利用扣囊复膜酵母菌A11菌株转化木薯淀粉生产海藻糖的研究
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摘要
海藻糖(Trehalose)广泛存在于微生物、无脊椎动物、脊椎动物和植物中。研究显示海藻糖不仅可以作为细胞碳源储存物,还可用作高效保护剂增加细胞组分对不良环境条件(如高温、饥饿、冷冻、脱水、辐射、有毒试剂、高渗透压和高酒精浓度)的抗性。海藻糖不仅可以在生物体内起作用,而且外加的海藻糖同样可以对生物大分子具有保护作用,这使其在很多方面具有广泛的应用,如在医学、分子生物学和微生物学中作为细胞防冻剂、化妆品的有效成分、诊断试剂和生物产品的稳定剂,以及用作新鲜食品的保鲜剂。因此,用廉价高效的的方法生产海藻糖已成为当前研究热点。
淀粉价格低廉,来源广泛,以淀粉为原料,酶法制备海藻糖,起始物价格便宜,转化率高,很有应用前景,被认为是用来生产海藻糖的最佳原料。我们在过去的研究中也发现Saccharomycopsis fibuligera sdu是利用淀粉来转化生产海藻糖的很好菌株。但该酵母菌体内能合成酸性和中性海藻糖酶,这些酶在细胞中可以分解合成的海藻糖,这将不利于海藻糖的累积。因此,为了提高海藻糖的产量,从该菌株中去除或降低酸性和中性海藻糖酶活力是必要的。后来,利用化学诱变技术获得酸性和中性海藻糖酶活力大大降低的A11突变株,该突变株在可溶性淀粉培养基中培养菌体内海藻糖的累积量达到19.1%(w／w),在玉米淀粉培养基中培养菌体内海藻糖的累积量达到24.0%(w／w)。本研究发现用木薯淀粉代替玉米淀粉来生产海藻糖能使海藻糖的生产成本更低,A11突变株在2.0%(w／v)木薯淀粉培养基2-L发酵罐中发酵48 h菌体内海藻糖的累积量达到25.8%(w／w)。
使用可溶性淀粉和玉米淀粉作为碳源来转化海藻糖是不经济的。木薯作为一种非粮作物,被用来当做食品和发酵工业的淀粉材料,在中国南方很多省市大范围种植。研究中,我们发现在摇瓶试验中,A11菌株利用木薯淀粉转化海藻糖量高于可溶性淀粉、玉米淀粉等。且在2.0%木薯淀粉、4.0%豆饼粉水解液、pH 5.5·和30℃条件下培养48 h,海藻糖累积量达24.8%(w／w)。
本论文还利用2-L全自动发酵罐研究该菌株发酵木薯淀粉生产海藻糖,A11菌株48 h发酵罐中海藻糖积累量为25.8%,残留还原糖浓度和残留总糖浓度分别为1.2 mg/mL和2.1 mg/mL。这是目前所报道的海藻糖积累量最高的菌株。
在海藻糖提取精制工艺方面,本论文主要用了三氯乙酸和乙醇沉淀法提取菌体中的海藻糖,提取液经过进一步分离纯化,得到海藻糖晶体。
针对目前海藻糖的提取方法都存在不足,本研究用化学诱变方法得到突变株A83,该菌株在37℃条件下细胞壁通透性发生变化能够自动释放海藻糖,且海藻糖释放率达到44.7%。此研究是在目前环境污染、资源消耗形势严峻的社会背景下,为响应节能环保的号召以及为低成本无毒性的海藻糖的工业生产做出了积极的探索和尝试。
Trehalose (α-D-glucopyranosyl-1-1-α-glucopyranose) is widely present in bacteria, yeasts, and fungi as well as some insects, invertebrates,vertebrates and plants. Recent results have shown that trehalose does not only primarily function as a reserve carbohydrate but also as a highly efficient protectant, enhancing the resistance of cellular components against adverse conditions such as high temperature, starvation, radiation, freezing, dehydration, high osmotic pressure,toxic reagents and high concentration of ethanol. These unique protective properties of trehalose make it an interesting compound for several applications, for example as cryoprotectant for cells in medicine, molecular biologyand microbiology, as an effective component in cosmetics, as a stabilizer for clinical reagents and bioproducts, and even as a preservative for fresh foodstuff. Therefore, much attention has been directed to inexpensive and effective means for producing trehalose.
It is thought that starch is the best substrate for production of trehalose due to its low price, high conversion rate and easily obtained raw material.It was shown that S. fibuligera is a good producer of trehalose from starch. But the yeast strain can synthesize acid trehalase and neutral trehalase which can mobilize trehalose accumulated by the cells. Therefore, it is very necessary to delete or decrease the activity of acid and neutral trehalase in the yeast in order to increased trehalose production. Later, using chemical mutagenesis techniques to obtain a mutant strain which acidic and neutral trehalase activity significantly reduce. Trehalose yield was 19.1 g per 100g of cell dry weight used use soluble starch as the substrate, and trehalose yield was 24.0 g per 100g of cell dry weight used use corn starch as the substrate.
It is not economical to use soluble starch or corn starch as the substrate for production of trehalose in a large scale. As one of the non-food crops, cassava is widely cultivated in some of the southern provinces of China as a source of starchy material for food and fermentation industry. In this study, we found that strain A11 accumulated more trehalose in the media containing corn starch than in those containg souble starch and corn strach. The results indicated that trehalose accumulation by S. fibuliger a A11 was optimal in the medium comprised of 2.0% cassava starch,4.0% hydrolysate of soybean cake at pH 5.5 and 30℃for 48 h. At the flask level, trehalose yield was 24.8 g per 100g of cell dry weight.
At the end of 2-Lfermation, trehalose yield was 25.8 g per 100 g of cell dry weight. This is the highest trehalose yield accumulated in the yeast cells reported so far. At the same time,0.12 mg/mL of reducing sugar and 0.21 mg/mL of total sugar were left in the fermented medium. In this paper, TCA and alcohol used to extract trehalose from the cells of strain All. After isolation and purification, the crystal trehalose was obtained from the culture.
