细菌碱性木聚糖酶菌种筛选、产酶条件与酶学性质研究
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摘要
从某造纸厂排水沟旁边的碱性土壤中，采集到要进行产酶菌种分离的土样，用RBB-Xylan选择培养基进行初筛，根据不同菌株所形成的透明圈的有无，决定需要复筛的菌株，经产酶基本培养基复筛后，筛选到一株碱性木聚糖酶高产菌株HNX01，其在基本产酶培养基中的产酶能力为80.39 IU/ml，根据其形态特征、运动性、培养特征及部分生理生化反应，初步鉴定该菌为芽孢杆菌（Bacillus.sp）。
经单因子试验和正交设计试验，得出HNX01产酶的最佳条件为：玉米芯4%、麸皮2%、NH4Cl 0.6%、NaNO3 0.2%、K2HPO41%、土温80 0.2%、FeCl3 2mmol/L、pH8.0、接种量为3%、250ml三角瓶装液量为50ml 。37℃、220r/min下用此培养基培养HNX01 24小时，木聚糖酶酶活力达高峰值198.41IU/ml，是未经优化的产酶基本培养基产酶能力的2.5倍。
HNX01所产木聚糖酶最适作用pH值为8.0，酶具有较宽的pH值耐受性，尤其是在pH7.0以上的碱性范围稳定性更好，在pH6.0~10.6
之间木聚糖酶酶活力都在80%以上；最适作用温度为50℃，酶在20℃~50℃下基本稳定， 50℃处理60分钟仍然具有70%以上的酶活力。金属离子除Zn2+对酶有轻微的激活作用外，其它对酶活力都存在不同程度的抑制作用，其中Cu2+对木聚糖酶酶活力的抑制最强烈。
According to RBB-xylan-hydrolyzed halos method , A bacterial strain which can produce extra-cellular alkaline xylanase, was isolated from alkaline soil near the drain ditch of a paper-producing factory .The strain was named as HNX01.The activity of xylanase produced by HNX01 was 89.39 IU/ml. According to its morphological characterization, physiological and biochemical experiments, HNX01 should belong to Bacillus.sp.
After single factor tests and orthogonal array ,the optimal medium consisted of 4% corn power, 2% wheat bran, 0.6% NH4Cl, 0.2% NaNO3, 1% K2HPO4, 0.2% tween-80, 2mmol/L FeCl3.The initial pH of medium was 8.0.The percent of inoculation was 3%.The volume was 50ml in 250ml flask.After shaken at 220rpm and cultured in this optimal medium for about 24 hours at 37℃ , the xylanase activity is 198.41 IU/ml, which is 2.5 times of original medium.
The xylanase from HNX01 displayed an optimal temperature at 50℃ and optimal pH at 8.0,a pH stability range from 5.0 to 10.6 and thermal stability up to 50℃.After incubated for 60 minute at 50℃,it still had 70% activity. In the metal ions investigated, only Zn2+ promoted effects on Xylanase, other ions all inhabited its activity more or less. Especially, Cu2+ inhabited almost all the activity. Fe3+ inhabited 50% activity in the enzyme reaction.
经单因子试验和正交设计试验，得出HNX01产酶的最佳条件为：玉米芯4%、麸皮2%、NH4Cl 0.6%、NaNO3 0.2%、K2HPO41%、土温80 0.2%、FeCl3 2mmol/L、pH8.0、接种量为3%、250ml三角瓶装液量为50ml 。37℃、220r/min下用此培养基培养HNX01 24小时，木聚糖酶酶活力达高峰值198.41IU/ml，是未经优化的产酶基本培养基产酶能力的2.5倍。
HNX01所产木聚糖酶最适作用pH值为8.0，酶具有较宽的pH值耐受性，尤其是在pH7.0以上的碱性范围稳定性更好，在pH6.0~10.6
之间木聚糖酶酶活力都在80%以上；最适作用温度为50℃，酶在20℃~50℃下基本稳定， 50℃处理60分钟仍然具有70%以上的酶活力。金属离子除Zn2+对酶有轻微的激活作用外，其它对酶活力都存在不同程度的抑制作用，其中Cu2+对木聚糖酶酶活力的抑制最强烈。
According to RBB-xylan-hydrolyzed halos method , A bacterial strain which can produce extra-cellular alkaline xylanase, was isolated from alkaline soil near the drain ditch of a paper-producing factory .The strain was named as HNX01.The activity of xylanase produced by HNX01 was 89.39 IU/ml. According to its morphological characterization, physiological and biochemical experiments, HNX01 should belong to Bacillus.sp.
