海藻糖合酶N端第3位-第543位氨基酸残基属于α淀粉酶家族成员，拥有类似于β/α的结构，该模拟结构中含有一个“口袋”区域，其中含有4个α淀粉酶家族保守区及关键氨基酸残基。通过定点突变，能够确定位于保守区中的H104、D200以及第三保守区对海藻糖合酶的活性起至关重要的作用，当将这些关键位点突变后，蛋白彻底失去活性。此外，位于口袋结构中的Y135、R388、R392位点也影响蛋白的功能，将它们突变为丙氨酸后，蛋白发生了活性丧失或下降。R392A的突变还会影响蛋白的嗜热性，使其在50℃失活，而在30℃具有活性，但与野生型蛋白相比，催化能力大大降低。说明R392位点或附近相关区域能够影响蛋白活性及嗜热性。
第四，对红色亚栖热菌海藻糖合酶的定向进化进行了研究。确立了利用甲苯透性化细胞制备粗酶，经过DNS反应后在570nm处测定吸光度，以检验红色亚栖热菌海藻糖合酶活性高低的高通量筛选方法。运用易错PCR以及分段DNA改组法对红色亚栖热菌海藻糖合酶进行定向进化的初步研究，经过一轮易错PCR和一轮分段DNA改组筛选得到一株粗酶活力为野生型红色亚栖热菌海藻糖合酶1.6倍的突变株，该突变子共发生了6个氨基酸位点的突变。将突变蛋白纯化后，研究该酶的催化动力学常数，发现其对麦芽糖为底物时的Km值约是野生型的一半，表示突变子对底物的亲和能力提高了一倍，而突变子的kcat/Km值是野生型的2倍，说明该突变子的催化效率则是野生型的2倍。
Trehalsoe is a non-reducing disaccharide widely used in pharmaceutical industry,cosmetic industry, food, agriculture, and many other fields. Because of its ability ofprotection, trehalose attracts lots of interest. Nowadays, trehalose is mainlymanufactured through enzymatic pathways. Among the enzymes which couldproduce trehalose, trehalose synthase (TreS) could convert maltose into trehalose inone step reaction, and the raw material is cheap, which makes it get more and moreconcerning. However, the relationship between the structure and function of TreS hasnot been investigated deeply. Moreover, the efficiency of some native TreS is not veryhigh. So it is important to study about the relationship between the structure andfunction and construct the method for directed evolution of TreS.
In our previous study, the treS gene had been cloned from a thermophilicMeiothermus ruber strain CBS-01and expressed in Escherichia coli to characterizeits properties. Because of its thermophilicity and thermostability, TreS from M. ruber(TSM) was fit to produce trehalose in industry. However, the activity of TSM was nothigh enough to be applied in industry. It was necessary to improve the efficiency ofTSM for application and investigate the thermoadapatation mechanism of theenzyme.
Firstly, the N-and C-terminal domains of TSM were constructed and expressedin E. coli. The C-termianl domain and wild-type TSM shared the similar secondarystructure, both of which were α/β type，a typical structure of α-amylase super family.While the N-terminal domain without C-terminal region underwent the change insecondary structure. It implied that the C-terminal domain was important for themaintenance of the structure of protein.
Moreover, a series of proteins with different deletion at C terminus of TSM wereconstructed to find the region which affected the function of TSM. In a result, thetruncated protein ΔC44, which was deleted44amino acid residues from C terminus,changed the affinity to the substrates. It preferred maltose to trehalose as its substrate. Meanwhile, the truncated protein ΔC68and more amino acid residues missed from Cterminus could not fold correctly in solution form. The result showed that the44amino acid residues from C terminus may affect the linkage between the protein andsubstrate, while the region from the residue68from C terminus played a key role inthe protein folding.
Secondly, the N-terminal and C-terminal domains of TSM and trehalosesynthase from Thermus thermophilus (TST) were switched to ascertain which of themplayed the important role in the characteristics and function of TreS. Two fusionproteins TSTtMr (N-terminal domain of TST fused with C-terminal domain of TSM)and TSMrTt (N-terminal domain of TSM and C-terminal domain of TST) wereconstructed. The enzymes with the same N-terminal domains shared the similarkinetics parameters and optimum temperature, indicating that the N-terminal domainsthemselves also play a major role in determining the thermostability and activity ofenzymes. Additionally, the fusion protein TSTtMr displayed the higher kcat/Kmvaluethan that of TSM, indicating that it could convert maltose to trehalose moreefficiently from a kinetic point of view.
Thirdly, the three-dimensional structure of the N-terminal domain (3-543residues) of TSM was predicted at the tertiary level as a GH13domain of α-amylasesuper family. There was a putative catalytic cleft in the N-terminal domain. Fourconserved region and key sites of α-amylase super family were located in the cleft.Through site-directed mutagenesis, H104, D200, and the third conserved region werefound to play important roles in the activity of TSM. Besides, Y135, R388, and R392,which were also located in the cleft, could affect the function of TSM as well. Themutagenesis at these sites led to the complete loss or sharp decrease in the activity.The mutant R392A was inactive at50℃and kept a little activity at30℃, suggestingthat R392or the region near R392could influence the activity and thermophilicity ofTSM.
Fourthly, the method for the directed evolution of TSM was constructed. Aproper process to prepare the crude enzyme was achieved with cell suspension treatedby2%toluene to get permeabilized cells. After DNS analysis, the absorbance at570nm was used to compare the activity of the mutants. Moreover, error-prone PCR and DNA shuffling for the directed evolution of TSM were performed to construct thelibrary of mutants. After screening the library, a mutant with6site substitution wasselected. The activity of the mutant was1.6fold of that of wild-type TSM. The Kmvalue of the mutant was a half of TSM, implying the affinity of mutant was2-foldhigher. And the catalytic efficiency was2-fold of wild-type TSM.
引文
[1] Elbein AD, Pan YT, Pastuszak I, et al. New insights on trehalose: a multifunctional molecule.Glycobiology,2003,13:17R-27R.
[2] Berthelot M. Sur le trehalose, nouvelle espece de sucre. Compt. Rend. Hebd. Seanc Acad Sci.,Paris1858,46:1276-1279.
[3] Elbein AD. The metabolism of α, α-trehalose. Adv. Carbohydr Chem Biochem,1974,30:227-256.
[4] Hudson CS. Some numerical relations among the rotatory powers of the compound sugars. JAm Chem Soc,1916,38:1566-1575.
[5] Taga T, Senma M, Osaki K. The crystal and molecular structure of trehalose dihydrate. ActaCrystallog,.1972, B28:3258-3263
[6] Teramoto N, Sachinvala ND, Shibata M. Trehalose and trehalose-based polymers forenvironmentally benign, biocompatible and bioactive material. Molecules,2008,13:1773-1816
[7] Roser B. Trehalose, a new approach to premium dried foods. Trends Food Sci Technol,1991,2:166-169.
[8] Wyatt GR, Kalf GF. The chemistry of insect hemolymph: II. Trehalose and othercarbohydrates. J Gen Physiol,1957,40:833-847.
[9] Cher B, and Klingenberg M. Wege des Wasserstoffs in der lebendigen Organisation. AngewChem,1958,70:552-570.
[10] Evans DR, Dethier VG. The regulation of taste thresholds for sugars in the blowfly. J Insecthysiol,1957,1:3-17
[11] Becker A, Schloeder P, Steele JE, and Wegener G. The regulation f trehalose metabolism ininsects. Experientia,1996,52:433-439.
[12] Juan Carlos Argüelles. Physiological roles of trehalose in bacteria and yeasts: a comparativeanalysis. Arch Microbiol,2000,174:217-224
[13] Spargo BJ, Crowe LM, Ioneda T, et al. Cord factor (α,α-trehalose6,6’ dimycolate) inhibitsfusion between phospholipid vesicles. Proc Natl Acad Sci,1991,88:737–740
[14]Reed RH, Richardson, DL, Warr SR, Stewart WD. Carbohydrate accumulation and osmoticstress in Cyanobacteria. J Gen Microbiol,1984,130:1–4
[15]Mikkat S, Effmert U, Hagemann M. Uptake and use of the osmoprotective compoundstrehalose, glucosylglycerol, and sucrose by the Cyanobacterium synechocystis sp.PCC6803.Arch Microbiol,1997,167(2-3):223-8
[16] Welsh DT, Guyoneaud T, Caumette P. Utilization of the compatible solutes sucrose andtrehalose by purple sulphur and nonsulphur bacteria. Can J Microbiol,1998,4:974–979
[17] Galinski EA, Herzog RM The role of trehalose as a substitute for nitrogen-containingcompatible solutes (Ectothiorhodospira halochloris) Arch Microbiol,1990,153:607–613
[18] Roser B. Trehalose, a new approach to premium dried foods. Trends Food Sci Tech,19912:166–169.
[19] Adams RP, Kendall E, Kartha KK. Comparison of free sugars in growing and desiccatedplants of Selaginella lepidophylla. Biochem Syst Ecol,1990,18:107–110.
[20] McBride MJ, Ensign JC. Effects of intracellular trehalose content on Streptomyces griseusspores. J Bacteriol,1987,169(11):4995–5001.
[21] Van Dijck P, Colavizza D, Smet P, et al. Differential importance of trehalose in stressresistance in fermenting and nonfermenting Saccharomyces cerevisiae cells. Appl EnvironMicrobiol,1995,61(1):109–115.
[22] Clegg JS. The origin of trehalose and its significance during the formation of encysteddormant embryos of Artemia salina. Comp Biochem Physiol,1965,14:135–143.
[23] Womersley C, Smith L. Anhydrobiosis in nematodes. I. The role of glycerol, myo-inositoland trehalose during dessication. Comp Biochem Physiol,1981,70B:579–586.
[24] Newman YM, Ring SG, Colaco C. The role of trehalose and other carbohydrates inbiopreservation. Biotechnol Genet Eng Rev,1993,11:263–294.