Based on the shortcomings of current extraction methods of trehalose from the yeast cells by using the toxic chemicals, a mutant A83 which was thermosensitive autolytic was obatined by chemical mutagenesis, the trehalose could be automatically releasedfrom the mutant cells under the conditions of the cell wall permeability changes at 37℃, and trehalose release rate was 44.7%.
淀粉价格低廉,来源广泛,以淀粉为原料,酶法制备海藻糖,起始物价格便宜,转化率高,很有应用前景,被认为是用来生产海藻糖的最佳原料。我们在过去的研究中也发现Saccharomycopsis fibuligera sdu是利用淀粉来转化生产海藻糖的很好菌株。但该酵母菌体内能合成酸性和中性海藻糖酶,这些酶在细胞中可以分解合成的海藻糖,这将不利于海藻糖的累积。因此,为了提高海藻糖的产量,从该菌株中去除或降低酸性和中性海藻糖酶活力是必要的。后来,利用化学诱变技术获得酸性和中性海藻糖酶活力大大降低的A11突变株,该突变株在可溶性淀粉培养基中培养菌体内海藻糖的累积量达到19.1%(w／w),在玉米淀粉培养基中培养菌体内海藻糖的累积量达到24.0%(w／w)。本研究发现用木薯淀粉代替玉米淀粉来生产海藻糖能使海藻糖的生产成本更低,A11突变株在2.0%(w／v)木薯淀粉培养基2-L发酵罐中发酵48 h菌体内海藻糖的累积量达到25.8%(w／w)。
使用可溶性淀粉和玉米淀粉作为碳源来转化海藻糖是不经济的。木薯作为一种非粮作物,被用来当做食品和发酵工业的淀粉材料,在中国南方很多省市大范围种植。研究中,我们发现在摇瓶试验中,A11菌株利用木薯淀粉转化海藻糖量高于可溶性淀粉、玉米淀粉等。且在2.0%木薯淀粉、4.0%豆饼粉水解液、pH 5.5·和30℃条件下培养48 h,海藻糖累积量达24.8%(w／w)。
本论文还利用2-L全自动发酵罐研究该菌株发酵木薯淀粉生产海藻糖,A11菌株48 h发酵罐中海藻糖积累量为25.8%,残留还原糖浓度和残留总糖浓度分别为1.2 mg/mL和2.1 mg/mL。这是目前所报道的海藻糖积累量最高的菌株。
在海藻糖提取精制工艺方面,本论文主要用了三氯乙酸和乙醇沉淀法提取菌体中的海藻糖,提取液经过进一步分离纯化,得到海藻糖晶体。
针对目前海藻糖的提取方法都存在不足,本研究用化学诱变方法得到突变株A83,该菌株在37℃条件下细胞壁通透性发生变化能够自动释放海藻糖,且海藻糖释放率达到44.7%。此研究是在目前环境污染、资源消耗形势严峻的社会背景下,为响应节能环保的号召以及为低成本无毒性的海藻糖的工业生产做出了积极的探索和尝试。
Trehalose (α-D-glucopyranosyl-1-1-α-glucopyranose) is widely present in bacteria, yeasts, and fungi as well as some insects, invertebrates,vertebrates and plants. Recent results have shown that trehalose does not only primarily function as a reserve carbohydrate but also as a highly efficient protectant, enhancing the resistance of cellular components against adverse conditions such as high temperature, starvation, radiation, freezing, dehydration, high osmotic pressure,toxic reagents and high concentration of ethanol. These unique protective properties of trehalose make it an interesting compound for several applications, for example as cryoprotectant for cells in medicine, molecular biologyand microbiology, as an effective component in cosmetics, as a stabilizer for clinical reagents and bioproducts, and even as a preservative for fresh foodstuff. Therefore, much attention has been directed to inexpensive and effective means for producing trehalose.
It is thought that starch is the best substrate for production of trehalose due to its low price, high conversion rate and easily obtained raw material.It was shown that S. fibuligera is a good producer of trehalose from starch. But the yeast strain can synthesize acid trehalase and neutral trehalase which can mobilize trehalose accumulated by the cells. Therefore, it is very necessary to delete or decrease the activity of acid and neutral trehalase in the yeast in order to increased trehalose production. Later, using chemical mutagenesis techniques to obtain a mutant strain which acidic and neutral trehalase activity significantly reduce. Trehalose yield was 19.1 g per 100g of cell dry weight used use soluble starch as the substrate, and trehalose yield was 24.0 g per 100g of cell dry weight used use corn starch as the substrate.
It is not economical to use soluble starch or corn starch as the substrate for production of trehalose in a large scale. As one of the non-food crops, cassava is widely cultivated in some of the southern provinces of China as a source of starchy material for food and fermentation industry. In this study, we found that strain A11 accumulated more trehalose in the media containing corn starch than in those containg souble starch and corn strach. The results indicated that trehalose accumulation by S. fibuliger a A11 was optimal in the medium comprised of 2.0% cassava starch,4.0% hydrolysate of soybean cake at pH 5.5 and 30℃for 48 h. At the flask level, trehalose yield was 24.8 g per 100g of cell dry weight.
At the end of 2-Lfermation, trehalose yield was 25.8 g per 100 g of cell dry weight. This is the highest trehalose yield accumulated in the yeast cells reported so far. At the same time,0.12 mg/mL of reducing sugar and 0.21 mg/mL of total sugar were left in the fermented medium. In this paper, TCA and alcohol used to extract trehalose from the cells of strain All. After isolation and purification, the crystal trehalose was obtained from the culture.
Based on the shortcomings of current extraction methods of trehalose from the yeast cells by using the toxic chemicals, a mutant A83 which was thermosensitive autolytic was obatined by chemical mutagenesis, the trehalose could be automatically releasedfrom the mutant cells under the conditions of the cell wall permeability changes at 37℃, and trehalose release rate was 44.7%.
