【作者】 李剑芳；

【导师】 夏文水；

【作者基本信息】 江南大学 ， 食品科学， 2007， 博士

【摘要】 β-甘露聚糖酶[β-1,4-D-mannan mannanohydrolase, EC 3.2.1.78]是一种能水解甘露聚糖、葡甘露聚糖、半乳甘露聚糖和半乳葡萄甘露聚糖的主链β-1,4-D-甘露吡喃糖苷键的半纤维素酶,主要来自于微生物。它是一种有一定应用前景的新型工业用酶,目前存在发酵酶活性偏低的缺陷,导致其生产和应用成本高,限制了该酶的广泛应用。本论文较系统研究了发酵培养基组成和发酵条件对黑曲霉Aspergillus niger LW-1固态发酵产酶的影响,大幅度提高了发酵酶活性,同时对酶性质和酶法水解工艺进行了研究,为指导酶的工业化生产和应用以及进一步的分子生物学研究奠定了基础。通过单因素试验、Plackett-Burman实验和响应面分析法,确定了黑曲霉Aspergillus niger LW-1菌株锥形瓶固态发酵的最佳产酶培养基组成。为麸皮:豆饼粉= 7.5:2.5（g/g）,其它物质相对于固体物料的质量分数分别为魔芋粉4%,KH2PO40.3%,玉米浆5%,CaCl2 0.1%,MgSO40.1%,（NH4）2SO4 1%,液固比为1.5:1。通过发酵条件优化得到了该菌株的最优培养条件,即培养基起始pH自然,培养温度32℃,静置培养84h。在上述最优条件下,该菌株产酸性β-甘露聚糖酶的最高酶活达24879 IU/g干曲,比未优化前发酵酶活提高了2.27倍,是迄今为止所有报道中酶活水平最高的。通过曲盘（大小φ20 cm×4 cm）固态发酵扩大培养试验,获得了酸性β-甘露聚糖酶固态发酵的最优工艺,即培养基最初料水比为1:1.8,起始pH自然,接种量10%（相对于干物料）,曲盘中装料150g（以干料计）,分别于发酵28h、45h补水和翻曲,32℃培养72 h。在该条件下最高发酵酶活可达22250 IU/g干曲。采用磷酸缓冲液浸提、硫酸铵分部盐析、Phenyl Sepharose CL-4B疏水层析——阶段洗脱、Sephadex G-75凝胶过滤层析、DEAE Sepharose Fast Flow阴离子交换层析以及Phenyl-Sepharose CL-4B疏水层析——梯度洗脱的手段方法,从黑曲霉Aspergillus niger LW-1固态发酵曲中分离到相对分子质量和疏水性质非常相近的A、B两种β-甘露聚糖酶组分,其中组分B所占酶活比例较大。纯化酶的回收率为14.3%,纯化倍数为38.6。采用SDS-PAGE电泳和HPLC两种不同方法对纯化的主要组分B进行鉴定,表明其为一种纯酶组分。运用Sephadex G-75凝胶过滤层析和SDS-PAGE电泳测得纯化酶的相对分子质量分别为41000和40000,两种方法测定结果基本一致,表明该酶以单体形式存在;IEF-PAGE测得该酶的等电点为4.3,苯酚-硫酸法测得该酶含糖量为21.7%;氨基酸含量测定显示,纯化酶中含量较高的前五种氨基酸分别为Asp、Gly、Ser、Glu和Thr,含有较多酸性氨基酸,为酸性蛋白。对纯化酶酶学性质研究显示,该酶最适反应温度为70℃,60℃以下稳定;在测试的三个温度范围内,纯化酶最适反应pH值均为3.5,表明该酶为酸性酶,该酶在pH3.5⁸范围稳定。Zn²⁺、Ca²⁺、EDTA对酶有明显的激活作用,Mn²⁺对酶有较强的抑制作用,Fe²⁺、Pb²⁺、Sn²⁺、Fe³⁺、Cu²⁺、Al³⁺、Ag⁺对酶的抑制作用较弱;其它金属离子对酶活影响不大。以上性质与粗酶试验结果基本一致。纯化酶对槐豆胶半乳甘露聚糖和魔芋葡甘露聚糖的Km值分别为5.1 mg/ml和1.4 mg/ml,对两种多糖的Vmax值分别为1429μmol·min^-1·mg^-1和625μmol·min^-1·mg^-1,表明魔芋粉与酶的亲和力大些。采用HPLC法和HPLC-MS法对纯化酶水解槐豆胶半乳甘露聚糖的产物进行定性和定量分析,发现产物除少量单糖甘露糖外,主要含有甘露二糖、甘露三糖,其二糖、三糖、四糖三类寡糖占总水解产物的49.75%。该结果与众多文献报道的相似,表明该酶为内切β-甘露聚糖酶。以魔芋胶葡甘露聚糖为原料,采用黑曲霉酸性β-甘露聚糖酶粗酶液进行酶法水解条件的研究,得到的优化工艺条件为:魔芋胶（水溶液）浓度150 g/L,加酶量50 IU/g魔芋胶,50℃水解6 h。所获酶解产物经薄板层析和HPLC检测表明主要为低聚糖以及少量单糖。利用酵母发酵法去除其中的可发酵性单糖,可以大大提高酶解产物中魔芋葡甘露低聚糖的含量。本研究试验成功了一种新的魔芋胶酶解工艺,突破了魔芋胶粘度大所带来的屏障,解决了甘露低聚糖生产工艺中最关键的问题,即将魔芋胶浓度从10³0 g/L提高至150 g/L,使工业化规模酶解魔芋胶生产甘露低聚糖成为可能。该魔芋甘露低聚糖的生产工艺尚未见报道。更多还原