[25]Gadd GM, Chalmers K, Reed RH. The role of trehalose in dehydration resistance ofSaccharomyces cerevesiae. FEMS Microbiol Lett,1987,48:249–254.
[26] Crowe,L.M., Crowe,J.H. Trehalose and dry dipalmitoyl phosphatidylcholine revisited.Biochim Biophys Acta,1988,946,193-201.
[27] Rudolph AS., ChandrasekharE, Gaber BP, et al. Molecular modeling of saccharide-lipidinteractions. Chem Phys Lipids,1990,53,243-261.
[28] Alex patist, Hans Zoerb. Preservation mechanisms of trehalose in food and biosystem.Colloids and surfaces B,2005,40:107-113.
[29] Satoshi Ohtake, Wang Y.John Trehalose: current use and future applications. J Pharm. Sci,2011,100:2020-2053
[30] Kandror O, DeLeon A, Goldberg AL Trehalose synthesis is induced upon exposure ofEscherichia coli to cold and is essential for viability at low temperatures. Proc Natl Acad Sci,2002,99:9727–9732
[31] Kandror O, Bretschneider N, Kreydin E, et al. Yeast adapt to near-freezing temperatures bySTRE/Msn2,4-dependent induction of trehalose synthesis and certain molecular chaperones.Mol Cell,2004,13:771–781
[32] Chen XM，Jiang Y, Li YT, et al. Regulation of expression of trehalose-6-phosphate synthaseduring cold shock in Arthrobacter strain A3. Extremophiles,2011,15:499-508
[33] Asahina E, Tanno K.. A large amount of trehalose in a frost-resistant insect. Nature,1964,204:1222.
[34] Kaplan F. Exploring the temperature-stress metabolome of Arabidopsis. Plant Physiol.2004,136:4159-4168
[35] Pramanik MHR. and Imai R. Functional identification of a trehalose6-phosphate phosphatasegene that is involved in transient induction of trehalose biosynthesis during chilling stress inrice. Plant Mol Biol,2005,58,751–762
[36] Shima, S. Biochemical characterization of rice trehalose-6-phosphate phosphatases supportsdistinctive functions of these plant enzymes. FEBS J,2006,274:1192–1201
[37] Mahmud SA, Nagahisa K, Hirasawa T, Yoshikawa K, Ashitani K, Shimizu H Effect oftrehalose accumulation on response to saline stress in Saccharomyces cerevisiae.Yeast,200926(1):17-30.
[38] Silva Z., Alarico S., Nobre A., et al. Osmotic adaptation of Thermus thermophilsRQ1:lesson from a mutant deficiedt in synthesis of trehalose. J Bacteriol,2003,185:5943-5952
[39] Makihara F, Tsuzuki M, Masuda KSS, et al. Role of trehalose synthesis pathways in salttolerance mechanism of Rhodobacter sphaeroides f.sp.denitrificans IL106.Arch Microbiol,2005,184:56-65
[40] Crowe, J.H. and Crowe, L.M. Lyotropic effects of water on phospholipids. In: F. Franks (ed.),Water science reviews. Cambridge University Press, Cambridge, England,1990, pp.1-23.
[41] Singer MA. and Lindquist S. Thermotolerance in Saccharomyces cerevesiae: the yin andyang of trehalose. TIB Tech,1998,16,460-468.
[42] Banaroudj, N., Lee, D.H., and Goldberg, A.L. Trehalose accumulation during cellular stressprotects cells and cellular proteins from damage by oxygen radicals. J Biol Chem,2001,276:24261-24267.
[43]Vogel G, Aeschbacher RA, Muller J, et al. Trehalose-6-phosphate phosphatases fromArabidopsis thaliana: identification by functional complementation of the yeast tps2mutant.Plant J,1998,13,673-683.
[44] Muller J, Wiemken A, and Aeschbacher, R. Trehalose metabolism in sugar sensing and plantdevelopment. Plant Sci,1999,147:37-47.
[45] Xie G, Timasheff SN. The thermodynamic mechanism of protein stabilization by trehalose.Biophys Chem,1997,4(1–3):25–43.
[46] Kaushik JK, Bhat R. Why is trehalose an exceptional protein stabilizer? An analysis of thethermal stability of proteins in the presence of the compatible osmolyte trehalose. J BiolChem,2003,278(29):26458–26465.
[47] Lin TY, Timasheff SN. On the role of surface tension in the stabilization of globular proteins.Protein Sci,1996,5(2):372–381.
[48] Kreilgaard L, Frokjaer S, Flink JM, et al. Effects of additives on the stability of Humicolalanuginosa lipase during freeze-drying and storage in the dried solid. J Pharm Sci,1999,88(3):281–290.
[49]Yoshioka S, Miyazaki T, Aso Y. Beta-relaxation of insulin molecule in lyophilizedformulations containing trehalose or dextran as a determinant of chemical reactivity. PharmRes,2006,23(5):961–966.
[50] Katayama DS, Carpenter JF, Menard KP, et al. Mixing properties of lyophilized proteinsystems: A spectroscopic and calorimetric study. J Pharm Sci,2009,98(9):2954–2969.
[51] Garzon-Rodriguez W, Koval RL, Chongprasert S, et al. Optimizing storage stability oflyophilized recombinant human interleukin-11with disaccharide/hydroxyethyl starchmixtures. J Pharm Sci,2004,93(3):684–696.
[52] Abdul-Fattah AM, Lechuga-Ballesteros D, Kalonia DS, et al. The impact of drying methodand formulation on the physical properties and stability of methionyl human growth hormonein the amorphous solid state. J Pharm Sci,2008,97(1):163–184.
[53] Conrad PB, Miller DP, Cielenski PR, et al. Stabilization and preservation of Lactobacillusacidophilus in saccharide matrices. Cryobiology,2000,41(1):17–24.
[54] Draber P, Draberova E, NovakovaM. Stability of monoclonal IgM antibodies freeze-dried inthe presence of trehalose. J Immunol Methods,1995,181(1):37–43.
[55] Andya JD, Maa YF, Costantino HR, et al. The effect of formulation excipients on proteinstability and aerosol performance of spray-dried powders of a recombinant humanizedanti-IgE monoclonal antibody. Pharm Res,1999,16(3):350–358.
[56] Li S, Patapoff TW, Overcashier D, et al. Effects of reducing sugars on the chemical stabilityof human relaxin in the lyophilized state. J Pharm Sci,1996,85(8):873–877.
[57] Miller DP, Anderson RE, de Pablo JJ. Stabilization of lactate dehydrogenase followingfreeze–thawing and vacuum-drying in the presence of trehalose and borate. Pharm Res,199815(8):1215–1221.
[58] Carpenter JF, Crowe JH. An infrared spectroscopic study of the interactions of carbohydrateswith dried proteins. Biochemistry,1989,28(9):3916–3922.
[59] Carpenter JF, Crowe LM, Crowe JH. Stabilization of phosphofructokinase with sugars duringfreeze-drying: Characterization of enhanced protection in the presence of divalent cations.Biochim Biophys Acta,1987,923(1):109–115.
[60] Yoshioka S, Miyazaki T, Aso Y, Kawanishi T.. Significance of local mobility in aggregationof beta-galactosidase lyophilized with trehalose, sucrose or stachyose. Pharm Res,2007,24(9):1660–1667.
[61] Crowe JH, Hoekstra FA, Nguyen KH, et al. Is vitrification involved in depression of thephase transition temperature in dry phospholipids? Biochim Biophys Acta,1996,1280(2):187–196.
[62] Anchordoguy TJ, Rudolph AS, Carpenter JF, Crowe JH. Modes of interaction ofcryoprotectants withmembrane phospholipids during freezing. Cryobiology,1987,24(4):324–331.
[63] Ricker JV, Tsvetkova NM, Wolkers WF, et al. Trehalose maintains phase separation in anair-dried binary lipid mixture. Biophys J,2003,84(5):3045–3051.
[64] Leidy C, Gousset K, Ricker J, et al. Lipid phase behavior and stabilization of domains inmembranes of platelets. Cell Biochem Biophys,2004,40(2):123–148.
[65] Miwa Y. Possibility of the use of trehalose in the field of alternative medicine. New FoodIndustry,2007,49(4):20–30.
[66] Beattie GM, Crowe JH, Lopez AD, et al. Trehalose: A cryoprotectant that enhances recoveryand preserves function of human pancreatic islets after longterm storage. Diabetes,1997,46(3):519–523
[67] Saha S, Rajamahendran R, Boediono A, et al. Viability of bovine blastocysts obtained after7,8or9days of culture in vitro following vitrification and one-step rehydration.Theriogenology,1996,46(2):331–343.
[68] Hirata T, Yokomise H, Fukuse T, et al. Effects of trehalose in preservation of canine lung fortransplantation. J Thorac Cardiovasc Surg,1993,41:59–63.
[69] Yokomise H, Inui K, Wada H, et al. Reliable cryopreservation of trachea for one month in anew trehalose solution. J Thorac Cardiovasc Surg,1995,110(2):382–385.
[70] Tanaka K.. Development of Treha(R) and its properties. Food Industry2009,52(10):45–51.
[71] Ohtake S, Martin RA, Yee L, et al.. Heatstable measles vaccine produced by spray drying.Vaccine,2010,28(5):1275–1284.
[72] Leslie SB, Israeli E, Lighthart B, et al. Trehalose and sucrose protect both membranes andproteins in intact bacteria during drying. Appl Environ Microbiol,1995,61(10):3592–3597.
[73] Zeng Y, Fan H, Chiueh G, et al. Towards development of stable formulations of a liveattenuated bacterial vaccine: A preformulation study facilitated by a biophysical approach.Hum Vaccin,2009,5(5):322–331.
[74] Nishizaki Y, Yoshizane C, Toshimori Y, et al. Disaccharidetrehalose inhibits bone resorptionin ovariectomized mice. Nutr Res,2000,20:653–664.
