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1 40 1 Vol.40, No OCEANOLOGIA ET LIMNOLOGIA SINICA Jan., 2009 α- * 1, ( ; ; ) (Thermococcus sp. HJ21) α- 9 h, 60 90, 80 ph , ph 7.5 NaCl 0.5% 4.0%, 2.5 % NaCl α kda, 95, % 90 5 h, h 40%, Ca 2+ ph 5.0, ph %, ph (80 4 h) 1 mmol/l Mg 2+ Co 2+ Sr 2+ Ba 2+ K + Na +, Cu 2+ Pb 2+ Hg 2+ Zn 2+ Al 3+,, α-,, TQ925 (Hyperthermophiles) (Archaea), , 4000 m,,,, (Vieille et al, 2001) α- α-1, (α-1,4- glucan-4-glucanohydrolase EC ),, (Gupta et al, 2003) α- (Geobacillus stearothermophilus) (Bacillus licheniformis), 90 ph 6.0,, 100 ph 6.0, Ca 2+ (Marc et al, 2002) , α-, α- (Pyrococcus woesei) (P. furiosus) (Thermococcus hydrothermalis) Thermococcus profundus Desulfurococcus mucosus Pyrodictium abyssi * 06-A-017, 06KJB550004, 2006HS008, KK06076,, shujunwang86@ hotmail.com :,,, luzhaoxin@njau.edu.cn : , :

2 20 40 (Staphylo thermus marinus) (Dictyoglomus thermophilum), (Thermotoga maritime)(vieille et al, 2001; Bertoldo et al, 2002), Pyrococcus woesei Pyrococcus furiosus Thermococcus hydrothermalis Thermococcus profundus Thermotoga maritime α- (Léveque et al, 2000a) α-, α- (, 2003;, 2005), α- (, 2005) HJ21 α-, 菌株 (Thermococcus sp. HJ21), 1.2 YPS 1000 ml, 10 ml, 1% CaCl 2 H 2 O 5 ml, N-P 10 ml, 500 Fe EDTA 2 ml, 0.02% 5 ml, PIPE 3.35 g, 3 g, 3 g, 5 g, 5 g, ph 6.5 (g/l) NaCl 19.6, NaSO 4 3.3, KCl 0.5, KBr 0.05, 8.8; (g/l) CuSO 4 5H 2 O 0.01, ZnSO 4 7H 2 O 0.1; CoCl 2 6H 2 O 0.005; MnCl 2 4H 2 O 0.2; Na 2 MoO 4 2H 2 O 0.1; KBr 0.05; KI 0.05; H 3 BO 3 0.1; NaF 0.05; LiCl 0.05; Al 2 (SO 4 ) , NiCl 2 6H 2 O 0.01, VoSO 4 2H 2 O 0.005, H 2 WO 4 2H 2 O 0.002, Na 2 SeO , SrCl 6H 2 O 0.005, BaCl 0.005; 100 N-P (g/l) (NH 4 ) 2 SO , NaNO , KH 2 PO 4 3.6; 500 Fe EDTA (g/l) FeSO 4 7H 2 O 1.54; Na-EDTA 培养基的制备和产酶发酵方法,,, Na 2 S 9H 2 O 5% (W/V) 0.025%, min, 5% YPS, 88 9 h(jolivet et al, 2004) YPS, 88, 3 h 1.4.2, 9 h ph YPS (10 mmol/l): (MES ), ph (PIPES ), ph (HEPES ), ph (Tris-HCl ), 88 9 h, NaCl NaCl, 0.5% 1% 1.5% 2% 2.5% 3% 4% 5%, 88 9 h, % 1% 2% 3% 4% 5% 7% 10% 1.4.6, 0.5%, 0.3% 1.5 酶的纯化 (Kwak et al, 1998) 65%, (FPLC, Bio-Rad, USA) DEAE G SDS, 0.3% (W/V) 0.1% SDS-10% PAGE( 4%, 10%)(Brown et al, 1964 ), R-250(Bio-Rad, USA), 2.5% (V/V) Triton X mmol/l Na 2 HPO 4 (ph7.5), 1 h, (ph5.0), h, 0.15% (W/V)I 2 1.5% (W/V) KI 2 min, ( ) 110 ( ), ph5.0 ph5.5 (50 mmol/l) 1%

