Microsoft Word 李鹏_new_.doc

Size: px

Start display at page:

Download "Microsoft Word 李鹏_new_.doc"

嗣洒完颜
7 years ago
Views:

1 45 5 Vol.45, No OCEANOLOGIA ET LIMNOLOGIA SINICA Sep., 2014 * ( ), Folin-,, DES, : 2,, Folin-, A20( 104.7U/mL), 16S rdna Streptomyces roseus A20 (DES),, 227.5U/mL, 117.3%, 50 C ph h ; ; ; 16S rdna Q936 doi: /hyhz (Alkaline protease) ph,,, (Bhaskar et al, 2007; Wang et al, 2008; Jellouli et al, 2009), 65% (Banik et al, 2004), (Gupta et al, 2002),,,, (, 2005) 3 : ; ;, ph (, 2010), (, 2001), (Kumar et al, 1999), (Banerjee et al, 1999; Johnvesly et al, 2001) (Bacillus),,,,,,, Sreeja (2011) Engyodontium album BTMFS10, *, ;, LY12C03003,,, lpqp @163.com :,, zswjx2575@163.com : , :

2 (60 C )(ph 11 ),,, Anjali (2014) Bacillus tequilensis P15,,,,,, : (1) : 20g, KNO 3 1g, K 2 HPO 4 0.5g, MgSO 4 7H 2 O 0.5g, NaCl 0.5g, FeSO g, 20g, 1000mL, ph (2) (, 1999): 5.0g, 10.0g, 1.0g, 20.0g, ph ,, 121 C 20min, 10.0g,, 115 C 30min 1000mL (3) : 4g, CaCl g, MgSO 4 7H 2 O 0.5g, K 2 HPO 4 7H 2 O 1.07g, KH 2 PO g, ZnCl g, NaCl 0.16g, FeSO g, 0.05g, 18g, 1000mL, ph 8.0 (4), 1 6 : 5g, 10g, KH 2 PO 4 1g, 1000mL, ph g, 5g, 5, Na 2 HPO 4 12H 2 O 4g, KH 2 PO 4 0.3g, Na 2 CO 3 1g, 1000mL, ph g, 3.0g, NaCl 5.0g, K 2 HPO 4 2.0g, 1000mL, ph %, 0.5%, 1.0% 1.5%, NaCl 0.5%, K 2 HPO %, NaH 2 PO %, Na 2 CO %, 1000mL, ph 8.5 (, 1999) 5.0g, 3.0g, 5.0g, 1000mL, ph 8.5 (, 2006) 5.0g, 10.0g, NaCl 1.0g, 10.0g, 1000mL, ph DNA (TIANamp BacteriaDNA Kit) ( ), DNA Marker TaKaRa, , I, I, 25 C 3 5d,, 2 3, , 25 C, /250mL, 25 C, 120r/min 3 5d μg/mL, 275nm, 275nm, 275nm,, 1 680nm (A),, 1, 680nm A,, r/min, 2, :, 1μg 1 U p = (A 680 *K*4) / (10*n), U p, A 680

3 5 : 1129 表 1 标准曲线的制作 Tab.1 Data for making standard curves (mg/l) (ml) (ml) (ml) Folin-(mL) , 40 C 20min 表 2 测样品酶活的步骤 Tab.2 Steps of measuring enzyme activity in the samples (ml) (ml) %(mL) 1 1 1, 40 C 10min (ml) 0 2 2, 40 C 10min, (ml) (ml) Folin-(mL) 1 1 1, K ( ), 4, 10, n A20,, A20 16S rdna DNA DNA, DNA PCR (Kunamneni et al, 2003), PCR 1%, PCR 1500bp PCR, Seq Forward Seq Reverse Seq Internal 16S rdna, ( ) A20 Blast, (97% ), (Clustalx 1.8), (PhyloDraw V8.0) A20, 1.2.9, DES, mL 6cm, 30cm 1min,,, s 100μL, 10 3, 0.1mL, 3,,, = (1 0.1mL / 0.1mL ) 100% 3 5d, HC ( / ), 25 C, 120r/min, 50mL/ 250mL, 3, DES 1, 50mL/250mL