[75] Yoshizane C, Arai N, Arai C, et al. Trehalose suppresses osteoclast differentiation inovariectomized mice: Correlation with decreased in vitro interleukin-6production by bonemarrow cells. Nutr Res,2000,1485–1491.
[76] Seyffart G, Rothe W, Bartz V. Dialysis solution for use in intraperitoneal dialysis. Patent US1985:4879280.
[77] Ukawa Y, Gu Y, Ohtsuki K, et al. Antitumor effect of trehalose on Sarcoma180in ICR mice.J Appl Glycosci,2005,367–368.
[78] McGinnis LK, Zhu L, Lawitts JA,et al. Mouse sperm desiccated and stored in trehalosemedium without freezing. Biol Reprod,2005,73(4):627–633.
[79] Matsuo T.. Trehalose protects corneal epithelial cells from death by drying. Br J Ophthalmol,2001,85:610–612.
[80] Colaco CALS, Roser B. Trehalose—A multifunctional additive for food preservation. InFood packaging and preservation; Mathlouthi M, Ed. London: Blackie Professional,1995,pp123–140.
[81] Himei S. New developments in the use of Treha(R) in the food industry. Food Chem,2008,7:25–29.
[82] Takeuchi K, Banno N. Function and application of trehalose: Application of cosmetical andpharmaceutical field. Fragrance J,2000,5:101–103
[83]朱玥明，张峻，邢来君，等。海藻糖合酶的分子生物学研究进展。微生物学报,2009,49(1):6-12。
[84] Meleiro, C.R.M. et al. Patent9303490, INPI, Brazil,1993.
[85] Fernandez O, Béthencourt L, Quero A,et al. Trehalose and plant stress responses: friend orfoe? Trens in Plant Sci,2010,15:409-417
[86] Leloir LF; Cabib E. The enzymic synthesis of trehalose phosphate. J Am Chem Soc,1953,75:5445-5446.
[87] Lapp D, Patterson BW, Elbein AD. Properties of a trehalose phosphate synthetase fromMycobacterium smegmatis. Activation of the enzyme by polynucleotides and otherpolyanions. J Biol Chem,1971,246:4567-4579.
[88] Kaasen I, Falkenberg P, Styrvold OB, et al.. Molecular cloning and physical mapping of theotsBA genes, which encode the osmoregulatory trehalose pathway of Escherichia coli:evidence that transcription is activated by katF (AppR). J Bacteriol,1992,174,889-898.
[89] Chary SN, Hicks G.R, Choi YG et al. Trehalose-6-phosphate synthase/phosphatase regulatescell shape and plant architecture in Arabidopsis. Plant Physiol,2008,146,97-107.
[90] Romero C, Bellés JM, Vayá JL, et al. Expression of the yeast trehalose-6-phosphate synthasegene in transgenic tobacco plants: pleiotropic phenotypes includedrought tolerance. Planta,1997,201:293-297.
[91] Gibson RP, Turkenburg JP, Charnock SJ, et al. Insights into trehalose synthesis provided bythe structure of the retaining glucosyltransferase OtsA. Chem Biol,2002,9:1337-1346.
[92] Rao KN, Kumaran D, Seetharaman J, et al. Crystal structure of trehalose-6-phosphatephosphatase-related protein: Biochemical and biological implications. Protein Sci,2006,15:1735-1744.
[93] Maruta K, Nakada T, Kubota M, et al. Formation of trehalose from maltooligosaccharides bya novel enzymatic system. Biosci Biotechnol Biochem,1995,59:1829-1834.
[94] Maruta K, Kubota M, Fukuda S, et al. Cloning and nucleotide sequence of a gene encoding aglycogen debranching enzyme in the trehalose operon from Arthrobacter sp. Q36. BiochimBiophys Acta,2000,1476,377-381.
[95] Nakada T, Ikegami S, Chaen H, et al. Purification and characterization of thermostablemaltooligosyl trehalose synthase from the thermoacidophilic archaebacterium Sulfolobusacidocaldarius. Biosci Biotechnol Biochem,1996,60:263-266.
[96] Kato M, Miura Y, Kettoku M, et al. Purification and characterization of newtrehalose-producing enzymes isolated from the hyperthermophilic archae, Sulfolobussolfataricus KM1. Biosci Biotechnol Biochem,1996,60,546-550.
[97] Feese M D, Kato Y, Tamada T, et al.. Crystal structure of glycosyltrehalose trehalohydrolasefrom the hyperthermophilic archaeum Sulfolobus solfataricus. J Mol Biol,2000,301:451–464.
[98] Cielo CBC, Okazaki S, Suzuki A, et al. Structure of ST0929, a putative glycosyl transferasefrom Sulfolobus tokodaii. Acta Crystallogr Sect F Struct Biol Cryst Commun,2010,66(Pt4):397-400
[99] Nishimoto T, Nakano M, Ikegami S, et al. Existence of a novel enzyme converting maltoseinto trehalose. Biosci Biotechnol Biochem,1995,59,2189-2190.
[100] Nishimoto T, Nakada T, Chaen H, et al. Purification and characterization of a thermostabletrehalose synthase from Thermus aquaticus. Biosci Biotechnol Biochem,1996,60:835-839.
[101] Qu Q, Lee S J, and Boos W. TreT, a novel trehalose glycosyltransferring synthase of thehyperthermophilic archaeon Thermococcus litoralis. J Biol Chem,2004,279:47890–47897.
[102] Ryu S I, Park C S, Cha J, et al. A novel trehalose-synthesizing glycosyltransferase fromPyrococcus horikoshii: molecular cloning and characterization. Biochem Biophys ResCommun,2005,329:429–436.
[103] Kouril T, Zaparty M, Marrero J, et al. A novel trehalose synthesizing pathway in thehyperthermophilic crenarchaeon Thermoproteus tenax: the unidirectional TreT pathway.Arch. Microbiol.2008,190:355–369.
[104] Nobre A., Alarico S, Fernandes C, et al.. A unique combination of genetic systems for thesynthesis of trehalose in Rubrobacter xylanophilus: properties of a rare actinobacterial TreT.J Bacteriol,2008,190:7939–7946
[105] Woo EJ, Ryu SI, Song HN, et al. Structural insights on the new mechanism of trehalosesynthesis by trehalose synthase TreT from Pyrococcus horikoshii. J Mol Biol.2010,404(2):247-59.
[106] Saito K, Kase T, Takahashi E. Purification and characterization of a trehalose synthasefrom the basidiomycete Grifola frondosa. Appl Environ Microbiol,1998,64,4340-4345.
[107] Nishimoto T, Nakano M, Nakada T, et al Purification and properties of a novel enzyme,trehalose synthase, from Pimelobacter sp.R48. Bioscience Biotechnology andBiochemistry,1996,60:640-644
[108] Wei YT, Zhu QX, Luo ZF, et al. Cloning, expression and identification of a new trehalosesynthase gene from Thermobifida fusca genome. Acta biochimica et Biophysica Sinica,2004,36:477-484
[109] Lee JH, Lee KH, Kim CG, et al. Cloning and expression of a trehalose synthase fromPseudomonas stutzeri CJ38in Excherichia coli for the production of trehalose. ApplMicrobiol Biotechnol,2005,68:213-219
[110] Koh S, Kim J, Lee DS, et al. Mechanistic study of the intramolecular conversion of maltoseto trehalose by Thermus caldophilus GK24trehalose synthase. Carbohydrate Research,2003,338:1339-1343
[111] Tsusaki K, Nishimoto T, Nakada T, et al. Cloning and sequencing of trehalose synthasegene from Thermus aquaticus ATCC33923. Biochim Biophys Acta,1997,1334,28-32.
[112] Wang JH, Tsai MY, Chen JJ, et al. Role of the C-terminal domain of Thermusthermophilus trehalose synthase in the thermophilicity, thermostability, and efficientproduction of trehalose. J Agric Food Chem,2007,55:3435-3443
[113] Zhu Y, Wei D, Zhang J, Wang Y, Xu H, Xing L, Li M. Overexpression andcharacterization of a thermostable trehalose synthase from Meiothermus ruber.Extremophiles.2010,14:1-8
[114] Cardoso FS, Castro RF, Santos H. Biochmical and genetic characterization of the pathwaysfor trehalose metabolism in Propionibacterium freudenreichii, and their role in stressresponse. Microbiology,2007,153:270-280
[115] Chen YS, Lee GC, Shaw JF. Gene cloning expression and biochemical characterization ofa recombinant trehalose synthase from Picrophilus torridus in Escherichia coli. J AgricFood Chem,2006,54:7098-7104
[116] Pan YT, Koroth EV, Jourdian WJ, et al. Trehalose synthase of Mycobacterium smegmatis:purification, cloning, expression, and properties of the enzyme. Eur J Biochem,2004,271:4259-4269
[117] Yue M, Wu XL, Gong WN, Ding HB. Molecular cloning and expression of a noveltrehalose synthase genefrom Enterobacter hormaechei. Microb Cell Fact.2009,doi:10.1186/1475-2859-8-34
[118] Wu X, Ding H, Yue M, Qiao Y. Gene cloning, expression, and characterization of a noveltrehalose synthase from Arthrobacter aurescens. Appl Microbiol Biothchnol,2009,83:477-482
[119] Besra GS., Bolton RC., McNeil MR, et al. Structural elucidation of a novel family ofacyltrehaloses from Mycobacterium tuberculosis. Biochemistry,1992,31:9832-9837.
[120]韦宇拓,朱绮霞,罗兆飞,陈发忠,李桂媛,黄鲲,黄日波。耐放射异常球菌海藻糖合成酶基因的克隆及功能鉴定。生物化学与生物物理进展，2004,31:1018-1023.
[121] Alugupalli S, Laneelle MA, Larsson L, et al. Chemical characterization of the ester-linked3-hydroxy fatty acyl-containing lipids in Mycobacterium tuberculosis. J Bacteriol,1995,177,4566-4570.