引文
程池.天然生物保存物质——海藻糖的特性和应用.食品与发酵工业,1996,1：59-64
丁振.海藻糖合酶高产菌株的选育及酶学特性研究.山东轻工业学院硕士论文,2007
高峻,池振明.耐高浓度酒精菌与酒精敏感酵母菌中海藻糖含量与耐酒精的关系.食品与发酵工业,2001,27：4-8
葛宇,袁勤生.海藻糖对生物活性物质的保护作用机理研究进展.药物生物技,2002,9：297-300
胡宗利,夏玉先,陈国平等.海藻糖的生产制备及其应用前景.中国生物工程杂志,2004,24：44-48
黄鸣,黎锡流.海藻糖独特的生物学功能及其广泛应用.食品研究与开发,2002,23：56
黄成垠,戴秀玉.海藻糖对医用诊断工具酶活性保护作用研究.微生物学通报,1997,4：341-34
黄平.奇妙的双糖—海藻糖.生命的化学,1995,15：26-28
简文杰,江滨伟,庞杰等。新型食品添加剂,海藻糖的生产及其应用.现代商贸工业,2003,6：41-50
赖承兴,葛宇,袁勤生等. 海藻糖的MTSase及MTHase酶促合成条件的研究.中国医药工业杂志,2003,34：433-436
李静,谭海刚,宫春波,李凤梅,陈勇,于翠芳.从面包酵母中提取海藻糖的研究. 食品与药品,2008,10：24-26
李浩明等.海藻在生物制品干燥与保存中得应用研究.食品与发酵工业,1994,4：49-51
刘传斌,谢健,苗蔚荣等.海藻糖的性质及其广阔应用前景.中国微生态学杂志,1998,10：58-62
刘传斌,云战友等.海藻糖的分析方法.第八届全国生物化工学术会议论文集,化学工业出版社,1998,414-416
刘洋,张红缨,高俊芳,张今.酵母菌中海藻糖的几种提取方法比较.中国生化药物杂志,1999,20:16
罗明典.微生物生产海藻糖及其应用前景.微生物学通报,1996,4：252-254
牟禹.海藻糖合成酶基因转化玉米的研究.四川农业大学硕士论文,2007
聂凌鸿,宁正祥.海藻糖的生物保护作用.生命的化学,2001,21：206-209
聂凌鸿,宁正祥.海藻糖研究现状及其在食品工业中的应用.食品与发酵工业,2002,28：71-74
任媛媛,刘景芳.海藻糖代谢途径相关基因及生物工程.微生物学报,2003,43：821-824
史戈峰等.酒精发酵过程中酿酒酵母海藻糖代谢的研究,食品与发酵工业,1999,25：8-10
孙长慧.以淀粉为原料酶法制备海藻糖的研究. 北京化工大学硕士论文,2002
唐传核,葛文光.从活性干酵母中提取海藻糖的工艺.无锡轻工业大学学报,1998,3：3
唐传核, 葛文光. 海藻糖的开发现状及应用前景.中国食品用化学品,1997,3：1-4
童桥,聂松奇,林克椿.海藻糖对载药脂质体在干燥—再水化过程中的保护作用.生物化学杂志,1992,8：711-714
王岁楼.食品生物技术.海洋出版社,1998
肖国驱,黄晓兰,蔡小伟等.海藻糖产生菌的筛选及其发酵条件研究.生物技术,2002,12：15-17
宣劲松.日本长海带DNA文库构建、EST序列分析及海带6-磷酸海藻糖合成酶基因的克隆和分离.中国科学院遗传与发育生物学研究所硕士论文,2005
姚汝华.海藻糖及其应用前景.广州食品工业科技,1995,11：29-31
叶芳.高产海藻糖酵母菌株利用玉米淀粉一步转化生产海藻糖的工艺研究.中国海洋大学硕士论文,2003
尤新.功能性发酵制品.中国轻工业出版社,2000
于春燕,郎刚华.海藻糖研究进展. 青岛大学学报,2000,6
袁勤生.隐生生物中的典型化合物.中国生化药物杂志,1999,20：48-50
张红缨,刘洋,张今.海藻糖的生物合成和相关酶的特性.微生物学通报,1998,25.236-238
张树珍,郑学勤.海藻糖合酶基因的克隆及其序列的分析.热带作物学报,1999
赵晓峰,吴荣书.海藻糖的功能特性及应用.广州食品与科技,2004,20：18-23
Adridge S. Trehalose boots prospects for improved biopharmaceuticals and Vaccines. Genetic Engineer News,1995,15:10-11
Attfield PV. Trehalose accumulate in Saceharomyees cerevisiae during exposure to agents that include heat shock response. FEBS Lett,1987,225:259-263
Bleha M. Berwent Biotechnol Abst,1995,14:07223
Beattie GM, Crowe JH, Lopez AO. Trehalose:a cryoprotectant that enhances recovery and preserves function of human pancreatic islets after long-term storage. Diabetes,1997,46: 519-523
Bell W, Klaassen P, Ohnacker M, Boller T, et al. Characterization of the 56 kDa subunit of yeast trehalose-6-phosphate synthase and cloning its gene reveal its identity with the product of CIF1, a regulator of carbon catabolite inactivation. Eur J Biochem,1992,209:951-959
Bhandal IS, Hauptmann RM, Widholm JM. Trehalose as cryoprotectant for the freeze preservation of carrot and tobacco cells. Plant Physio,1985,78:430-432
Cabib E and Duran A. Simple and sensitive procedure for screening yeast mutants that lyse at nonpermissive temperatures. J Bacteriol,1975,124:1604-1606
Charlemagne GH, et al. Microbiol,1998,144:671-680
Chi ZM, Chi Z, Liu GL, Wang F, Ju L, Zhang T. Saccharomycopsis fibuligera and its applications in biotechnology.Biotecnol Adv,2009,27,423-431
Chi ZM. Enhanced conversation of soluble starch to trehalose by a mutant of Saccharomycopsis fibuligera sdu. J Biotechnol,2003,102:135-141
Colaco C. Extraordinary stability of enzymes dried in trehalose:simplified molecular biology. Biotechnol,1992,10:701-703
Colaco C. Food packaging and preservation. London:Applied Science Publishers LTD,1994, 123-139
Collins EB. Bifid bacteria in dairy products. Cult Dai Prod J,1993,28:1620-1627
Crowe JH,Crowe LM, Chapman D. Preservation of membranes in anhydrobiotic organisms:The role of trehalose. Scienee,1984,223:701-703
Crowe JH. Anhydrobiosis:An unsolved problem.American Naturalist,1971,105:563