4 , 25 C 120r/min 5d 5000r/min 10min,, 10mL 0.1 mol/l (ph 7.0) 2, ph 7.0, 10 8 /ml( OD 600 = 0.5 ) 4mL 16mL (ph 7.0), 0.2mL, min, 0.5mL 25% min 10 3, 3, 0.1mL,, 25 C 5d (, 2008),, HC (), 3, , C, ph ph , ph 7.5, 28 C h , A8 A μg/mL 表 3 Tab.3 2 A20 Fig.2 Shape of Strain A20 两株放线菌所形成透明圈直径大小 The size of diameter of transparent zone (mm) (mm) / A A , OD 680nm, 4 (A),,, 3 y = x , R 2 = , 表 4 不同含量的酪氨酸吸光度值结果 Tab.4 Results of different levels of tyrosine absorbance (μg/ml) (A) Fig.3 Standard curve of tyrosine 1 A8 Fig.1 Shape of Strain A A8 A20,, 5

5 5 : 1131 表 5 碱性蛋白酶活力测量结果 Tab.5 Results of alkaline protease activity (U/mL) (U/mL) A A A20U A20U A20, A20, DES 2.3 A20 A20,,, A20 A20 3d 6, 7, 8, A20,, 4 A20 Fig.4 Hyphal morphology of Strain A20 表 6 菌株在不同培养基上培养 3d 的生长情况 Tab.6 Strain A20 in different culture media on the growth after 3d Strain A PDA 表 7 菌株 A20 对不同碳源的利用情况 Tab.7 Strain A20 on the utilization of different carbon sources A 表 8 菌株 A20 的生理生化试验结果 Tab.8 Physiological and biochemical tests on Strain A20 H 2 S A S rdna 16S rdna A bp, NCBI GenBank GU Blast, GenBank,, 5 A20 Streptomyces roseoviridis JS-9() Streptomyces roseus JS-18( ), 99.8%, A20, PDA, A A A ( ) , s 120s HC (HC 5.3), 182.8U/mL 104.7U/mL, 74.6%, DES

6 S rdna A20 Fig.5 The NJ evolutionary tree based on completed sequence of Strain A20 16S rdna 表 9 碱性蛋白酶活力测量结果 Tab.9 Alkaline protease activity measurement results (U/mL) 表 10 紫外线诱变结果 Tab.10 The result of ultraviolet mutation (s) 10 3 (%) DES DES 11, 30 50min (min) 表 11 DES 处理结果 Tab.11 The result of DES mutation 10 3 (%)

7 5 : min HC (HC 5.9),, 227.5U/mL, 51.8% 2.9 DES 227.5U/mL A20, 10, 12 表 12 传代 10 次的酶活测试结果 Tab.12 Results of enzyme activity test after ten times of subculture (U/mL) U/mL, 8.17%,, C, 227.9U/mL 2.11 ph ph 7 ph Fig.8 8 Testing for the optimal culture time 6 Fig.6 The optimal temperature test 7 ph Fig.7 The optimal ph test h,,, 48h,,, 72h,, 228.5U/mL 8 A20, 16S rdna Streptomyces roseus, Streptomyces roseus A20, -, s, 76.5% 90.1%,, 182.8U/mL, 74.6% 30 50min DES, 68.8% 93.8%, 227.5U/mL, 104.7U/mL 117.3%,, C ph 9.0, 72h,, :,,, :,,,