[122]韦宇拓，黄日波，蒙健宗，卢福燊，庞中文，朱绮霞，陈发忠，罗兆飞，卢运琨，王青艳，黄鲲。谷氨酸棒杆菌海藻糖合成酶基因及海藻糖制造方法。中国：200410013006.9。2005.
[123] Kim HJ，Kim AR, Jeon SJ Immobilization on chitosan of a thermophilic trehalosesynthase from Thermus thermophilus HJ6. J Microbiol Biotechnol,2010,20:513-517
[124] MacGregor EA，Janecek S, Svensson B.Relationship of sequence and structure to specificityin the α-amylase family of enzyme. Biochimica et Biophysica Acta,2001,1546：1-20
[125] Janecek S, α-Amylase family: molecular biology and evolution, Prog Biophys Mol Biol,1997,67:67-97.
[126] Svensson B. Protein engineering in the α-amylase family: catalytic mechanism,substratespecificity,and stability. Plant Molecular Biology,1994,25:141-157
[127] Janecek S, Svensson B, Henrissat B. Domain evolution in the α-amylase family. J Mol Evol,1997,45:322-331.
[128] Park KH, Kim TJ, Cheong TK, et al. Structure, specificity and function ofcyclomaltodextrinase, a multispecific enzyme of the α-amylase family. Biochem BiophysActa,2000,1478:165-185.
[129] MacGregor EA, Jespersen HM, Svensson B. A circularly permuted α-amylase-typeα/β-barrel structure in glucan-synthesizing glucosyltransferases. FEBS Lett,1996,378:263-266.
[130] Katsuya Y, Mezaki Y, Kubota M, et al. Three-dimensional structure of Pseudomonasisoamylase at2.2resolution. J Mol Biol,1998,281:885-897
[131] Kamitori S, Kondo S, Okuyama K, et al. Crystal structure of Thermoactinonyces vulgarisR-47α-amylase II (TVAII) hydrolyzing cyclodextrins and pullulan at2.6resolution, JMol Biol,1999,287:907-921.
[132] Pan YT, Carroll JD, Asano N, et al. Trehalose synthase converts glycogen to trehalose.FEBS Journal,2008,275(13):3408-3420.
[133] van der Maarel MJEC, van der Veen B, Uitdehaag JCM, et al. Properties and applications ofstarch-converting enzymes of the α-amylase family. J Biotechnol,2002,94:137-155
[134] Svensson B. Protein engineering in the α-amylase family: catalytic mechanism, substratespecificity, and stability. Plant Mol Biol,1994,25:141-157
[135] Zhang R, Pan YT, He S, Lam M, Brayer GD, Elbein AD, Withers SG.2011Mechanisticanalysis of trehalose synthase from Mycobacterium smegmatis. J Biolog Chem,286:35601-35609
[136] ZollerM J, SmithM. Oligonucleotide-directed mutagenesis using M13-derived vectors: anefficient and general procedure for the production of point mutations in any fragment ofDNA. Nucleic Acids Res,1982,10(20):6487-6500
[137]陶苏丹,刘佳,陈喜文,陈德富点饱和突变技术及其在蛋白质工程中的应用.中国生物工程杂志,2007,27(8):82-86
[138] Kegler-Ebo D, Pollack G, DiMaio D. Use of codon cassette mutagenesis for saturationmutagenesis in: In Vitro Mutagenesis Protocols. Totowa, N J: Humana Press,1996.297-311
[139] Hill CM, Li WS, Thoden JB, et al. Enhanced degradation of chemical warfare agentsthrough molecular engineering of the phosphotriesterase active site. J Am Chem,2003,125(30):8990-8991
[140] Doucet N, Wals PD, Pelletier JN. Site-saturation mutagenesisof Tyr-105reveals itsimportance in substrate stabilization and discrimination in TEM-1beta-lactamase. J BiolChem,2004,279(44):46295-46303
[141] Vandeyar MA, Weiner MP, Hutton CJ, et al. A simple and rapid method for the selection forthe selection of oligodeoxy-nucleotide-directed mutants. Gene,1988,65:129-133
[142] Fang TY, Tseng WC, Wang MY, et al. Protein engineering of Sulfolobus solfataricusmaltooligosyltrehalose synthase to alter its selectivity. J Agric Food Chem,2007,55:5588-5594
[143]齐向辉，韦宇拓，黄日波等.甘油脱水酶的理性设计：基因杂合改善酶的性质.科学通报，2006，51（20）：2372-2380
[144] Caldwell RC, Joyee GF. Randomization of genes by PCR mutagenesis. In PCR MethodsApplic,1992,2:28-33
[145] Fujii R, Kitaoka M, Hayashi K. One-step random mutagenesis by error-prone rolling circleamplification. Nucleic Acids Res,2004,19: e145
[146] Chirumamilla RR, Muralidhar R, Nigam P, et al.Improving the quality of industriallyimportant enzymes by directed evolution. Molecular and Cellular Bochemistry,2001,224:159-168
[147]吴梧桐。蛋白质工程技术与新型生物催化剂设计.药物生物技术，2004，11：1-6
[148] Stemmer W P C. DNA shuffling by randomfragmentation and reassembly:in vitrorecombination for molecular evolution. Proc Natl Acad Sci, USA,1994,91(22):10747-107511
[149] Crameri A, Raillard S A, Bermudez E, et al. DNA shuffling of a family of genes fromdiverse species accelerate directed evolution. Nature,1998,391:288
[150] Volkov AA, Arnold FH. Methods in enzyme. Elservier,2000,447-456.
[151] Schmidt C, Umeno D, Armoldz F H. Molecular breeding of carotenoid biosyntheticpathway. Nature Biotechnol,2000,18:750
[152] Zhao H, Giver L, Shao Z, et al. Molecular evolution by staggered extension process (StEP)in vitro recombination. Nature Biotechnology,1998,16(3):258-261.
[153] Leung D, Chen E, Goeddel D. A method for random mutagenesis of a defined DNA segmentusing a modified polymerase chain reaction. Technique,1989,2:302-303
[154] Kikuchi M, Ohnishi K, Harayama S. Novel family shuffling methods for the in vitroevolution of enzymes.Gene,1999,236:159-167
[155] Park YM, Ghim SY. Enhancement of the activity and pH-performance of chitosanase fromBacillus cereus strains by DNA shuffling. Biotechnol Lett,2009,31:1463-1467
[156] Lutz S, Ostermeier M, Benkovic SJ, et al. Creating multiple-crossover DNA librariesindependent of sequence identity. PNAS,2001,98(20):11248-11253
[157]胡光星，郭美锦，储炬等，DNA改组技术发展与应用。中国生物工程杂志，2002，22（3）：9-12
[158] Grace DeSantis， J Bryan Jones． Chemical modification of enzymes for enhancedfunctionality．Current Opinion in Bintechnology，1999，10：324-330．
[159] Ueji S L，UedaA，TanakaH，et al．Chemical modification of lipase with various hydrophobicgroups improves their enantioselectivity in hydrolytic reantions．Biotechnol Lett，2003，25：83-87
[160] Zhu Y, Zhang J, Wei D, et al. Isolation and identification of a thermophilic strain producingtrehalose synthase from geothermal water in China. Biosci Biotechnol Biochem,2008,72(8):2019-2024
[161]卢圣栋，现代分子生物学实验技术.北京：高等教育出版社，1993.
[162]萨木布鲁克J,弗里奇EF,曼尼阿蒂斯T.金冬雁等,译.分子克隆试验指南.第二版.北京：科学出版社，1993.
[163]沈萍，范秀容，李广武.微生物学实验(第三版).北京：高等教育出版社，1999.
[164] Arnold K, Bordoli L, Kopp J, et al. The Swiss-Model Workspace:a web-based environmentfor protein structure homology modeling. Bioinformatics,2006,22:195-201
[165] Kiefer F, Arnold K, Künzli M, et al. The Swiss-Model Repository and Associated resources.Nucleic Acids Res,2009,37:D387-D392
[166] Cristianini N and Shawe-Taylor J An Introduction to Support Vector Machines and OtherKernel-based Learning Methods. Cambridge University Press, Cambridge, UK.2000
[167] Krishnan VG, and Westhead DR A comparative study of machine-learning methods topredict the effects of single nucleotide polymorphisms on protein function. Bioinformatics,2003,19:2199-2209.
[168] Gao S, Zhang N, Duan GY, Yang Z, Ruan JS, Zhang T. Prediction of function changesassociated with single-point protein mutations using support vector machines (SVMs). HumMutat,2009,30:1161-1166
[169]宋卓，张宁，阮吉寿，杨卓，张涛。2007基于氨基酸序列预测蛋白质功能性点突变位点。生物物理学报23:134-138
[170] Andrade MA, Chacón P, Merelo JJ and Morán F. Evaluation of secondary structure ofproteins from UV circular dichroism using an unsupervised learning neural network. ProtEng,1993,6:383-390
[171] Merelo JJ, Andrade MA, Prieto A and Morán F Proteinotopic Feature MapsNeurocomputing,1994,6:443-454
[172]沈星灿，梁宏，何锡文，王新省2004圆二色光谱分析蛋白质构象的方法及研究进展分析化学评述与进展。3:388-394
[173] Greenfield NJ Methods to Estimate the Conformation of Proteins and Polypeptides fromCircular Dichroism Data. Anal Bioch,1996,235:1-10
[174] PerczelA, Park K, Fasm GD. Deconvolution of the circular dichroism spectra of proteins:The circular dichroism spectra of the antiparallel and sheet in proteins. Proteins Struct FunctGenet,1992:57-69.
[175] Chang CT, Wu CS, Yang JT. Circular dichroic analysis of protein conformation inclusion ofthe beta-turns. Anal Biochem,1978,91:13-31.
[176]丁晓岚,高红旗2008圆二色光谱技术应用和实验方法实验技术与管理25:48-52
[177] Levitt, M.; Chothia, C. Structural patterns in globular proteins. Nature,1976,261(5561):552–558.