3 1 : α ( Ca 2+, 5 mmol/l) ( ) 5 h, h, 4,, 100% ph ph 1%, 95, ph 50 mmol/l (ph ); 50 mmol/l (ph ); 50 mmol/l (ph ); 50 mmol/l Tris- (ph ) ph 50 μl 200 μl 0.04 mol/l ph Britton-Robinson (ph4 11) (Rauen et al, 1964), h,, 100% 50 μl 150 μl ph, 50 mmol/l (ph4.0); 50 mmol/l (ph ); 50 mmol/l (ph )50 mmol/l Tris- (ph ), 80, 0.5 h 1 h 2 h 3 h 4 h, 100% 1.6.6, 1.0 mmol/l 5.0 mmol/l, 95, 1 mmol/l 5 mmol/l,, 100% 1.6.7, min,, K m V max (Li et al, 2007) 1.7 淀粉酶活力的测定 10µl 190 μl 1% (50 mmol/l, ph 5.0), min, 3,5- (DNS), 1 μg 2 结果与分析 2.1 菌株产酶条件 ph NaCl, 3 h, 9 h HJ21,,, 60 90, 60 90, 80 ( 1a); ph , ph 4 10, ph 7.5( 1b); NaCl 0.5% 4.0%, 2.5% NaCl ( 1c); 5% (a) ph(b) NaCl (c) Fig.1 Effect of temperature(a), ph(b) and NaCl(c) on α-amylase production

22 40,, ( 1),,, ( 2), HJ21 2.2 酶学性质 2.2.1 HJ21 α- DEAE G-75 ( 3), SDS-PAGE α-, 51.4 kda ( 2) 2.2.2 HJ21 α- ph 5.0 5.5, 95, 100 60% (ph 5.0)( 3a), 2 SDS-PAGE Fig.2 SDS-PAGE of the α-amylase 1.

8 14.1 13.6 30.1 100 10.8 32.3 81.5 17.2 22.6 表 3 α- 淀粉酶纯化结果 Tab.3 Purification result of α-amylase (U/mg) (mg) (U/mg) (%) 300 000 600 500 1 100 65% 242 000 400 605 1.2 80.

4 22 40,, ( 1),,, ( 2), HJ 酶学性质 HJ21 α- DEAE G-75 ( 3), SDS-PAGE α-, 51.4 kda ( 2) HJ21 α- ph , 95, % (ph 5.0)( 3a), 2 SDS-PAGE Fig.2 SDS-PAGE of the α-amylase 1. ; 2. ; M. 表 1 培养基中氮源对产酶的影响 Tab.1 Effect of nitrogen source on α-amylase production (%) 表 2 培养基中碳源对产酶的影响 Tab.2 Effect of carbon source on α-amylase production (%) 表 3 α- 淀粉酶纯化结果 Tab.3 Purification result of α-amylase (U/mg) (mg) (U/mg) (%) % DEAE-Sepherose Fast Flow G (a) (b) Fig.3 Effects of temperature on enzyme activity (a) and stability (b) 5 mmol/l Ca 2+ ( ), 5 mmol/l Ca 2+ ( )