Crowe JH. Hoekstra. F. Anhydrobiosis. Annual Review Physiol,1992,54:579-599
De Virgilio, C, Hottinger, T, Dominiguez J, Boller T and Wiemken A. The role of trehalose synthesis for the acquisition of thermotolerance in yeast. Eur J Biochem,1994,219:179-186
Dijck VP. Differential importance of trehalose in stress resistance in fermenting and nonfermenting Saccharomyces cerevisiae cells. Appl Environ Microbiol,1995,61:109-115
Diniz-Mendes L. Biotechnol Bioeng,1999,65:572-578
Dunean K,Sam T,Vineent M. Organization and Mobility of water in amorphous and crystalline trehalose. Nature,2006:1-4
Eduarda D, Vadym S, Tetyana S, Konrad J, Pavel D. Recovery of tubulin functions after freeze-drying in the presence of trehalose. Anal Biochem,2010,397:67-72
Elbein AD, Pan YT, Pastuszak I, Carroll D. New insights on trehalose:a multifunction molecule. Glycobio,2003,13.:17-27
Gadd GM, Chalmers K and Reed RH. The role of trehalose in dehydration resistance of Saccharomyces cerevesiae. FEMS Microbiol Lett,1987,48:249-254
Galinski EA, Herzog RM.The role of trehalose as a substitute for nitrogen-containing compatile solutes. Archieve Microbiol,1990,153:607-613
Gareia A,Engler J. Effeets of osmoprotectants upon NaCL stress in riee. Plant Microbiol, 1997,144:671-680
Goddijn DK. Trehalose metabolism in Plants. Tre Plant Sci,1999,4:315-319
Goddijn,Smeekens S.Sensing trehalose biosynthesis in piants. Plants J,1998,14:143-146
Guo N, Puhlev I, Brown DR, Mansbridge J and Levine F. Trehalose expression confers desiccation tolerance on human cells. Nature Biotechnol,2000,18:168-171
Hakkok. Derwent Biotechnol Albst,1995,14:07216
Hoelzle I,Streeter JG. Increased accumulation of trehalose in Rhlizibia cultured under 1% oxygen. Appl. Environ Microbiol,1990,56:3213-3215
Holmstrom KO. Engineering plant adaptation to water-stress. Acta-Universitatis-Agriculture-Sueciae-Agraia,1998,84:49
Hottiger T, Boller T, Wiemken A. Correlation of trehalose and heat resistance in mutants altered in the RAS/adenylate cyclase pathway is trehalose a themoprotectant. FEBS Lett,1989,225:431-434
Jang IC, Seo JS. Expression of a bifunctional fusion of the Escherichia coli genes for trehalose-6- phosphate synthase and trehalose -6-phosphatephosphatase in transgenic rice plants increases trehlose accumulation and a biotitics stress tolerance without stunting growth. Plant Physiol,2003,131:516-524
Jeong SC, Pack IS, Cho EY. Molecular analysis and quantitative detection of a transgenic rice line expressing a bifunctional fusion TPSP. Food Control,2007,18:1434-1442
John M. Thevelein Regulation of trehalose mobilization in fungi. Microbiol Reviews,1984,48(3):42-59
Kandror O, DeLeon A and Goldberg AL. Trehalose synthesis is induced upon exposure of Escherichia coli to cold and is essential for viability at low temperatures. Proc Natl Acad Sci USA,2002,99:9727-9732
Karl W,Iris K,Markus B.Metabolic regulation of the trehalose content of vegetative yeast.FEBS Lett,1991,291:269-272
Kaushik JK, Bhat R. Why is trehalose an exceptional protein stabilizer? Biol Chem,2003,278: 6458-26465
Kazubiko M. Cloning and sequencing of Trehalose Biosynthesis Genes from Rhizobium. Sp.,M-11. Bioscience. Biotechnol. Biochem,1996,60:717-720
Kjell H,Einar M,Biorn W.Droughtt tolerance in tobacco.Nature,1996,379:683-684
Koichi Y,Hiroe Y. Proteetion by trehalose of DNA from Radiation Damage. Biochem,1997,84:157-1540
Lebo Z. Analysis of PFK3 a gene involved in part-culate phso1 yeast.1994,10:199-204
Lederer E. Cord factor and related trehalose esters. Chem Phys Lipids,1976,16:91-106.