8 ,,,, Ahmed (2013) EMS Penicillium chrysogenum NRRL 792, EMS-1, (ph, ),, Wang (2010) Pseudomonas sp., PCR, 1443bp OFR 463,, Escherichia coli Jasmin (2010) Engyodontium album (Eap), Tsuyoshi (2004), Kunamneni (2003) PE-11, 60 C 350min 100% Sudhir (2010) Paenibacillus tezpurensis sp. nov. AS-S24-II WANG (2012) Brevibacillus sp. PLI-1, 70 C Raja (2006),, 124 Subramani (2009) MML1614,, Hames- Kocabas (2007) MA1-1, ph C, Jignasha (2009) Mit-1, (70 C ),, Iram (2012) (Stenotrophomonas sp.), (15 C),, (Anil, 2010), (Madhuri et al, 2012) Feng (2013) Kocuria kristinae F7,,,, Vijay (2011) Bacillus altitudinis GVC11,,, : ph ( ),,,,,,, XE , 41(5): ,,, L-., 03: ,,, :, 45 46, :, 75 76,,, , 50(1): 54 59,,, , 35(4): Ahmed F Afifi, Heba I Abo-Elmagd, Manal M Housseiny, Improvement of alkaline protease production by Penicillium chrysogenum NRRL 792 through physical and chemical mutation, optimization, characterization and genetic variation between mutant and wild-type strains. Ann Microbiol, 35: Anil Kumar Singh H S, Optimization of protease production by Streptomyces sp. A6 using statistical approach for reclamation of shellfish waste. World J Microbiol Biotechnol, 26: Anjali Bose, Vishal Chawdhary, Haresh Keharia, Production and characterization of a solvent-tolerant protease from

9 5 : 1135 a novel marine isolate Bacillus tequilensis P15. Ann Microbiol, 64: Banerjee U C, Sani R K, Azmi W, Thermostable alkalineprotease from Bacillus brevis and its characterizationas a laundry detergent additive. Process Biochemistry, 35(1/2): Banik R M, Prakash M, Laundry detergent compatibility of the alkaline protease from Bacillus cereus. Microbiol Res, 159: Bhaskar N, Sudeepa E S, Rashmi H N et al, Partial purification and characterization of protease of Bacillus proteolyticus CFR3001 isolated from fish processing waste and its antibacterial activities. Bioresour Technol, 98: Feng Zhen, Chen Xi, Li Juan-juan et al, An alkaline protease from Kocuria kristinae F7: properties and characterization of its hydrolysates from soy protein. Eur Food Res Technol, 236: Gupta R, Beg Q K, Lorenz P, Bacterial alkaline proteases: molecular approaches and industrial applications. Applied Microbiology and Biotechnology, 59(1): Hames-Kocabas E Esin, Atac Uzel, Alkaline protease production by an actinomycete MA1-1 isolated from marine sediments. Annals of Microbiology, 57(1): Iram Saba Parvaiz H, Qazi Shabir A, Rather Refaz A et al, Purification and characterization of a cold active alkaline protease from Stenotrophomonas sp., isolated from Kashmir, India. World J Microbiol Biotechnol, 28: Jasmin C, Sreeja Chellappan, Rajeev K Sukumaran et al, Molecular cloning and homology modeling of a subtilisin-like serine protease from the marine fungus, Engyodontium album BTMFS10. World J Microbiol Biotechnol, 26: Jellouli K, Bougatef A, Manni L et al, Molecular and biochemical characterization of an extracellular serineprotease from Vibrio metschnikovii J1. J Ind Microbiol Biotechnol, 36: Jignasha T Thumar, Satya P Singh, Organic solvent tolerance of an alkaline protease from salt-tolerant alkaliphilic Streptomyces clavuligerus strain Mit-1. J Ind Microbiol Biotechnol, 36: Johnvesly B, Naik G R, Studies on production of thermostable alkaline protease from thermophilic and alkaliphilic Bacillus sp. JB-99 in a chemically defined medium. Process Biochemistry, 37(2): Kumar C G, Tiwari M P, Jany K D, Novel alkaline serineproteases from alkalophilic Bacillus spp. purification and some properties. Process Biochemistry, 34(5): Kunamneni Adinarayana, Poluri Elllaiah, Davuluri Siva Prasad, Purification and partial characterization of thermostable serine alkaline protease from a newly isolated Bacillus sublitis PE-11. AAPS Pharm Sci Tech, 4(4): Madhuri A, Nagaraju B, Harikrishna N et al, Production of Alkaline Protease by Bacillus altitudinis GVC11 using Castor Husk in Solid-State Fermentation. Appl Biochem Biotechnol, 167: Raja Noor Zaliha Raja Abd Rahman, Lee Poh Geok, Mahiran Basri, An organic solvent-stable alkaline protease from Pseudomonas aeruginosa strain K: Enzyme purification and characterization. Enzyme and Microbial Technology, 7: Sreeja Chellappan C, Jasmin Soorej M, Basheer Archana Kishore K et al, Characterization of an extracellular alkaline serine protease from marine Engyodontium album BTMFS10. Microbiol Biotechnol, 38: Subramani Ramesh, Mahalingam Rajesh, Narayanasamy Mathivanan, Characterization of a thermostable alkaline protease produced by marine Streptomyces fungicidicus MML1614. Bioprocess Biosyst Eng, 32: Sudhir K Rai, Jetendra K Roy, Ashis K Mukherjee, Characterisation of a detergent-stable alkaline protease from a novel thermophilic strain Paenibacillus tezpurensis sp. nov. AS-S24-II. Appl Microbiol Biotechnol, 85: Tsuyoshi Nonaka, Masahiro Fujihashi, Akiko Kita Katsuhisa, The crystal structure of an oxdatively-stable subtilisin alkaline serine protease, KP-43, with a C-terminal -barrel domain. J Biol Chem, 45: Vijay Kumar E, Srijana M, Kiran Kumar K et al, A novel serine alkaline protease from Bacillus altitudinis GVC11 and its application as a dehairing agent. Bioprocess Biosyst Eng, 34: Wang Fang, Hao Jianhua, Yang Chengye et al, Cloning, expression, and identification of a novel extracellular cold-adapted alkaline protease gene of the marine bacterium strain YS Appl Biochem Biotechnol, 162: Wang S L, Yang C H, Liang T W et al, Optimization of conditions for protease production by Chryseobacterium taeanense TKU001. Bioresour Technol, 99: WANG Shuai, LIN Xuezheng, HUANG Xiaohang et al, Screening and characterization of the alkaline protease isolated from PLI-1, a strain of Brevibacillus sp. collected from Indonesia, hot springs. Oceanic and Coastal Sea Research, 11(2):