5 1 : α- 23 Ca 2+,, 90 5 h, h 40% ( 3b) ph ph HJ21 α- ph 5.0, ph %, ( 4a) HJ21 α- ph Britton-Robinson h, ph ( 4b), ph, 80 2 h, ph 5.0 ; 80 4 h, ph , ph 6.0 ( 4c) mmol/l Ca 2+, 5 mmol/l Ca 2+ ( 4) 1 mmol/l 5 mmol/l Mn 2+ Ni 2+ Ag + Al 3+ Fe 2+ Zn 2+ Cu 2+ Hg 2+, Zn 2+ Cu 2+ Hg 2+ 1 mmol/l, 1 mmol/l 5 mmol/l N- (61% 13.5%)( 5) K m V max 1.6 mg/ml mg/(ml min) 3 讨论, min ph , ph ,, ph α- ( Geobacillus stearothermophilus Bacillus licheniformis) 90, ph 6, 100 ph 6, (Marc et al, 2002) ph 4.5, ph 5.8, 90, Ca 2+,, 60, ph , 90 60, ph , ph,, (Vieille et al, 2001) 1983 Vulcano (Zillig et al, 1983), 16S rdna Genbank 180, 30 ((Jolivet et al, 4 ph (a) ph (b,c) Fig.4 Effect of ph on enzyme activity and the stability

6 24 40 表 4 金属离子对 α- 淀粉酶活的影响 Tab.4 The effect of metal ions on α-amylase activity Tab.5 (%) 1 mmol/l (%) 5 mmol/l Ca Co Ba Hg K Pb Mn Ni Mg Sr Li Ag Al Cu Na Fe Zn 表 5 化学试剂对 α- 淀粉酶活的影响 Effect of chemical reagents on the amylase activity (%) 1 mmol/l (%) 5 mmol/l N ; Cambon-Bonavita et al, 2003; Atomi et al, 2004; Kuwabara et al, 2005; Bae et al, 2006; Kuwabara et al, 2007; Pikuta et al, 2007 ) Thermococcus sp. HJ21, α-,,, (Gupta et al, 2003), HJ21 (Thermococcus),, 55 94, 88 ; ph , ph 6.5; NaCl 1.0% 5.0%, 2%( ), HJ21α- ph NaCl α-,,,, Kwak T. profunds (Kwak et al, 1998) HJ21,,,,,,, (Bertoldo et al, 2002; Léveque et al, 2000) 16S rdna Genebank 180, α- T. liter, T. guaymasensis, T. aggregans, T. celer, T. profundus, T. fumicolans, T. hydrothermalis T. thioreducens (Léveque et al, 2000; Chung et al, 1995; Lee et al, 1996; Bernhardsdotter et al, ) ),, T. profundus, T. hydrothermalis T. Thioreducens(Kwak et al, 1998; Chung et al, 1995; Lee et al, 1996; Bernhardsdotter et al, ) ) α- (Pyrococcus furiosus) (Thermotoga maritime) (, 2003 a,b;, 2005;, 2005), (Thermococcus) HJ21 α kda, T. hydrothermalis (53.6 kda)(léveque et al, 2000), T. profundus(42 kda)(kwak et al, 1998; Chung et al,1995; Lee et al, 1996) T. thioreducens(43 kda)(bernhardsdotter et al, ) ) HJ21 α- (THJA) T. profundus, T. hydrothermalis T. Thioreducens, HJ21 1) THJA 95, , T. profundus (80 ), T. hydrothermalis (75 85 ) T. thioreducens (90 )