Leopold AC. Membranes, metabolism, and dry organisms. Cornell University Press, Ithaca, NY, 1986,1-377
Leslie B.Trehalose lowers membrane phase transitions in dry yeast cells. Biochim Biophy Acta,1987,1192:7-13
Li AZ. Chan-Halbrendt C. Ethanol production in China:Potential and technologies. Appl Energ, 2009,04:047
Lvine H,Sladc I.Another view of trehalose for drying and stabilizing biological materials. Bipoharm,1992,5:36-40
Madin, KAC and Crowe JH. Anhydrobiosis in nematodes:carbohydrate and lipid metabolism during dehydration. J Exp Zool.,1975,193:335-342
Marutu K, Nakada T, Kubota M,et al. Formation of trehalose from maltooligosaccharides by a novel enzymatic system.Biosci Biotech Biochem,1995,59:1829-1834
Masaru K. Purification and characteriazation of New Trehalose-producing Enzymes Isolated from the Hyperthermophilic Archae sulfolobus solfataricus KM1. Bioscience Biotechnology and Biochemistry,1996,60:546-550
Mauro S, Jose RM. Stabilization against thermal inactivation promoted by sugars on enzyme struture and function:why is trehalose more effective than other sugars. Archive of Biochemistry and Biophysics,1998,360:10
Mohamad RG, Bijan R, Saman H,Khosrow K, Leila H. Roles of trehalose and magnesium sulfate on structural and functional stability of firefly luciferase. J Mole Cataly B: Enzymatic,2010,62:127-132
Mozhaer VV, Structure-stability relationships in proteins:new approaches to stabilizing Enzy. Microb Technol,1984,6
Murao S. Enzymatic synthesis of trehalose from maltose. Agric Biol Chem, 1985,49:2113-2118
Nicolaus B, Gambacorta A, Basso AL, Riccio R, DeRosa M and Grant WD. Trehalose in Archaebacteria systems. Appl Microbiol,1988,10:215-217
Nishant K. Jain, IR. Role of trehalose in moisture-induced aggregation of bovine serum albumin. Euro J Pharma Biopharma,2008,69:824-834
Nishimoto T. Purification and properties of a novel enzyme, trehalose synthase, from Pimeloba-cter sp.R48. Biochem Biosci Biotech,1996,60:640-644
Ohguchi M,Kubota N,Wada K,Uritani M,et al.Purification and properties of trehalose synthesizing enzyme from Pseudomomonas sp. Ferment Bioeng,1997,84:358-360
Pan YT,Koroth EW,Jourdian WJ,EdmondsonR,Carroll JD,Pastuszak I,ElbeinAD.Trehalose synthase of Mycobacterium smegmatis. Purification,cloning,expression and preperties of the enzyme.Euro Biochem,2004,271:4259-4269
Paul M, Pellny T. Enhancing photosynthesis with sugar signals.Trends Plant Sci.2001,6:197-200
Pilon-Smits EAH., Terry N, Sears T, Kim H, Zayed A, Hwang S, Dun K, Voogd E,Verwoerd TC, Krutwagen RWHH and Goddijn OJM. Trehalose-producing transgenic tobacco plants show improved growth performance under drought stress. Plant Physiol,1998,152:525-532
Ribeiro MJS, Reinders A, Boller T, et al.Trehalose synthesis is important for the acquisition of thermolerance in Schizosaccharomyces pombe.Mol Microbiol,1997,25;571-581
Richards AB. Food and Chemical Toxicology,2002,40:871-898
Saha S,Rajamahendran-R,Boediono A,et al. Viability of bovine blastocystsobtained after 7,8, or 9 days of culture in vitro following vitrication and one-step rehydration. Theriogenol,1996,46:331-343
Saito K, Kase T. Purification and characterization of a trehalose synthase from the basidiomycete Grifola frondosa. Appl Environ Microbiol,1998,64:4340-4345
Saito K,Yamazaki H, Ohnishi Y, et al. Production of trehalose synthase from a basidiomycete Grifola frondose in Escherichia coli. Appl Microbil Bioteehnol,1998,50:193-198
Schiiffner A, Krumey FU. Hoppe Seyler's Z. Physiol. Chem.1938,255:145-158
Singer MA and Lindquist S. Thermotolerance in Saccharomyces cerevesiae:the yin and yang of trehalose. TIB Tech,1998,16:460-468
Siraje AM, Takashi H. Hiroshi S. Differential importance of trehalose accumulation in Saccharomyces cerevisiae in response to various environmental stresses. Biosci Bioeng,2009
Spiro RG. Analysis of sugars found in glycoproteins. Methods Enzymol,1966,8:3-26
Suomalainen H, Linko M, Oura E. Changes in the phosphatase activity of baker's yeast during the growth phase and location of the phosphatases in the yeast cell. Biochim Biophys Acta,1960, 37:482-490
Tanghe A, Dijek P, DumortierF, Teunissen A, Hohmannh S, Thevelein JM.Aquaporin expression correlates with freeze tolerance in bake,a yeast and overexpression improves freeze tolerance in industrialstrains. Appl Enviro Microbio,2002,68:5981-5989
Tetsuya N, Shoji I, Hiroto C, Michio K, Shigeharu F, Thermoacidophilic Archaebacterium Sulfolobus acidocaldarius. Biosci Biotech Biochem,1996,60:263-266