After single factor tests and orthogonal array ,the optimal medium consisted of 4% corn power, 2% wheat bran, 0.6% NH4Cl, 0.2% NaNO3, 1% K2HPO4, 0.2% tween-80, 2mmol/L FeCl3.The initial pH of medium was 8.0.The percent of inoculation was 3%.The volume was 50ml in 250ml flask.After shaken at 220rpm and cultured in this optimal medium for about 24 hours at 37℃ , the xylanase activity is 198.41 IU/ml, which is 2.5 times of original medium.
The xylanase from HNX01 displayed an optimal temperature at 50℃ and optimal pH at 8.0,a pH stability range from 5.0 to 10.6 and thermal stability up to 50℃.After incubated for 60 minute at 50℃,it still had 70% activity. In the metal ions investigated, only Zn2+ promoted effects on Xylanase, other ions all inhabited its activity more or less. Especially, Cu2+ inhabited almost all the activity. Fe3+ inhabited 50% activity in the enzyme reaction.
引文
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[3 ] Sunna A, Antranikian G Xylanolytic enzymes from fungi and bacteria. Crit Rev Biotechnol (1997) 17:39–67
[4 ] Chanda SK, Hirst EL, Jones JKN, Percival EGVThe constitution of xylan from esparto grass (Stipa tenacissima). J Chem Soc (1950) 50:1287–1289
[5 ] Eda S, Ohnishi A, Kato K Xylan isolated from the stalks of Nicotiana tabacum. Agric Biol Chem (1976) 40:359–364
[6 ] Montgomery R, Smith F, Srivastava HCStructure of cornhull hemicellulose. I. Partial hydrolysis and identification of 2-0-(?-D-glucopyranosyluronic acid)-D-xylopyranose. J Am
Chem Soc (1956) 78:2837–2839
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[9 ] Uffen RLXylan degradation: a glimpse at microbial diversity. J Ind Microbiol Biotechnol (1997) 19:1–6
[10] Dekker R F H.ACS Symp. SER., 1989, 399:619~629
[11] Dekker R F H. et al. Adv . Carbohydr. Chem. Biochem., 1976,3:277~252
[12] Wood W A et al .Methods in Enzymeology. San Diego: Academic Press Inc., 1988.160：511~725
[13] Belancic A, Scarpa J, Peirano A, Diaz R, Steiner J, Eyzayuirre Penicillium purpurogenum produces several xylanases: purification and properties of two of the enzymes. J Biotechnol(1995) 41:71–79
[14] Biely P Microbial xylanolytic systems. Trends Biotechnol(1985):286–290
[15] Elegir G, Szakacs G, Jeffries TW Purification, characterization and substrate specificities of multiple xylanases from Streptomyces sp. strain B-12–2. Appl Environ Microbiol (1994) 60:2609–2615
[16] Biely P, Mackenzie CR, Puls J, Schneider H Cooperativity of esterases and xylanases in the enzymatic degradation of acetyl xylan. Biotechnology (1986)4:731–733
[17] Wong KKY, Tan LUL, Saddler JN Multiplicity of β-1,4-xylanase in microorganisms: functions and applications. Microbiol Rev(1988) 52:305–317
[18] Shao W, DeBlois S, Wiegel J (1995) A high molecular weight, cell-associated xylanase isolated from exponentially growingThermoanaerobacterium sp. strain JW/SL-YS485. Appl Environ Microbiol 61:937–940
[19] Hall J , Durrant A J, Mol Microbiol, 1989, 3:1211~1219
[20] Grosalbes M J, Perez J A ,Gonzalez R . Bacteriol, 1991, 173:7705~7710
[21]Wong KKY, Tan LUL, Saddler JN (1988) Multiplicity of ?-1,4-xylanase in microorganisms: functions and applications.Microbiol Rev 52:305–317
[22] Okazaki W, Akiba T, Horikoshi K, Akahoshi R (1985) Purification and characterization of xylanases from alkalophilic thermophilic Bacillus spp. Agric Biol Chem 49:2033–2039
[23]Esteban R, Villanueva JR, Villa TG (1982) ?-D-xylanases of Bacillus circulans WL-12. Can J Microbiol 28:733–739
[24] Khasin A, Alchanati I, Shoham Y (1993) Purification and characterization of a thermostable xylanase from Bacillus stearothermophilus T-6. Appl Environ Microbiol 59:1725–1730