7 1 : α- 25 (Kwak et al, 1998; Léveque et al, 2000; Lee et al, 1996; Bernhardsdotter et al, ) ) THJA 90, 5 h h 3 h 40% 30%, T. profundus ( 90, min)(kwak et al, 1998; Lee et al, 1996) 2) Ca 2+ (Gupta et al, 2003), Ca 2+ T. hydrothermalis T. profundus (Léveque et al, 2000),, 1 mmol/l Ca 2+ THJA, 5 mmol/l Ca 2+ THJA Ca 2+, 3) THJA ph 5.0, ph %, T. profundus T. thioreducens (Kwak et al, 1998; Chung et al,1995; Bernhardsdotter et al, ) ) Thermococcus, T. sp. HJ21, T. hydrothermalis T. celer (Kwak et al, 1998, Legin et al, 1998; Canganella et al, 1994) α-, (PCR) Thermococcus sp. HJ21 α- ( 1164 bp), 388 Swiss Model, α- (β/α) 8 HJ21 α- Geobacillus stearothermophilus Bacillus licheniformis α-, 95 ph 5.0, Ca 2+, HJ21 α-, α-, α- α- ( ),,,, α- E-coli., 32(4) 25 30,,, α-., 45(4) ,,, 2003a. Pyrococcus furiosus α-., 30 (3) 22 25,,, 2003b. Pyrococcus furiosus α-., 29(3) Atomi H, Fukui T, Tanaka T et al, Description of Thermococcus kodakaraensis sp. nov., a well studied hyperthermophilic archaeon previously reported as Pyrococcus sp. KOD1. Archaea, 1: Bae S S, Kim Y J, Yang S H et al, Thermococcus onnurineus sp. nov., a hyperthermophilic archaeon Isolated from a deep-sea hydrothermal vent area at the Pacmanus Field. J Microbiol Biotechnol, 11: Bertoldo C, Antranikian G, Starch-hydrolyzing enzymes from thermophilic archaea and bacteria. Curr Opin Chem Biol, 6: Brown S H, Costantino H R, Kelly R M, Characterization of amylolytic enzyme activities associated with the hyperthermophilic archaebacterium Pyrococcus furiosus. Appl Environ Microbiol, 56 : Canganella F, Andrade CM, Antranikian G, Characterization of amylolytic and pullulytic enzymes from thermophilic archaea and from a new Fervidobacterium species. Appl Microbiol Biotechnol, 42: Cambon-Bonavita M A, Lesongeur F, Pignet P et al, Extremophiles, thermophily section, species description Thermococcus atlanticus sp. nov., a hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent in the Mid-Atlantic Ridge. Extremophiles, 7: Chung Y C, Kobayashi T, Kanai H et al, Purification and properties of extracellular amylase from the hyperthermophilic archaeon Thermococcus profundus DT5432. Appl Environ Microbiol, 61: Gupta R, Gigras P, Mohapatra H et al, Microbial α-amylases: a biotechnological perspective. Process Biochemistry, 38: Jolivet E, Corre E, L Haridon S et al, Thermococcus marinus sp. nov., and Thermococcus radiotolerans sp. nov., two hyperthermophilic archaea from deep-sea hydrothermal vents that resist ionizing radiation. Extremophiles, 8: Kuwabara T, Minaba M, Iwayama Y et al, Thermococcus coalescens sp. nov., a cell-fusing hyperthermophilic archaeon from Suiyo Seamount. Int J Syst Evol Microbiol, 55: ) Bernhardsdotter E C M J, Pusey M L, Ng J D et al, Cloning and Characterization of an alpha-amylase Gene from the Hyperthermophilic Archaeon Thermococcus thioreducens. Marshall Space Flight Center