Thevelein JH.Trehalose synthase:guard to the gate of glycolysis in yeast. Trends Biochem Sci.1995,20:3-10
Thevelein JM. Signal transduction in yeast. Yeast,1994,10:1751-90
Timesheff SN. The control of protein stability and association by weak interactions with water: how do solvents affect these processes. Annu Rev Biophys Biomol Struct,1993,22:67
Toshiyuki S, Michio K. Tetsuya from starch with novel enzyme, Nippon Nogeikagaka Kaishi,1998,72:915-922
Tschirch A. Handbuch der Pharmakognoise Ball(Stuttgart:Julius Weise Hofbuckhandlung),1912,147
Ulrik S. Determination of Intracellular Trehalose and Glycogen in Saccharomyces cerevisiae.Anal Biochem,1995,228:143-149
Vuorio OE, Kalkkinen N, Londesborough J. Cloning of two related genes encoding the 56-Kda and 123 Kda subunits of trehalose synthase from the yeast Saccharomyces Ceretisiae. Euro Biochem,1993,216:849-861
Wingler A,Frit ZT.Trehalose indueed the ADP-glueose pyrophospborylase gene APL3 and starch synthesis in Arabidopsis.Plant Physiol,2000,124:105-114
Womersley C. Biochemical and physiological aspects of anhydrobiosis. Comp Biochem Physiol, 1981,70B:669-678
Yannick G,Jean-Luc R,Silke S,Didier F,Sophie D,Marie-Helene S,Jacques D.Characterization of the maltooligosyl trehalose synthase from the thermophilic archaeon Sulfolobus acidocaldarius. FEMS Microbiol Lett,2001,1994:201-206
Yoshida M. Production of trehalose by a dual enzyme system of immobilized maltose phosphorylase and trehalose phosphorylase. Enzy Microb Technol,1998,22:71-75
Zaved G and Roos YH. Influence of trebalose and moisture content on survival of Lactobacillus salivarius subjected to freeze-drying and storage. Proc Biochem.2004,39: 1081-1086
Zentella R, Mascorro-Gallardo JO, Van DP, Folch-Mallol J, Bonini B, VanVaeck C, Gaxiola R, Covarrubias AA, Nieto-Sotelo J, Thevelein JM and Iturriaga GA. Selaginella lepidophylla trehalose-6-phosphate synthase complements growth and stress-tolerance defects in a yeast tps1 mutant. Plant Physiol,1999,119:1473-1482
丁振.海藻糖合酶高产菌株的选育及酶学特性研究.山东轻工业学院硕士论文,2007
高峻,池振明.耐高浓度酒精菌与酒精敏感酵母菌中海藻糖含量与耐酒精的关系.食品与发酵工业,2001,27：4-8
葛宇,袁勤生.海藻糖对生物活性物质的保护作用机理研究进展.药物生物技,2002,9：297-300
胡宗利,夏玉先,陈国平等.海藻糖的生产制备及其应用前景.中国生物工程杂志,2004,24：44-48
黄鸣,黎锡流.海藻糖独特的生物学功能及其广泛应用.食品研究与开发,2002,23：56
黄成垠,戴秀玉.海藻糖对医用诊断工具酶活性保护作用研究.微生物学通报,1997,4：341-34
黄平.奇妙的双糖—海藻糖.生命的化学,1995,15：26-28
简文杰,江滨伟,庞杰等。新型食品添加剂,海藻糖的生产及其应用.现代商贸工业,2003,6：41-50
赖承兴,葛宇,袁勤生等. 海藻糖的MTSase及MTHase酶促合成条件的研究.中国医药工业杂志,2003,34：433-436
李静,谭海刚,宫春波,李凤梅,陈勇,于翠芳.从面包酵母中提取海藻糖的研究. 食品与药品,2008,10：24-26
李浩明等.海藻在生物制品干燥与保存中得应用研究.食品与发酵工业,1994,4：49-51
刘传斌,谢健,苗蔚荣等.海藻糖的性质及其广阔应用前景.中国微生态学杂志,1998,10：58-62
刘传斌,云战友等.海藻糖的分析方法.第八届全国生物化工学术会议论文集,化学工业出版社,1998,414-416
刘洋,张红缨,高俊芳,张今.酵母菌中海藻糖的几种提取方法比较.中国生化药物杂志,1999,20:16
罗明典.微生物生产海藻糖及其应用前景.微生物学通报,1996,4：252-254
牟禹.海藻糖合成酶基因转化玉米的研究.四川农业大学硕士论文,2007
聂凌鸿,宁正祥.海藻糖的生物保护作用.生命的化学,2001,21：206-209
聂凌鸿,宁正祥.海藻糖研究现状及其在食品工业中的应用.食品与发酵工业,2002,28：71-74
任媛媛,刘景芳.海藻糖代谢途径相关基因及生物工程.微生物学报,2003,43：821-824
史戈峰等.酒精发酵过程中酿酒酵母海藻糖代谢的研究,食品与发酵工业,1999,25：8-10
孙长慧.以淀粉为原料酶法制备海藻糖的研究. 北京化工大学硕士论文,2002
唐传核,葛文光.从活性干酵母中提取海藻糖的工艺.无锡轻工业大学学报,1998,3：3
唐传核, 葛文光. 海藻糖的开发现状及应用前景.中国食品用化学品,1997,3：1-4
童桥,聂松奇,林克椿.海藻糖对载药脂质体在干燥—再水化过程中的保护作用.生物化学杂志,1992,8：711-714
王岁楼.食品生物技术.海洋出版社,1998
肖国驱,黄晓兰,蔡小伟等.海藻糖产生菌的筛选及其发酵条件研究.生物技术,2002,12：15-17
宣劲松.日本长海带DNA文库构建、EST序列分析及海带6-磷酸海藻糖合成酶基因的克隆和分离.中国科学院遗传与发育生物学研究所硕士论文,2005
姚汝华.海藻糖及其应用前景.广州食品工业科技,1995,11：29-31
叶芳.高产海藻糖酵母菌株利用玉米淀粉一步转化生产海藻糖的工艺研究.中国海洋大学硕士论文,2003
尤新.功能性发酵制品.中国轻工业出版社,2000
于春燕,郎刚华.海藻糖研究进展. 青岛大学学报,2000,6
袁勤生.隐生生物中的典型化合物.中国生化药物杂志,1999,20：48-50
张红缨,刘洋,张今.海藻糖的生物合成和相关酶的特性.微生物学通报,1998,25.236-238
张树珍,郑学勤.海藻糖合酶基因的克隆及其序列的分析.热带作物学报,1999
赵晓峰,吴荣书.海藻糖的功能特性及应用.广州食品与科技,2004,20：18-23
Adridge S. Trehalose boots prospects for improved biopharmaceuticals and Vaccines. Genetic Engineer News,1995,15:10-11
Attfield PV. Trehalose accumulate in Saceharomyees cerevisiae during exposure to agents that include heat shock response. FEBS Lett,1987,225:259-263
Bleha M. Berwent Biotechnol Abst,1995,14:07223
Beattie GM, Crowe JH, Lopez AO. Trehalose:a cryoprotectant that enhances recovery and preserves function of human pancreatic islets after long-term storage. Diabetes,1997,46: 519-523
Bell W, Klaassen P, Ohnacker M, Boller T, et al. Characterization of the 56 kDa subunit of yeast trehalose-6-phosphate synthase and cloning its gene reveal its identity with the product of CIF1, a regulator of carbon catabolite inactivation. Eur J Biochem,1992,209:951-959
Bhandal IS, Hauptmann RM, Widholm JM. Trehalose as cryoprotectant for the freeze preservation of carrot and tobacco cells. Plant Physio,1985,78:430-432