[25] Blanco A, Vidal T, Colon JF, Pastor FIJ (1995) Purification and properties of xylanase a from alkali-tolerant Bacillus sp. StrainBP-23. Appl Environ Microbiol 61:4468–4470
[26] Lopez-Fernandez C, Rodriguez J, Ball AS, Lopa-Patino JL, Periz-Lebic MI, Arias ME (1998) Application of the affinity binding of xylanases to oat-spelt xylan in the purification of endoxylanase CM-2 from Streptomyces chattanoogensis CECT 3336. Appl Microbiol Biotechnol 50:284–287
[27] Morales P, Madrarro A, Flors A, Sendra JM, Gonzalez JAP (1995) Purification and characterization of a xylanase and arabinofuranosidase from Bacillus polymyxa. Enzyme Microb Technol 17:424–429
[28] Ratankhanokchai K, Kyu KL, Tantichareon M (1999) Purification and properties of a xylan-binding endoxylanase from alkaliphilicBacillus sp. strain K-1. Appl Environ Microbiol 65:694–697
[29] Gupta N, Vohra RM, Hoondal GS (1992) A thermostable extracellular xylanase from alkalophilic Bacillus sp. NG-27. Biotechnol Lett 14:1045–1046
[30] Bataillon M, Cardinali APN, Duchiron F (1998) Production of xylanases from a newly isolated alkalophilic thermophilic Bacillus sp. Biotechnol Lett 20:1067–1071
[31] Gessesse A, Mamo G (1998) Purification and characterization of an alkaline xylanase from alkaliphilic Micrococcus sp.AR-135. J Ind Microbiol Biotechnol 20:210–214
[32] Dey D, Hinge J, Shendye A, Rao M (1992) Purification and properties of extracellular endo-xylanases from alkalophilic thermophilic Bacillus sp. Can J Microbiol 38:436–442
[33] Khanna S, Gauri P (1993) Regulation, purification and properties of xylanase from
Cellulomonas fimi. Enzyme Microb Technol 15:990–995
[34] Chaudhary P, Deobagkar D (1997) Purification and characterization of xylanase from Cellulomonas sp. N.C.I.M. 2353. Biotechnol Appl Biochem 25:127–133
[35] Gessesse A (1998) Purification and properties of two thermostable alkaline xylanases from an alkalophilic Bacillus sp. Appl Environ Microbiol 64:3533–3535
[36] Gupta S, Bhushan B, Hoondal GS (2000) Isolation, purification and characterization of xylanase from Staphylococcus sp.SG-13 and its application in biobleaching of kraft pulp. J Appl Microbiol 88:325–334
[37] Winterhalter C, Liebel W (1995) Two extremely thermostable xylanases of the hyperthermo philic bacterium Thermotoga maritama MSB8. Appl Environ Microbiol 61:1810–1815
[38] Shao W, DeBlois S, Wiegel J (1995) A high molecular weight, cell-associated xylanase isolated from exponentially growingThermoanaerobacterium sp. strain JW/SL-YS485. Appl Environ Microbiol 61:937–940
[39] Frederick MM, Kiang C, Frederick JR, Reilly PJ (1985) Purification and characterization of endo-xylanases from Aspergillusniger. I. Two isozymes active on xylan backbones near branch points. Biotechnol Bioeng 27:525–532
[40] Ito K, Ogasawara H, Sugimoto T, Ishikawa T (1992) Purification and properties of acid stable xylanases from Aspergillus kawachii. Biosci Biotechnol Biochem 56:547–550
[41] Fernandez-Epsinar MT, Ramon D, Pinaga F, Valles S (1992) Xylanase production by Aspergillus nidulans. FEMS MicrobiolLett 91:91–96
[42] Raj KC, Chandra TS (1996) Purification and characterization of xylanase from alkalitolerant Aspergillus fischeri Fxn 1. FEMS Microbiol Lett 145:457–461
[43] Kimura I, Sasahar, H, Tajima S (1995) Purification and characterization of two xylanases and an arabinofuranosidase from Aspergillus sojae. J Ferment Bioeng 80:334–339
[44] Ghosh M, Nanda G (1994) Purification and some properties of xylanase from Aspergillus sydowii MG 49. Appl Environ
Microbiol 60:4620–4623
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