8 26 40 Kuwabara T, Minaba M, Ogi N et al, Thermococcus celericrescens sp. nov., a fast-growing and cell-fusing hyperthermophilic archaeon from a deep-sea hydrothermal vent. Int J Syst Evol Microbiol, 57: Kwak Y S, Akiba T, Kudo T, Purification and characterization of α-amylase from hyperthermophilic archaeon Thermococcus profundus, which hydrolyzes both α-1, 4 and α-1, 6 glucosidic linkages. J Ferment Bioeng, 86: Lee J T, Kanai H, Kobayashi T et al, Cloning, nucleotide sequence, and hyperexpression of α-amylase Gene from an archaeon, Thermococcus profundus. J Ferment Bioeng, 82: Legin E, Copinet A, Duchiron F, 1998, Production of thermostable amylolytic enzymes by Thermococcus hydrothermalis. Biotechnology Letters, 20(4): Léveque E, Janeček S, Haye B et al, 2000a. Thermophilic archaeal amylolytic enzymes. Enz Microbiol Technol, 26: 3 14 Léveque E, Haye B, Belarbi A, 2000b. Cloning and expression of an α-amylase encoding gene from the hyperthermophilic archaebacterium Thermococcus hydrothermalis and biochemical characterization of the recombinant enzyme. FEMS Microbio Lett, 186: Li H F, Chi Z M, Wang X H, Purification and characterization of extracellular amylase from the marine yeast Aureobasidium pullulans N13d and its raw potato starch digestion. Enz Microbiol Technol, 40: Marc J E C, Maarel v d, Veen B v d et al, Properties and applications of starch-converting enzymes of the α-amylase family. J Biotech, 94: Pikuta E V, Marsic D, Itoh T et al, Thermococcus thioreducens sp. nov., a novel hyperthermophilic, obligately sulfur-reducing archaeon from a deep-sea hydrothermal vent. Int J Syst Evol Microbiol, 57: Rauen H M, Biochemisches Taschenbuch, Springer Verlag, Berlin, Germany, 2: Vieille C, Zeikus G J, Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiol. Mol Biol Rev, 65: 1 43 Zillig W, Holz I, Janekovic D et al, The archaebacterium Thermococcus celer represents a novel genus within the thermophilic branch of the archaebacteria. Syst Appl Microbiol, 4: PRODUCTION AND CHARACTERIZATION OF THERMOSTABLE AND ACID-STABLE α-amylase FROM HYPERTHERMOPHILIC ARCHAEON THERMOCOCCUS SP. WANG Shu-Jun 1,2, LU Zhao-Xin 1, QIN Song 3, LU Ming-Sheng 2, LI Fu-Chao 3, LIU Hong-Fei 2, DENG Xiang-Yuan 3 (1.College of Food Science and Technology, Nanjing Agricultural University, Nanjing, ; 2.Jiangsu key Laboratory of Marine Biotechnology, Huaihai Institute of Technology, Lianyungang, ; 3.Institute of Oceanology, Chinese Academy of Sciences, Qingdao, ) Abstract The production and characterization of α-amylase from Thermococcus sp. HJ21 are studied. The maximum enzyme production is achieved after 9 h cultivation. The ranges and the best points of α-amylase production are 60 90, 80 ; , 7.5 in ph; and 0.5% 4.0%, 2.5% for NaCl concentration, respectively. The result shows that adding yeast extract, peptone, glycogen, starch, and maltose could enhance the enzyme production, and the strain HJ21 could produce extracellular thermostable and acid-stable α-amylase. In addition, the molecular weight of the enzyme is estimated 51.4 kda by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The maximal activity of α-amylase occurs at 95 ; the half-life of the enzyme is 5 h at 90 ; and over 40% of them remain active 2 h after incubation at 100. Meanwhile, no Ca 2+ is required for thermostability. The enzyme remain stable in a wide ph range of 5.5 to 7.0 (at 80 4 h), and best in Moreover, the enzyme can be activated by Mg 2, Co 2+, Sr 2, Ba 2+, K, and Na +, and inhibited by Cu 2, Pb 2, Hg 2, Zn 2, and Al 3+. Key words Hyperthermophilic Archaeon, Thermococcus, α-amylase, Production, Characterization

第 33 卷第 2 期 2010 年 4 月 - 149 - 脱蛋白较好的方法是 Sevag 法, 它是根据蛋白质在氯仿等有机溶剂中变性的特点, 使蛋白质变性成胶状, 此法条件温和, 可避免多糖的降解

第 33 卷第 2 期 2010 年 4 月 - 149 - 脱蛋白较好的方法是 Sevag 法, 它是根据蛋白质在氯仿等有机溶剂中变性的特点, 使蛋白质变性成胶状, 此法条件温和, 可避免多糖的降解 - 148 - Drug Evaluation Research Vol.33 No.2 April 2010 植物多糖的研究进展陶遵威 1, 郑夺 1, 邸明磊 2, 单晓菊 1 天津市医药科学研究所, 天津 300020; 2 天津中医药大学, 天津 300073 2 摘要多糖是由单糖连接而成的多聚物,