[178] Nakai K, Kidera A, Kanehisa M, Cluster analysis of amino acid indices for prediction ofprotein structure and function. Protein Eng,1988,224:437-449
[179]李军、张莉娜、温珍昌生物软件的选择与使用指南北京：化学工业出版社2008
[180] Sippl JM Calculation of conformational ensembles from potetials of mean force: anapproach to the knowledge based prediction of local structures in globular proteins. J MolBiol,1990,213:859-883
[181] Charkravarty S, Varadarajun R. Elucidation of determineants of protein stability throughgenome sequence analysis. FEBS Lett,2000,470:65-69.
[182] Kumar S, Tsai CJ, Nussinov R. Factors enhancing protein thermostability. Protein Eng,2000,13:179-191.
[183] Sen S, Venkata Dasu V, Mandal B. Developments in directed evolution fro improvingenzyme functions. Appl Biochem Biotechnol,2007,143:212-223
[184] Vartanian JP, Henry M, Wain-Hobson S. Hypermutagenic PCR involving all four transitionsand a sizeable proportion of transversions. Nucleic Acids Res,1996,24(14):2627-2631
[185] Shafikhani S, Siegel RA, Ferrari E, et al. Generation of large libraries of random mutants inBacillus subtilis by PCR-based plasmid multimerization. BioTechniques,1997,23(2):304-310
[186] Suzuki M, Christians FC, Kim B, et al. Tolerance of different proteins for amino aciddiversity. Mol Divers,1997,2(1-2):111-118.
[187] Lin L, Meng X, Liu P, et al. Improved catalytic efficiency of endo-β-1,4-glucanase fromBacillus subtilis BME-15by directed evolution. Appl Microbiol Biotechnol,2009,82:671-679
[188] Rabbani M, Mirmohammad Sadeghi H, Moazen F, et al. Cloning and expression ofrandomly mutated Bacillus subtilis α-amylase genes in HB101. Biotechnol Res Int,2011,doi:10.4061/2011/305956
[189] Caldwell RC, Joyce GF. Mutagenic PCR.PCR Meth Appl,1994,3:136-140
[190]李晶琴基于PCR技术的蛋白质改造策略。国际检验医学杂志，2006,27（12）：1129-1131
[191] Wang JH, Tsai MY, Shaw JF, et al. Construction of a recombinant thermostableβ-amylase-trehalose synthase bifunctional enzyme for facilitating the conversion of starch totrehalose. J Agric Food Chem,2007,55:1256-1263
[192] Chou HH, Chang SW, Lee GC，et al. Site-directed mutagenesis improves the thermostabilityof a recombinant Picrophilus torridus trehalose synthase and efficiency for the productionof trehalose from sweet potato starch. Food Chem,2010,119:1017–1022.
[193]王青艳，陈发忠，黄日波等。Thermobifida fusca海藻糖合成酶的定点突变及其动力学性质研究。广西农业生物科学，2007,26（2）：115-119
[2] Berthelot M. Sur le trehalose, nouvelle espece de sucre. Compt. Rend. Hebd. Seanc Acad Sci.,Paris1858,46:1276-1279.
[3] Elbein AD. The metabolism of α, α-trehalose. Adv. Carbohydr Chem Biochem,1974,30:227-256.
[4] Hudson CS. Some numerical relations among the rotatory powers of the compound sugars. JAm Chem Soc,1916,38:1566-1575.
[5] Taga T, Senma M, Osaki K. The crystal and molecular structure of trehalose dihydrate. ActaCrystallog,.1972, B28:3258-3263
[6] Teramoto N, Sachinvala ND, Shibata M. Trehalose and trehalose-based polymers forenvironmentally benign, biocompatible and bioactive material. Molecules,2008,13:1773-1816
[7] Roser B. Trehalose, a new approach to premium dried foods. Trends Food Sci Technol,1991,2:166-169.
[8] Wyatt GR, Kalf GF. The chemistry of insect hemolymph: II. Trehalose and othercarbohydrates. J Gen Physiol,1957,40:833-847.
[9] Cher B, and Klingenberg M. Wege des Wasserstoffs in der lebendigen Organisation. AngewChem,1958,70:552-570.
[10] Evans DR, Dethier VG. The regulation of taste thresholds for sugars in the blowfly. J Insecthysiol,1957,1:3-17
[11] Becker A, Schloeder P, Steele JE, and Wegener G. The regulation f trehalose metabolism ininsects. Experientia,1996,52:433-439.
[12] Juan Carlos Argüelles. Physiological roles of trehalose in bacteria and yeasts: a comparativeanalysis. Arch Microbiol,2000,174:217-224
[13] Spargo BJ, Crowe LM, Ioneda T, et al. Cord factor (α,α-trehalose6,6’ dimycolate) inhibitsfusion between phospholipid vesicles. Proc Natl Acad Sci,1991,88:737–740
[14]Reed RH, Richardson, DL, Warr SR, Stewart WD. Carbohydrate accumulation and osmoticstress in Cyanobacteria. J Gen Microbiol,1984,130:1–4
[15]Mikkat S, Effmert U, Hagemann M. Uptake and use of the osmoprotective compoundstrehalose, glucosylglycerol, and sucrose by the Cyanobacterium synechocystis sp.PCC6803.Arch Microbiol,1997,167(2-3):223-8
[16] Welsh DT, Guyoneaud T, Caumette P. Utilization of the compatible solutes sucrose andtrehalose by purple sulphur and nonsulphur bacteria. Can J Microbiol,1998,4:974–979
[17] Galinski EA, Herzog RM The role of trehalose as a substitute for nitrogen-containingcompatible solutes (Ectothiorhodospira halochloris) Arch Microbiol,1990,153:607–613
[18] Roser B. Trehalose, a new approach to premium dried foods. Trends Food Sci Tech,19912:166–169.
[19] Adams RP, Kendall E, Kartha KK. Comparison of free sugars in growing and desiccatedplants of Selaginella lepidophylla. Biochem Syst Ecol,1990,18:107–110.
[20] McBride MJ, Ensign JC. Effects of intracellular trehalose content on Streptomyces griseusspores. J Bacteriol,1987,169(11):4995–5001.
[21] Van Dijck P, Colavizza D, Smet P, et al. Differential importance of trehalose in stressresistance in fermenting and nonfermenting Saccharomyces cerevisiae cells. Appl EnvironMicrobiol,1995,61(1):109–115.
[22] Clegg JS. The origin of trehalose and its significance during the formation of encysteddormant embryos of Artemia salina. Comp Biochem Physiol,1965,14:135–143.
[23] Womersley C, Smith L. Anhydrobiosis in nematodes. I. The role of glycerol, myo-inositoland trehalose during dessication. Comp Biochem Physiol,1981,70B:579–586.
[24] Newman YM, Ring SG, Colaco C. The role of trehalose and other carbohydrates inbiopreservation. Biotechnol Genet Eng Rev,1993,11:263–294.
[25]Gadd GM, Chalmers K, Reed RH. The role of trehalose in dehydration resistance ofSaccharomyces cerevesiae. FEMS Microbiol Lett,1987,48:249–254.
[26] Crowe,L.M., Crowe,J.H. Trehalose and dry dipalmitoyl phosphatidylcholine revisited.Biochim Biophys Acta,1988,946,193-201.
[27] Rudolph AS., ChandrasekharE, Gaber BP, et al. Molecular modeling of saccharide-lipidinteractions. Chem Phys Lipids,1990,53,243-261.
[28] Alex patist, Hans Zoerb. Preservation mechanisms of trehalose in food and biosystem.Colloids and surfaces B,2005,40:107-113.
[29] Satoshi Ohtake, Wang Y.John Trehalose: current use and future applications. J Pharm. Sci,2011,100:2020-2053
[30] Kandror O, DeLeon A, Goldberg AL Trehalose synthesis is induced upon exposure ofEscherichia coli to cold and is essential for viability at low temperatures. Proc Natl Acad Sci,2002,99:9727–9732
[31] Kandror O, Bretschneider N, Kreydin E, et al. Yeast adapt to near-freezing temperatures bySTRE/Msn2,4-dependent induction of trehalose synthesis and certain molecular chaperones.Mol Cell,2004,13:771–781
[32] Chen XM，Jiang Y, Li YT, et al. Regulation of expression of trehalose-6-phosphate synthaseduring cold shock in Arthrobacter strain A3. Extremophiles,2011,15:499-508
[33] Asahina E, Tanno K.. A large amount of trehalose in a frost-resistant insect. Nature,1964,204:1222.
[34] Kaplan F. Exploring the temperature-stress metabolome of Arabidopsis. Plant Physiol.2004,136:4159-4168
[35] Pramanik MHR. and Imai R. Functional identification of a trehalose6-phosphate phosphatasegene that is involved in transient induction of trehalose biosynthesis during chilling stress inrice. Plant Mol Biol,2005,58,751–762
[36] Shima, S. Biochemical characterization of rice trehalose-6-phosphate phosphatases supportsdistinctive functions of these plant enzymes. FEBS J,2006,274:1192–1201
[37] Mahmud SA, Nagahisa K, Hirasawa T, Yoshikawa K, Ashitani K, Shimizu H Effect oftrehalose accumulation on response to saline stress in Saccharomyces cerevisiae.Yeast,200926(1):17-30.
[38] Silva Z., Alarico S., Nobre A., et al. Osmotic adaptation of Thermus thermophilsRQ1:lesson from a mutant deficiedt in synthesis of trehalose. J Bacteriol,2003,185:5943-5952
[39] Makihara F, Tsuzuki M, Masuda KSS, et al. Role of trehalose synthesis pathways in salttolerance mechanism of Rhodobacter sphaeroides f.sp.denitrificans IL106.Arch Microbiol,2005,184:56-65
[40] Crowe, J.H. and Crowe, L.M. Lyotropic effects of water on phospholipids. In: F. Franks (ed.),Water science reviews. Cambridge University Press, Cambridge, England,1990, pp.1-23.