10 BREEDING OF A MARINE ACTINOMYETES STRAIN PRODUCING ALKALINE PROTEASE LI Peng, MIAO Zeng-Liang, WANG Jian-Xin (Marine Science & Technology College, Zhejiang Ocean University, Zhoushan , China) Abstract Marine actinomycetes able to produce alkaline protease were isolated from an intertidal zone in Zhoushan Islands region. Enzyme activity was determined by the Lowry method, and the strain of highest enzyme activity was studied. After ultraviolet and DES mutation, a marine actinomycete Strain A20 in high yield of alkaline protease was obtained. We found that Strain A20 was more capable of producing alkaline protease. Morphological, physiological, and biochemical characteristics, as well as 16S rdna sequence homology show that Strain A20 is relative to Streptomyces roseus by sharing 99.8% in the sequence under phylogenetic tree. The result of mutation shows that enzyme activity increased by 117.3% from to U/mL. Genetic stability test showed the Strain A20 had good genetic stability. The optimal conditions for producing alkaline protease was temperature 50 C, ph 9.0, and culture period for 72h. Key words alkaline protease; phylogenetic tree; mutant; 16S rdna

L 8 0 g L ~0 700 g L % 6 00% 1 50% 1 3 NaCl 0 50% NH 4 2 SO % KH 2 PO % CaCO % 0 03% ~ 0 04% 2 24 h 0 02

L 8 0 g L ~0 700 g L % 6 00% 1 50% 1 3 NaCl 0 50% NH 4 2 SO % KH 2 PO % CaCO % 0 03% ~ 0 04% 2 24 h 0 02 38 5 Vol 38 No 5 2014 9 Journal of Jiangxi Normal UniversityNatural Science Sep 2014 1000-5862201405-0501-05 1 1* 2 2 1 3300222 330052 Streptomyces lincolnensis 13 8 6% 12 4% Q 93 A 0 1 2 Streptomyces

Microsoft Word 李 鹏_new_.doc

Microsoft Word 李鹏_new_.doc