Cabib E and Duran A. Simple and sensitive procedure for screening yeast mutants that lyse at nonpermissive temperatures. J Bacteriol,1975,124:1604-1606
Charlemagne GH, et al. Microbiol,1998,144:671-680
Chi ZM, Chi Z, Liu GL, Wang F, Ju L, Zhang T. Saccharomycopsis fibuligera and its applications in biotechnology.Biotecnol Adv,2009,27,423-431
Chi ZM. Enhanced conversation of soluble starch to trehalose by a mutant of Saccharomycopsis fibuligera sdu. J Biotechnol,2003,102:135-141
Colaco C. Extraordinary stability of enzymes dried in trehalose:simplified molecular biology. Biotechnol,1992,10:701-703
Colaco C. Food packaging and preservation. London:Applied Science Publishers LTD,1994, 123-139
Collins EB. Bifid bacteria in dairy products. Cult Dai Prod J,1993,28:1620-1627
Crowe JH,Crowe LM, Chapman D. Preservation of membranes in anhydrobiotic organisms:The role of trehalose. Scienee,1984,223:701-703
Crowe JH. Anhydrobiosis:An unsolved problem.American Naturalist,1971,105:563
Crowe JH. Hoekstra. F. Anhydrobiosis. Annual Review Physiol,1992,54:579-599
De Virgilio, C, Hottinger, T, Dominiguez J, Boller T and Wiemken A. The role of trehalose synthesis for the acquisition of thermotolerance in yeast. Eur J Biochem,1994,219:179-186
Dijck VP. Differential importance of trehalose in stress resistance in fermenting and nonfermenting Saccharomyces cerevisiae cells. Appl Environ Microbiol,1995,61:109-115
Diniz-Mendes L. Biotechnol Bioeng,1999,65:572-578
Dunean K,Sam T,Vineent M. Organization and Mobility of water in amorphous and crystalline trehalose. Nature,2006:1-4
Eduarda D, Vadym S, Tetyana S, Konrad J, Pavel D. Recovery of tubulin functions after freeze-drying in the presence of trehalose. Anal Biochem,2010,397:67-72
Elbein AD, Pan YT, Pastuszak I, Carroll D. New insights on trehalose:a multifunction molecule. Glycobio,2003,13.:17-27
Gadd GM, Chalmers K and Reed RH. The role of trehalose in dehydration resistance of Saccharomyces cerevesiae. FEMS Microbiol Lett,1987,48:249-254
Galinski EA, Herzog RM.The role of trehalose as a substitute for nitrogen-containing compatile solutes. Archieve Microbiol,1990,153:607-613
Gareia A,Engler J. Effeets of osmoprotectants upon NaCL stress in riee. Plant Microbiol, 1997,144:671-680
Goddijn DK. Trehalose metabolism in Plants. Tre Plant Sci,1999,4:315-319
Goddijn,Smeekens S.Sensing trehalose biosynthesis in piants. Plants J,1998,14:143-146
Guo N, Puhlev I, Brown DR, Mansbridge J and Levine F. Trehalose expression confers desiccation tolerance on human cells. Nature Biotechnol,2000,18:168-171
Hakkok. Derwent Biotechnol Albst,1995,14:07216
Hoelzle I,Streeter JG. Increased accumulation of trehalose in Rhlizibia cultured under 1% oxygen. Appl. Environ Microbiol,1990,56:3213-3215
Holmstrom KO. Engineering plant adaptation to water-stress. Acta-Universitatis-Agriculture-Sueciae-Agraia,1998,84:49
Hottiger T, Boller T, Wiemken A. Correlation of trehalose and heat resistance in mutants altered in the RAS/adenylate cyclase pathway is trehalose a themoprotectant. FEBS Lett,1989,225:431-434
Jang IC, Seo JS. Expression of a bifunctional fusion of the Escherichia coli genes for trehalose-6- phosphate synthase and trehalose -6-phosphatephosphatase in transgenic rice plants increases trehlose accumulation and a biotitics stress tolerance without stunting growth. Plant Physiol,2003,131:516-524
Jeong SC, Pack IS, Cho EY. Molecular analysis and quantitative detection of a transgenic rice line expressing a bifunctional fusion TPSP. Food Control,2007,18:1434-1442
John M. Thevelein Regulation of trehalose mobilization in fungi. Microbiol Reviews,1984,48(3):42-59
Kandror O, DeLeon A and Goldberg AL. Trehalose synthesis is induced upon exposure of Escherichia coli to cold and is essential for viability at low temperatures. Proc Natl Acad Sci USA,2002,99:9727-9732
Karl W,Iris K,Markus B.Metabolic regulation of the trehalose content of vegetative yeast.FEBS Lett,1991,291:269-272
Kaushik JK, Bhat R. Why is trehalose an exceptional protein stabilizer? Biol Chem,2003,278: 6458-26465
Kazubiko M. Cloning and sequencing of Trehalose Biosynthesis Genes from Rhizobium. Sp.,M-11. Bioscience. Biotechnol. Biochem,1996,60:717-720
Kjell H,Einar M,Biorn W.Droughtt tolerance in tobacco.Nature,1996,379:683-684
Koichi Y,Hiroe Y. Proteetion by trehalose of DNA from Radiation Damage. Biochem,1997,84:157-1540
Lebo Z. Analysis of PFK3 a gene involved in part-culate phso1 yeast.1994,10:199-204
Lederer E. Cord factor and related trehalose esters. Chem Phys Lipids,1976,16:91-106.