[41] Singer MA. and Lindquist S. Thermotolerance in Saccharomyces cerevesiae: the yin andyang of trehalose. TIB Tech,1998,16,460-468.
[42] Banaroudj, N., Lee, D.H., and Goldberg, A.L. Trehalose accumulation during cellular stressprotects cells and cellular proteins from damage by oxygen radicals. J Biol Chem,2001,276:24261-24267.
[43]Vogel G, Aeschbacher RA, Muller J, et al. Trehalose-6-phosphate phosphatases fromArabidopsis thaliana: identification by functional complementation of the yeast tps2mutant.Plant J,1998,13,673-683.
[44] Muller J, Wiemken A, and Aeschbacher, R. Trehalose metabolism in sugar sensing and plantdevelopment. Plant Sci,1999,147:37-47.
[45] Xie G, Timasheff SN. The thermodynamic mechanism of protein stabilization by trehalose.Biophys Chem,1997,4(1–3):25–43.
[46] Kaushik JK, Bhat R. Why is trehalose an exceptional protein stabilizer? An analysis of thethermal stability of proteins in the presence of the compatible osmolyte trehalose. J BiolChem,2003,278(29):26458–26465.
[47] Lin TY, Timasheff SN. On the role of surface tension in the stabilization of globular proteins.Protein Sci,1996,5(2):372–381.
[48] Kreilgaard L, Frokjaer S, Flink JM, et al. Effects of additives on the stability of Humicolalanuginosa lipase during freeze-drying and storage in the dried solid. J Pharm Sci,1999,88(3):281–290.
[49]Yoshioka S, Miyazaki T, Aso Y. Beta-relaxation of insulin molecule in lyophilizedformulations containing trehalose or dextran as a determinant of chemical reactivity. PharmRes,2006,23(5):961–966.
[50] Katayama DS, Carpenter JF, Menard KP, et al. Mixing properties of lyophilized proteinsystems: A spectroscopic and calorimetric study. J Pharm Sci,2009,98(9):2954–2969.
[51] Garzon-Rodriguez W, Koval RL, Chongprasert S, et al. Optimizing storage stability oflyophilized recombinant human interleukin-11with disaccharide/hydroxyethyl starchmixtures. J Pharm Sci,2004,93(3):684–696.
[52] Abdul-Fattah AM, Lechuga-Ballesteros D, Kalonia DS, et al. The impact of drying methodand formulation on the physical properties and stability of methionyl human growth hormonein the amorphous solid state. J Pharm Sci,2008,97(1):163–184.
[53] Conrad PB, Miller DP, Cielenski PR, et al. Stabilization and preservation of Lactobacillusacidophilus in saccharide matrices. Cryobiology,2000,41(1):17–24.
[54] Draber P, Draberova E, NovakovaM. Stability of monoclonal IgM antibodies freeze-dried inthe presence of trehalose. J Immunol Methods,1995,181(1):37–43.
[55] Andya JD, Maa YF, Costantino HR, et al. The effect of formulation excipients on proteinstability and aerosol performance of spray-dried powders of a recombinant humanizedanti-IgE monoclonal antibody. Pharm Res,1999,16(3):350–358.
[56] Li S, Patapoff TW, Overcashier D, et al. Effects of reducing sugars on the chemical stabilityof human relaxin in the lyophilized state. J Pharm Sci,1996,85(8):873–877.
[57] Miller DP, Anderson RE, de Pablo JJ. Stabilization of lactate dehydrogenase followingfreeze–thawing and vacuum-drying in the presence of trehalose and borate. Pharm Res,199815(8):1215–1221.
[58] Carpenter JF, Crowe JH. An infrared spectroscopic study of the interactions of carbohydrateswith dried proteins. Biochemistry,1989,28(9):3916–3922.
[59] Carpenter JF, Crowe LM, Crowe JH. Stabilization of phosphofructokinase with sugars duringfreeze-drying: Characterization of enhanced protection in the presence of divalent cations.Biochim Biophys Acta,1987,923(1):109–115.
[60] Yoshioka S, Miyazaki T, Aso Y, Kawanishi T.. Significance of local mobility in aggregationof beta-galactosidase lyophilized with trehalose, sucrose or stachyose. Pharm Res,2007,24(9):1660–1667.
[61] Crowe JH, Hoekstra FA, Nguyen KH, et al. Is vitrification involved in depression of thephase transition temperature in dry phospholipids? Biochim Biophys Acta,1996,1280(2):187–196.
[62] Anchordoguy TJ, Rudolph AS, Carpenter JF, Crowe JH. Modes of interaction ofcryoprotectants withmembrane phospholipids during freezing. Cryobiology,1987,24(4):324–331.
[63] Ricker JV, Tsvetkova NM, Wolkers WF, et al. Trehalose maintains phase separation in anair-dried binary lipid mixture. Biophys J,2003,84(5):3045–3051.
[64] Leidy C, Gousset K, Ricker J, et al. Lipid phase behavior and stabilization of domains inmembranes of platelets. Cell Biochem Biophys,2004,40(2):123–148.
[65] Miwa Y. Possibility of the use of trehalose in the field of alternative medicine. New FoodIndustry,2007,49(4):20–30.
[66] Beattie GM, Crowe JH, Lopez AD, et al. Trehalose: A cryoprotectant that enhances recoveryand preserves function of human pancreatic islets after longterm storage. Diabetes,1997,46(3):519–523
[67] Saha S, Rajamahendran R, Boediono A, et al. Viability of bovine blastocysts obtained after7,8or9days of culture in vitro following vitrification and one-step rehydration.Theriogenology,1996,46(2):331–343.
[68] Hirata T, Yokomise H, Fukuse T, et al. Effects of trehalose in preservation of canine lung fortransplantation. J Thorac Cardiovasc Surg,1993,41:59–63.
[69] Yokomise H, Inui K, Wada H, et al. Reliable cryopreservation of trachea for one month in anew trehalose solution. J Thorac Cardiovasc Surg,1995,110(2):382–385.
[70] Tanaka K.. Development of Treha(R) and its properties. Food Industry2009,52(10):45–51.
[71] Ohtake S, Martin RA, Yee L, et al.. Heatstable measles vaccine produced by spray drying.Vaccine,2010,28(5):1275–1284.
[72] Leslie SB, Israeli E, Lighthart B, et al. Trehalose and sucrose protect both membranes andproteins in intact bacteria during drying. Appl Environ Microbiol,1995,61(10):3592–3597.
[73] Zeng Y, Fan H, Chiueh G, et al. Towards development of stable formulations of a liveattenuated bacterial vaccine: A preformulation study facilitated by a biophysical approach.Hum Vaccin,2009,5(5):322–331.
[74] Nishizaki Y, Yoshizane C, Toshimori Y, et al. Disaccharidetrehalose inhibits bone resorptionin ovariectomized mice. Nutr Res,2000,20:653–664.
[75] Yoshizane C, Arai N, Arai C, et al. Trehalose suppresses osteoclast differentiation inovariectomized mice: Correlation with decreased in vitro interleukin-6production by bonemarrow cells. Nutr Res,2000,1485–1491.
[76] Seyffart G, Rothe W, Bartz V. Dialysis solution for use in intraperitoneal dialysis. Patent US1985:4879280.
[77] Ukawa Y, Gu Y, Ohtsuki K, et al. Antitumor effect of trehalose on Sarcoma180in ICR mice.J Appl Glycosci,2005,367–368.
[78] McGinnis LK, Zhu L, Lawitts JA,et al. Mouse sperm desiccated and stored in trehalosemedium without freezing. Biol Reprod,2005,73(4):627–633.
[79] Matsuo T.. Trehalose protects corneal epithelial cells from death by drying. Br J Ophthalmol,2001,85:610–612.
[80] Colaco CALS, Roser B. Trehalose—A multifunctional additive for food preservation. InFood packaging and preservation; Mathlouthi M, Ed. London: Blackie Professional,1995,pp123–140.
[81] Himei S. New developments in the use of Treha(R) in the food industry. Food Chem,2008,7:25–29.
[82] Takeuchi K, Banno N. Function and application of trehalose: Application of cosmetical andpharmaceutical field. Fragrance J,2000,5:101–103
[83]朱玥明，张峻，邢来君，等。海藻糖合酶的分子生物学研究进展。微生物学报,2009,49(1):6-12。
[84] Meleiro, C.R.M. et al. Patent9303490, INPI, Brazil,1993.
[85] Fernandez O, Béthencourt L, Quero A,et al. Trehalose and plant stress responses: friend orfoe? Trens in Plant Sci,2010,15:409-417
[86] Leloir LF; Cabib E. The enzymic synthesis of trehalose phosphate. J Am Chem Soc,1953,75:5445-5446.
[87] Lapp D, Patterson BW, Elbein AD. Properties of a trehalose phosphate synthetase fromMycobacterium smegmatis. Activation of the enzyme by polynucleotides and otherpolyanions. J Biol Chem,1971,246:4567-4579.
[88] Kaasen I, Falkenberg P, Styrvold OB, et al.. Molecular cloning and physical mapping of theotsBA genes, which encode the osmoregulatory trehalose pathway of Escherichia coli:evidence that transcription is activated by katF (AppR). J Bacteriol,1992,174,889-898.
[89] Chary SN, Hicks G.R, Choi YG et al. Trehalose-6-phosphate synthase/phosphatase regulatescell shape and plant architecture in Arabidopsis. Plant Physiol,2008,146,97-107.
[90] Romero C, Bellés JM, Vayá JL, et al. Expression of the yeast trehalose-6-phosphate synthasegene in transgenic tobacco plants: pleiotropic phenotypes includedrought tolerance. Planta,1997,201:293-297.
[91] Gibson RP, Turkenburg JP, Charnock SJ, et al. Insights into trehalose synthesis provided bythe structure of the retaining glucosyltransferase OtsA. Chem Biol,2002,9:1337-1346.
