【作者】 周芳；

【导师】 牟海津；

【作者基本信息】 中国海洋大学 ， 水产品加工及贮藏工程， 2007， 硕士

【摘要】 β-甘露聚糖酶是一类能够水解甘露聚糖、葡甘露聚糖、半乳甘露聚糖的生物活性酶。在食品、医药、饲料、造纸、石油等方面已得到广泛应用;近年来,它在降解植物胶生产功能性甘露低聚糖以及作为食品和饲料添加剂等方面倍受关注。本研究从土壤中分离、选育出β-甘露聚糖酶产生菌株,对其进行初步鉴定,考查了不同因素对菌株发酵产酶的影响,对酶进行了分离纯化与性质研究,并用此酶降解制备甘露低聚糖,对甘露低聚糖进行了分离纯化和结构分析鉴定。主要研究成果如下:(1)通过富集培养、分离纯化、选择性平板分离、平板水解圈初筛和摇瓶复筛,从土壤中筛选出一株具较高产β-甘露聚糖酶能力的菌株(编号为M-21),经初步鉴定为芽孢杆菌属(Bacillus sp.)。(2)通过单因素实验和正交优化实验,确定了Bacillus sp. M-21最佳发酵产酶条件。菌株的产酶最适培养基组成包括(g/L)碳源:瓜尔豆胶4,复合氮源:豆粉20、(NH4)2HPO4 5,其他无机盐组分:K2HPO4·3H2O 1、MgSO4·7H2O 0.5、NaCl 0.5、CaCl2 0.1、FeSO4·7H2O 0.001。产酶最适培养条件:培养基初始pH 8.0,接种量4%,装液量50 mL/250 mL三角瓶,32℃180 r/min振荡培养36 h。此条件下酶活力最高可达1487 U/mL。(3) Bacillus sp. M-21产β-甘露聚糖酶经1.4倍丙酮沉淀、Q-sepharose阴离子交换柱后分离纯化,SDS-PAGE电泳确定酶的分子量为33.4 kDa。相比之下,此β-甘露聚糖酶的分离步骤简单,酶的比活力和回收率(70.1%)都很高,具有工业上大量制备的前提。(4)此β-甘露聚糖酶最适反应的pH为7.0,且耐受的pH范围较宽(5.0-11.0),具有一定的耐碱性;最适反应温度为50℃,50℃以下酶活力稳定。金属离子Ba2+、Ca2+、K+、Mg2+、Na+、Mn2+对酶有不同程度的激活作用;Zn2+、Pb2+、Fe2+、Cu2+、Hg2+、Ag+有对酶有抑制作用。酶对魔芋胶、刺槐豆胶和瓜尔豆胶的Km值分别为3.97、8.79和17.56mg/mL,Vmax值分别为9.02、12.44和20.92μmol/(min·mL)。菌株M-21所分泌的β-甘露聚糖酶的最适底物为魔芋胶。(5)酶降解魔芋胶、刺槐豆胶、瓜尔豆胶制备甘露低聚糖,通过乙醇分级沉淀、凝胶过滤层析(Bio-Gel P-4 Extra Fine)对寡糖进行分离纯化,经TLC、质谱分析,魔芋胶酶解产物由二糖、三糖、四糖等一系列寡糖片段组成,单糖很少。本研究为β-甘露聚糖酶的工业化生产和甘露低聚糖的酶法制备提供了一定的理论和实践依据。更多还原

【Abstract】 β-mannanase is an enzyme capable of hydrolyzingβ-1,4-D-manno-pyanosyl linkages of mannans with main product manno-oligosaccharide. Mannanase has wide commercial applications in industry processes such as foodstuff, pharmaceutical, feed, paper and pulp, and oil industry.β-mannanase producing bacterial strain M-21 was isolated from the soil samples. After identification of the strain, the fermentation conditions were optimized and the purification and characterizations of theβ-mannanase were studied. Theβ-mannanase was also applied to hydrolyze several mannan gums yielding manno-oligosaccharides.The results showed that:1.β-mannanase producing strain M-21 was isolated from the soil samples using the methods of enriching culture, isolation and selective plate culture, first screening by clear circles on selective paltes, and second screening by shaking culture. It was identified primarily as Bacillus sp.2. The fermentation conditions were optimized in order to improve the production of the enzyme. The optimal liquid medium consisted of 4 g/L guar gum as carbon source, 20 g/L soybean powder and 5 g/L (NH4)2HPO4 as nitrogen source and other inorganic salts(g/L): K2HPO4·3H2O 1, MgSO4·7H2O 0.5, NaCl 0.5, CaCl2 0.1, FeSO4·7H2O 0.001. The optimal culture conditions were initial pH 8.0, inoculation volume 4%, medium volume 50 mL in 250 mL flask, agitation speed 180 r/min. After incubation at 32℃for 36h, the enzyme showed the highest activity of 1487 U/mL.3. Theβ-mannanase was purified to homogeneity by acetone precipitation and Q-sepharose ion exchange chromatogramphy. The enzyme was shown to have a relative molecular weight of 33.4 kDa by SDS polyacrylamide gel electrophoresis.4. The maximum enzyme activity existed at 55℃and the optimized pH was 7.0. It was stable between a pH range of 5.0 and 11.0,below 50℃. The metal ions Zn2+, Pb2+, Fe2+, Cu2+, Hg2+ and Ag+ inhibitedβ-mannanase activity strongly, but Ba2+, Ca2+, K+, Mg2+, Na+ and Mn2+ activated the enzyme. Kinetics ofβ-mannanase was examined. The Km values for konjak gum, locust bean gum and guar gum were 3.97, 8.79 and 17.56 mg/mL, and Vmax were 9.02, 12.44 and 20.92μmol/(min·mL). Its optimal substrate was konjak gum.5. Konjak gum, locust bean gum and guar gum were hydrolyzed by the crudeβ-mannanase from Bacillus sp. M-21 to yield serial manno-oligosaccharides as the main products in different velocity and degree. The hydrolysate was separated by ethanol grading precipitation and gel filtration chromatography (Bio-Gel P-4 Extra Fine). By means of TLC and MS methods, the konjak oligosaccharides were identified as a series of di-, tri- and tetra-saccharides with little monosaccharide .This work might be helpful for both the industrial production ofβ-mannanase and manno-oligosaccharide hydrolysis catalysed byβ-mannanase.更多还原