【Abstract】 β-Mannanase （β-1,4-D-mannan mannanohydrolase EC 3.2.1.78）, which catalyzes the random cleavage ofβ-D-1,4-mannopyranosyl linkages within the main chain of galactomannan, glucomannan, galactoglucomannan and mannan , is a kind of hemicellulase. It can be produced mainly by various microorganisms.β-mannanase can be useful in several processes in the food, feed, textile, as well as in the pulp and paper industries. Despite having high practical potentialities, the use of mannanase is still limited due to low yields and high-production costs . The researches on theβ-mannanase from Aspergillus niger LW-1 were very important for the manufacture of functional konjak glucomannan oligosaccharides , which provid an important base for industrialized production and application.Multivariant statistical approaches containing Plackett-Burman design and response surface methodology were employed to evaluate the effects of several variables on solid state fermentation in flask with the strain Aspergillus niger LW-1. The optimum medium ingredients for acidicβ-mannanase production in flask were as follows: the proportion of wheat bran to soybean flour 7.5 :2.5 ,initial pH natural, the ratio of water to draw medium 1.5:1, 4% konjak powder, 0.3%KH2PO4, 5%steep liquor, 0.1% CaCl2, 0.1%MgSO4, 1% （NH4）2SO4（all related to dry medium）. The cultivation was carried out at 32℃for 84h. With these conditions, the acidicβ-mannanase activity reached 24879 IU/g dry medium, being 3.27-fold as compared with control. It reaches the highest enzyme activity of all reports so far.Based on successful results of flask, acidicβ-mannanase scale-up production was carried out in koji plate（φ20 cm×4 cm）.The optimal koji plate fermentation conditions were as follows: the ratio of water to dry medium 1.8:1, initial pH natural, inoculum size 10%（related to dry medium）, dry medium content 150g/koji plate. With these conditions, the acidicβ-mannanase activity reached 22250 IU/g dry medium at 32℃for 72 h by adding water and turning koji.Two fractions A and B withβ-mannanase activity from solid-state fermentation culture were purified through sequential steps of buffer extraction, ammonium sulfate precipitation, Phenyl Sepharose CL-4B hydrophobic interaction chromatography （with stepwise gradient）, Sephadex G-75 gel filtration , DEAE Sepharose Fast Flow ion-exchange chromatography and Phenyl Sepharose CL-4B hydrophobic interaction chromatography （with Linear gradient）. Fraction A was similar to fraction B on molecular mass and hydrophobic property . After the steps the enzyme was purified by 38.6-fold with a recovery of 14.3%. The main fraction B was homogeneous as examined by SDS-PAGE and HPLC.The molecular mass of the purified enzyme was estimated to be 41 kD by Sephadex G-75 gel filtration and 40 kD by SDS-PAGE, which indicated that theβ-mannanase was a monomer. The isoelectric point was estimated to be 4.3 by IEF-PAGE. The enzyme was a glycoprotein with carbohydrate content of 21.7% by phenol-vitriol method. The composition of amino acids of the enzyme was analyzed. It showed that the 5 highest content of amino acids were Asp, Gly, Ser, Glu and Thr of all the amino acids. The content of acidic amino acid was higher than that of basic amino acid, indicating that the purified enzyme was an acidic protein.The enzymatic properties of the purifiedβ-mannanase were investigated. The optimal temperature for enzyme reaction was 70℃. The optimal pH was 3.5 at 50⁷0℃, which indicated that theβ-mannanase was an acidic enzyme. The enzyme was stable between pH5.0⁸.0 and below 60℃. The enzyme activity was stimulated by Zn²⁺、Ca²⁺、EDTA and partly inhibited by Mn²⁺、Fe²⁺、Pb²⁺、Sn²⁺、Fe³⁺、Cu²⁺、Al³⁺、Ag+. These properties were very similar to crude enzyme. The Michaelis constants（Km） of theβ-mannanase for locust bean gum and konjak gum were 5.1 mg/ml and 1.4 mg/ml, and maximum velocities（Vmax） for these saccharides were 1429μmol·min^-1·mg^-1 and 625μmol·min^-1·mg^-1, respectively. The result indicated that konjak gum was more suitable substrate than locust bean gum.The hydrolysate of locust bean gum galactomannan degraded by the purifiedβ-mannanase was analyzed by HPLC and HPLC-MS. Theβ-mannanase hydrolyzed locust bean gum galactomannan maimly to mannobiose and mannotriose. The content of mannobiose ,mannotriose and mannotetraose reached 49.75% of total hydrolysate. Above result was similar to many reports, indicating that the purifiedβ-mannanase was an endoenzyme.The hydrolysis conditions of konjak glucomannan were investigated with crude enzyme from Aspergillus niger LW-1. Under the conditions of konjak gum water solution 150 g/L,β-mannanase 50 IU/g konjak gum, hydrolytic temperature 50℃and hydrolytic time 6 h, the hydrolysate was mainly oligo-saccharides with only small traces of mono-saccharides analyzed by thin-layer chromatography and by HPLC. The content of oligo-glucomannan in hydrolysate was obviously increased through eliminating mono-saccharides with yeast fermentation.An unique enzymolysis process for konjak gum was obtained. Konjak gum concentration was obviously increased from 10³0 g/L to 150 g/L. This made it possible for industrialized production from konjak gum to mannooligosaccharide. So far no information is available on similar production process about konjak glucomannan.更多还原