[92] Rao KN, Kumaran D, Seetharaman J, et al. Crystal structure of trehalose-6-phosphatephosphatase-related protein: Biochemical and biological implications. Protein Sci,2006,15:1735-1744.
[93] Maruta K, Nakada T, Kubota M, et al. Formation of trehalose from maltooligosaccharides bya novel enzymatic system. Biosci Biotechnol Biochem,1995,59:1829-1834.
[94] Maruta K, Kubota M, Fukuda S, et al. Cloning and nucleotide sequence of a gene encoding aglycogen debranching enzyme in the trehalose operon from Arthrobacter sp. Q36. BiochimBiophys Acta,2000,1476,377-381.
[95] Nakada T, Ikegami S, Chaen H, et al. Purification and characterization of thermostablemaltooligosyl trehalose synthase from the thermoacidophilic archaebacterium Sulfolobusacidocaldarius. Biosci Biotechnol Biochem,1996,60:263-266.
[96] Kato M, Miura Y, Kettoku M, et al. Purification and characterization of newtrehalose-producing enzymes isolated from the hyperthermophilic archae, Sulfolobussolfataricus KM1. Biosci Biotechnol Biochem,1996,60,546-550.
[97] Feese M D, Kato Y, Tamada T, et al.. Crystal structure of glycosyltrehalose trehalohydrolasefrom the hyperthermophilic archaeum Sulfolobus solfataricus. J Mol Biol,2000,301:451–464.
[98] Cielo CBC, Okazaki S, Suzuki A, et al. Structure of ST0929, a putative glycosyl transferasefrom Sulfolobus tokodaii. Acta Crystallogr Sect F Struct Biol Cryst Commun,2010,66(Pt4):397-400
[99] Nishimoto T, Nakano M, Ikegami S, et al. Existence of a novel enzyme converting maltoseinto trehalose. Biosci Biotechnol Biochem,1995,59,2189-2190.
[100] Nishimoto T, Nakada T, Chaen H, et al. Purification and characterization of a thermostabletrehalose synthase from Thermus aquaticus. Biosci Biotechnol Biochem,1996,60:835-839.
[101] Qu Q, Lee S J, and Boos W. TreT, a novel trehalose glycosyltransferring synthase of thehyperthermophilic archaeon Thermococcus litoralis. J Biol Chem,2004,279:47890–47897.
[102] Ryu S I, Park C S, Cha J, et al. A novel trehalose-synthesizing glycosyltransferase fromPyrococcus horikoshii: molecular cloning and characterization. Biochem Biophys ResCommun,2005,329:429–436.
[103] Kouril T, Zaparty M, Marrero J, et al. A novel trehalose synthesizing pathway in thehyperthermophilic crenarchaeon Thermoproteus tenax: the unidirectional TreT pathway.Arch. Microbiol.2008,190:355–369.
[104] Nobre A., Alarico S, Fernandes C, et al.. A unique combination of genetic systems for thesynthesis of trehalose in Rubrobacter xylanophilus: properties of a rare actinobacterial TreT.J Bacteriol,2008,190:7939–7946
[105] Woo EJ, Ryu SI, Song HN, et al. Structural insights on the new mechanism of trehalosesynthesis by trehalose synthase TreT from Pyrococcus horikoshii. J Mol Biol.2010,404(2):247-59.
[106] Saito K, Kase T, Takahashi E. Purification and characterization of a trehalose synthasefrom the basidiomycete Grifola frondosa. Appl Environ Microbiol,1998,64,4340-4345.
[107] Nishimoto T, Nakano M, Nakada T, et al Purification and properties of a novel enzyme,trehalose synthase, from Pimelobacter sp.R48. Bioscience Biotechnology andBiochemistry,1996,60:640-644
[108] Wei YT, Zhu QX, Luo ZF, et al. Cloning, expression and identification of a new trehalosesynthase gene from Thermobifida fusca genome. Acta biochimica et Biophysica Sinica,2004,36:477-484
[109] Lee JH, Lee KH, Kim CG, et al. Cloning and expression of a trehalose synthase fromPseudomonas stutzeri CJ38in Excherichia coli for the production of trehalose. ApplMicrobiol Biotechnol,2005,68:213-219
[110] Koh S, Kim J, Lee DS, et al. Mechanistic study of the intramolecular conversion of maltoseto trehalose by Thermus caldophilus GK24trehalose synthase. Carbohydrate Research,2003,338:1339-1343
[111] Tsusaki K, Nishimoto T, Nakada T, et al. Cloning and sequencing of trehalose synthasegene from Thermus aquaticus ATCC33923. Biochim Biophys Acta,1997,1334,28-32.
[112] Wang JH, Tsai MY, Chen JJ, et al. Role of the C-terminal domain of Thermusthermophilus trehalose synthase in the thermophilicity, thermostability, and efficientproduction of trehalose. J Agric Food Chem,2007,55:3435-3443
[113] Zhu Y, Wei D, Zhang J, Wang Y, Xu H, Xing L, Li M. Overexpression andcharacterization of a thermostable trehalose synthase from Meiothermus ruber.Extremophiles.2010,14:1-8
[114] Cardoso FS, Castro RF, Santos H. Biochmical and genetic characterization of the pathwaysfor trehalose metabolism in Propionibacterium freudenreichii, and their role in stressresponse. Microbiology,2007,153:270-280
[115] Chen YS, Lee GC, Shaw JF. Gene cloning expression and biochemical characterization ofa recombinant trehalose synthase from Picrophilus torridus in Escherichia coli. J AgricFood Chem,2006,54:7098-7104
[116] Pan YT, Koroth EV, Jourdian WJ, et al. Trehalose synthase of Mycobacterium smegmatis:purification, cloning, expression, and properties of the enzyme. Eur J Biochem,2004,271:4259-4269
[117] Yue M, Wu XL, Gong WN, Ding HB. Molecular cloning and expression of a noveltrehalose synthase genefrom Enterobacter hormaechei. Microb Cell Fact.2009,doi:10.1186/1475-2859-8-34
[118] Wu X, Ding H, Yue M, Qiao Y. Gene cloning, expression, and characterization of a noveltrehalose synthase from Arthrobacter aurescens. Appl Microbiol Biothchnol,2009,83:477-482
[119] Besra GS., Bolton RC., McNeil MR, et al. Structural elucidation of a novel family ofacyltrehaloses from Mycobacterium tuberculosis. Biochemistry,1992,31:9832-9837.
[120]韦宇拓,朱绮霞,罗兆飞,陈发忠,李桂媛,黄鲲,黄日波。耐放射异常球菌海藻糖合成酶基因的克隆及功能鉴定。生物化学与生物物理进展，2004,31:1018-1023.
[121] Alugupalli S, Laneelle MA, Larsson L, et al. Chemical characterization of the ester-linked3-hydroxy fatty acyl-containing lipids in Mycobacterium tuberculosis. J Bacteriol,1995,177,4566-4570.
[122]韦宇拓，黄日波，蒙健宗，卢福燊，庞中文，朱绮霞，陈发忠，罗兆飞，卢运琨，王青艳，黄鲲。谷氨酸棒杆菌海藻糖合成酶基因及海藻糖制造方法。中国：200410013006.9。2005.
[123] Kim HJ，Kim AR, Jeon SJ Immobilization on chitosan of a thermophilic trehalosesynthase from Thermus thermophilus HJ6. J Microbiol Biotechnol,2010,20:513-517
[124] MacGregor EA，Janecek S, Svensson B.Relationship of sequence and structure to specificityin the α-amylase family of enzyme. Biochimica et Biophysica Acta,2001,1546：1-20
[125] Janecek S, α-Amylase family: molecular biology and evolution, Prog Biophys Mol Biol,1997,67:67-97.
[126] Svensson B. Protein engineering in the α-amylase family: catalytic mechanism,substratespecificity,and stability. Plant Molecular Biology,1994,25:141-157
[127] Janecek S, Svensson B, Henrissat B. Domain evolution in the α-amylase family. J Mol Evol,1997,45:322-331.
[128] Park KH, Kim TJ, Cheong TK, et al. Structure, specificity and function ofcyclomaltodextrinase, a multispecific enzyme of the α-amylase family. Biochem BiophysActa,2000,1478:165-185.
[129] MacGregor EA, Jespersen HM, Svensson B. A circularly permuted α-amylase-typeα/β-barrel structure in glucan-synthesizing glucosyltransferases. FEBS Lett,1996,378:263-266.
[130] Katsuya Y, Mezaki Y, Kubota M, et al. Three-dimensional structure of Pseudomonasisoamylase at2.2resolution. J Mol Biol,1998,281:885-897
[131] Kamitori S, Kondo S, Okuyama K, et al. Crystal structure of Thermoactinonyces vulgarisR-47α-amylase II (TVAII) hydrolyzing cyclodextrins and pullulan at2.6resolution, JMol Biol,1999,287:907-921.
[132] Pan YT, Carroll JD, Asano N, et al. Trehalose synthase converts glycogen to trehalose.FEBS Journal,2008,275(13):3408-3420.