Leopold AC. Membranes, metabolism, and dry organisms. Cornell University Press, Ithaca, NY, 1986,1-377
Leslie B.Trehalose lowers membrane phase transitions in dry yeast cells. Biochim Biophy Acta,1987,1192:7-13
Li AZ. Chan-Halbrendt C. Ethanol production in China:Potential and technologies. Appl Energ, 2009,04:047
Lvine H,Sladc I.Another view of trehalose for drying and stabilizing biological materials. Bipoharm,1992,5:36-40
Madin, KAC and Crowe JH. Anhydrobiosis in nematodes:carbohydrate and lipid metabolism during dehydration. J Exp Zool.,1975,193:335-342
Marutu K, Nakada T, Kubota M,et al. Formation of trehalose from maltooligosaccharides by a novel enzymatic system.Biosci Biotech Biochem,1995,59:1829-1834
Masaru K. Purification and characteriazation of New Trehalose-producing Enzymes Isolated from the Hyperthermophilic Archae sulfolobus solfataricus KM1. Bioscience Biotechnology and Biochemistry,1996,60:546-550
Mauro S, Jose RM. Stabilization against thermal inactivation promoted by sugars on enzyme struture and function:why is trehalose more effective than other sugars. Archive of Biochemistry and Biophysics,1998,360:10
Mohamad RG, Bijan R, Saman H,Khosrow K, Leila H. Roles of trehalose and magnesium sulfate on structural and functional stability of firefly luciferase. J Mole Cataly B: Enzymatic,2010,62:127-132
Mozhaer VV, Structure-stability relationships in proteins:new approaches to stabilizing Enzy. Microb Technol,1984,6
Murao S. Enzymatic synthesis of trehalose from maltose. Agric Biol Chem, 1985,49:2113-2118
Nicolaus B, Gambacorta A, Basso AL, Riccio R, DeRosa M and Grant WD. Trehalose in Archaebacteria systems. Appl Microbiol,1988,10:215-217
Nishant K. Jain, IR. Role of trehalose in moisture-induced aggregation of bovine serum albumin. Euro J Pharma Biopharma,2008,69:824-834
Nishimoto T. Purification and properties of a novel enzyme, trehalose synthase, from Pimeloba-cter sp.R48. Biochem Biosci Biotech,1996,60:640-644
Ohguchi M,Kubota N,Wada K,Uritani M,et al.Purification and properties of trehalose synthesizing enzyme from Pseudomomonas sp. Ferment Bioeng,1997,84:358-360
Pan YT,Koroth EW,Jourdian WJ,EdmondsonR,Carroll JD,Pastuszak I,ElbeinAD.Trehalose synthase of Mycobacterium smegmatis. Purification,cloning,expression and preperties of the enzyme.Euro Biochem,2004,271:4259-4269
Paul M, Pellny T. Enhancing photosynthesis with sugar signals.Trends Plant Sci.2001,6:197-200
Pilon-Smits EAH., Terry N, Sears T, Kim H, Zayed A, Hwang S, Dun K, Voogd E,Verwoerd TC, Krutwagen RWHH and Goddijn OJM. Trehalose-producing transgenic tobacco plants show improved growth performance under drought stress. Plant Physiol,1998,152:525-532
Ribeiro MJS, Reinders A, Boller T, et al.Trehalose synthesis is important for the acquisition of thermolerance in Schizosaccharomyces pombe.Mol Microbiol,1997,25;571-581
Richards AB. Food and Chemical Toxicology,2002,40:871-898
Saha S,Rajamahendran-R,Boediono A,et al. Viability of bovine blastocystsobtained after 7,8, or 9 days of culture in vitro following vitrication and one-step rehydration. Theriogenol,1996,46:331-343
Saito K, Kase T. Purification and characterization of a trehalose synthase from the basidiomycete Grifola frondosa. Appl Environ Microbiol,1998,64:4340-4345
Saito K,Yamazaki H, Ohnishi Y, et al. Production of trehalose synthase from a basidiomycete Grifola frondose in Escherichia coli. Appl Microbil Bioteehnol,1998,50:193-198
Schiiffner A, Krumey FU. Hoppe Seyler's Z. Physiol. Chem.1938,255:145-158
Singer MA and Lindquist S. Thermotolerance in Saccharomyces cerevesiae:the yin and yang of trehalose. TIB Tech,1998,16:460-468
Siraje AM, Takashi H. Hiroshi S. Differential importance of trehalose accumulation in Saccharomyces cerevisiae in response to various environmental stresses. Biosci Bioeng,2009
Spiro RG. Analysis of sugars found in glycoproteins. Methods Enzymol,1966,8:3-26
Suomalainen H, Linko M, Oura E. Changes in the phosphatase activity of baker's yeast during the growth phase and location of the phosphatases in the yeast cell. Biochim Biophys Acta,1960, 37:482-490
Tanghe A, Dijek P, DumortierF, Teunissen A, Hohmannh S, Thevelein JM.Aquaporin expression correlates with freeze tolerance in bake,a yeast and overexpression improves freeze tolerance in industrialstrains. Appl Enviro Microbio,2002,68:5981-5989
Tetsuya N, Shoji I, Hiroto C, Michio K, Shigeharu F, Thermoacidophilic Archaebacterium Sulfolobus acidocaldarius. Biosci Biotech Biochem,1996,60:263-266
Thevelein JH.Trehalose synthase:guard to the gate of glycolysis in yeast. Trends Biochem Sci.1995,20:3-10
Thevelein JM. Signal transduction in yeast. Yeast,1994,10:1751-90
Timesheff SN. The control of protein stability and association by weak interactions with water: how do solvents affect these processes. Annu Rev Biophys Biomol Struct,1993,22:67
Toshiyuki S, Michio K. Tetsuya from starch with novel enzyme, Nippon Nogeikagaka Kaishi,1998,72:915-922
Tschirch A. Handbuch der Pharmakognoise Ball(Stuttgart:Julius Weise Hofbuckhandlung),1912,147
Ulrik S. Determination of Intracellular Trehalose and Glycogen in Saccharomyces cerevisiae.Anal Biochem,1995,228:143-149
Vuorio OE, Kalkkinen N, Londesborough J. Cloning of two related genes encoding the 56-Kda and 123 Kda subunits of trehalose synthase from the yeast Saccharomyces Ceretisiae. Euro Biochem,1993,216:849-861
Wingler A,Frit ZT.Trehalose indueed the ADP-glueose pyrophospborylase gene APL3 and starch synthesis in Arabidopsis.Plant Physiol,2000,124:105-114
Womersley C. Biochemical and physiological aspects of anhydrobiosis. Comp Biochem Physiol, 1981,70B:669-678
Yannick G,Jean-Luc R,Silke S,Didier F,Sophie D,Marie-Helene S,Jacques D.Characterization of the maltooligosyl trehalose synthase from the thermophilic archaeon Sulfolobus acidocaldarius. FEMS Microbiol Lett,2001,1994:201-206
Yoshida M. Production of trehalose by a dual enzyme system of immobilized maltose phosphorylase and trehalose phosphorylase. Enzy Microb Technol,1998,22:71-75
Zaved G and Roos YH. Influence of trebalose and moisture content on survival of Lactobacillus salivarius subjected to freeze-drying and storage. Proc Biochem.2004,39: 1081-1086
Zentella R, Mascorro-Gallardo JO, Van DP, Folch-Mallol J, Bonini B, VanVaeck C, Gaxiola R, Covarrubias AA, Nieto-Sotelo J, Thevelein JM and Iturriaga GA. Selaginella lepidophylla trehalose-6-phosphate synthase complements growth and stress-tolerance defects in a yeast tps1 mutant. Plant Physiol,1999,119:1473-1482