[133] van der Maarel MJEC, van der Veen B, Uitdehaag JCM, et al. Properties and applications ofstarch-converting enzymes of the α-amylase family. J Biotechnol,2002,94:137-155
[134] Svensson B. Protein engineering in the α-amylase family: catalytic mechanism, substratespecificity, and stability. Plant Mol Biol,1994,25:141-157
[135] Zhang R, Pan YT, He S, Lam M, Brayer GD, Elbein AD, Withers SG.2011Mechanisticanalysis of trehalose synthase from Mycobacterium smegmatis. J Biolog Chem,286:35601-35609
[136] ZollerM J, SmithM. Oligonucleotide-directed mutagenesis using M13-derived vectors: anefficient and general procedure for the production of point mutations in any fragment ofDNA. Nucleic Acids Res,1982,10(20):6487-6500
[137]陶苏丹,刘佳,陈喜文,陈德富点饱和突变技术及其在蛋白质工程中的应用.中国生物工程杂志,2007,27(8):82-86
[138] Kegler-Ebo D, Pollack G, DiMaio D. Use of codon cassette mutagenesis for saturationmutagenesis in: In Vitro Mutagenesis Protocols. Totowa, N J: Humana Press,1996.297-311
[139] Hill CM, Li WS, Thoden JB, et al. Enhanced degradation of chemical warfare agentsthrough molecular engineering of the phosphotriesterase active site. J Am Chem,2003,125(30):8990-8991
[140] Doucet N, Wals PD, Pelletier JN. Site-saturation mutagenesisof Tyr-105reveals itsimportance in substrate stabilization and discrimination in TEM-1beta-lactamase. J BiolChem,2004,279(44):46295-46303
[141] Vandeyar MA, Weiner MP, Hutton CJ, et al. A simple and rapid method for the selection forthe selection of oligodeoxy-nucleotide-directed mutants. Gene,1988,65:129-133
[142] Fang TY, Tseng WC, Wang MY, et al. Protein engineering of Sulfolobus solfataricusmaltooligosyltrehalose synthase to alter its selectivity. J Agric Food Chem,2007,55:5588-5594
[143]齐向辉，韦宇拓，黄日波等.甘油脱水酶的理性设计：基因杂合改善酶的性质.科学通报，2006，51（20）：2372-2380
[144] Caldwell RC, Joyee GF. Randomization of genes by PCR mutagenesis. In PCR MethodsApplic,1992,2:28-33
[145] Fujii R, Kitaoka M, Hayashi K. One-step random mutagenesis by error-prone rolling circleamplification. Nucleic Acids Res,2004,19: e145
[146] Chirumamilla RR, Muralidhar R, Nigam P, et al.Improving the quality of industriallyimportant enzymes by directed evolution. Molecular and Cellular Bochemistry,2001,224:159-168
[147]吴梧桐。蛋白质工程技术与新型生物催化剂设计.药物生物技术，2004，11：1-6
[148] Stemmer W P C. DNA shuffling by randomfragmentation and reassembly:in vitrorecombination for molecular evolution. Proc Natl Acad Sci, USA,1994,91(22):10747-107511
[149] Crameri A, Raillard S A, Bermudez E, et al. DNA shuffling of a family of genes fromdiverse species accelerate directed evolution. Nature,1998,391:288
[150] Volkov AA, Arnold FH. Methods in enzyme. Elservier,2000,447-456.
[151] Schmidt C, Umeno D, Armoldz F H. Molecular breeding of carotenoid biosyntheticpathway. Nature Biotechnol,2000,18:750
[152] Zhao H, Giver L, Shao Z, et al. Molecular evolution by staggered extension process (StEP)in vitro recombination. Nature Biotechnology,1998,16(3):258-261.
[153] Leung D, Chen E, Goeddel D. A method for random mutagenesis of a defined DNA segmentusing a modified polymerase chain reaction. Technique,1989,2:302-303
[154] Kikuchi M, Ohnishi K, Harayama S. Novel family shuffling methods for the in vitroevolution of enzymes.Gene,1999,236:159-167
[155] Park YM, Ghim SY. Enhancement of the activity and pH-performance of chitosanase fromBacillus cereus strains by DNA shuffling. Biotechnol Lett,2009,31:1463-1467
[156] Lutz S, Ostermeier M, Benkovic SJ, et al. Creating multiple-crossover DNA librariesindependent of sequence identity. PNAS,2001,98(20):11248-11253
[157]胡光星，郭美锦，储炬等，DNA改组技术发展与应用。中国生物工程杂志，2002，22（3）：9-12
[158] Grace DeSantis， J Bryan Jones． Chemical modification of enzymes for enhancedfunctionality．Current Opinion in Bintechnology，1999，10：324-330．
[159] Ueji S L，UedaA，TanakaH，et al．Chemical modification of lipase with various hydrophobicgroups improves their enantioselectivity in hydrolytic reantions．Biotechnol Lett，2003，25：83-87
[160] Zhu Y, Zhang J, Wei D, et al. Isolation and identification of a thermophilic strain producingtrehalose synthase from geothermal water in China. Biosci Biotechnol Biochem,2008,72(8):2019-2024
[161]卢圣栋，现代分子生物学实验技术.北京：高等教育出版社，1993.
[162]萨木布鲁克J,弗里奇EF,曼尼阿蒂斯T.金冬雁等,译.分子克隆试验指南.第二版.北京：科学出版社，1993.
[163]沈萍，范秀容，李广武.微生物学实验(第三版).北京：高等教育出版社，1999.
[164] Arnold K, Bordoli L, Kopp J, et al. The Swiss-Model Workspace:a web-based environmentfor protein structure homology modeling. Bioinformatics,2006,22:195-201
[165] Kiefer F, Arnold K, Künzli M, et al. The Swiss-Model Repository and Associated resources.Nucleic Acids Res,2009,37:D387-D392
[166] Cristianini N and Shawe-Taylor J An Introduction to Support Vector Machines and OtherKernel-based Learning Methods. Cambridge University Press, Cambridge, UK.2000
[167] Krishnan VG, and Westhead DR A comparative study of machine-learning methods topredict the effects of single nucleotide polymorphisms on protein function. Bioinformatics,2003,19:2199-2209.
[168] Gao S, Zhang N, Duan GY, Yang Z, Ruan JS, Zhang T. Prediction of function changesassociated with single-point protein mutations using support vector machines (SVMs). HumMutat,2009,30:1161-1166
[169]宋卓，张宁，阮吉寿，杨卓，张涛。2007基于氨基酸序列预测蛋白质功能性点突变位点。生物物理学报23:134-138
[170] Andrade MA, Chacón P, Merelo JJ and Morán F. Evaluation of secondary structure ofproteins from UV circular dichroism using an unsupervised learning neural network. ProtEng,1993,6:383-390
[171] Merelo JJ, Andrade MA, Prieto A and Morán F Proteinotopic Feature MapsNeurocomputing,1994,6:443-454
[172]沈星灿，梁宏，何锡文，王新省2004圆二色光谱分析蛋白质构象的方法及研究进展分析化学评述与进展。3:388-394
[173] Greenfield NJ Methods to Estimate the Conformation of Proteins and Polypeptides fromCircular Dichroism Data. Anal Bioch,1996,235:1-10
[174] PerczelA, Park K, Fasm GD. Deconvolution of the circular dichroism spectra of proteins:The circular dichroism spectra of the antiparallel and sheet in proteins. Proteins Struct FunctGenet,1992:57-69.
[175] Chang CT, Wu CS, Yang JT. Circular dichroic analysis of protein conformation inclusion ofthe beta-turns. Anal Biochem,1978,91:13-31.
[176]丁晓岚,高红旗2008圆二色光谱技术应用和实验方法实验技术与管理25:48-52
[177] Levitt, M.; Chothia, C. Structural patterns in globular proteins. Nature,1976,261(5561):552–558.
[178] Nakai K, Kidera A, Kanehisa M, Cluster analysis of amino acid indices for prediction ofprotein structure and function. Protein Eng,1988,224:437-449
[179]李军、张莉娜、温珍昌生物软件的选择与使用指南北京：化学工业出版社2008
[180] Sippl JM Calculation of conformational ensembles from potetials of mean force: anapproach to the knowledge based prediction of local structures in globular proteins. J MolBiol,1990,213:859-883
[181] Charkravarty S, Varadarajun R. Elucidation of determineants of protein stability throughgenome sequence analysis. FEBS Lett,2000,470:65-69.
[182] Kumar S, Tsai CJ, Nussinov R. Factors enhancing protein thermostability. Protein Eng,2000,13:179-191.
[183] Sen S, Venkata Dasu V, Mandal B. Developments in directed evolution fro improvingenzyme functions. Appl Biochem Biotechnol,2007,143:212-223
[184] Vartanian JP, Henry M, Wain-Hobson S. Hypermutagenic PCR involving all four transitionsand a sizeable proportion of transversions. Nucleic Acids Res,1996,24(14):2627-2631
[185] Shafikhani S, Siegel RA, Ferrari E, et al. Generation of large libraries of random mutants inBacillus subtilis by PCR-based plasmid multimerization. BioTechniques,1997,23(2):304-310
[186] Suzuki M, Christians FC, Kim B, et al. Tolerance of different proteins for amino aciddiversity. Mol Divers,1997,2(1-2):111-118.
[187] Lin L, Meng X, Liu P, et al. Improved catalytic efficiency of endo-β-1,4-glucanase fromBacillus subtilis BME-15by directed evolution. Appl Microbiol Biotechnol,2009,82:671-679
[188] Rabbani M, Mirmohammad Sadeghi H, Moazen F, et al. Cloning and expression ofrandomly mutated Bacillus subtilis α-amylase genes in HB101. Biotechnol Res Int,2011,doi:10.4061/2011/305956
[189] Caldwell RC, Joyce GF. Mutagenic PCR.PCR Meth Appl,1994,3:136-140
[190]李晶琴基于PCR技术的蛋白质改造策略。国际检验医学杂志，2006,27（12）：1129-1131
[191] Wang JH, Tsai MY, Shaw JF, et al. Construction of a recombinant thermostableβ-amylase-trehalose synthase bifunctional enzyme for facilitating the conversion of starch totrehalose. J Agric Food Chem,2007,55:1256-1263
[192] Chou HH, Chang SW, Lee GC，et al. Site-directed mutagenesis improves the thermostabilityof a recombinant Picrophilus torridus trehalose synthase and efficiency for the productionof trehalose from sweet potato starch. Food Chem,2010,119:1017–1022.
[193]王青艳，陈发忠，黄日波等。Thermobifida fusca海藻糖合成酶的定点突变及其动力学性质研究。广西农业生物科学，2007,26（2）：115-119