Phylogenetic analysis of inuin-degrading enzymes and efficient expression

1. 克隆并测定了青霉n -88株内生菊氨酸酶基因的序列。该基因的开放阅读框未被内含子打断，编码25个氨基酸的信号肽和490个氨基酸的成熟蛋白。成熟蛋白含有3个胱氨酸残基和10个潜在的n -连接糖基化位点。推导出的氨基酸序列与黑曲霉和紫青霉内生酶基因的同源性分别为72%和85%。邻居连接树显示，真菌内生酶与细菌左旋酶关系密切。从丝状真菌黑霉突变体817的培养滤液中纯化了部分内生酶。酶制剂以共价固定在多孔纤维素衍生物氨基纤维素上。在含有固定化酶的填充床塔式反应器中，以5% (w/v) （pH 5.0）的菊粉溶液连续水解。水解产物大部分为低聚低聚糖，聚合度为3 ~ 5。体外实验表明，双歧杆菌对F_3和F_4均有较强的利用能力。采用DEAE-Cellulofine A-500、Sephadex G-100和Sephadex G-200层析对黑霉菌株12菌丝体内的胞外酶和内生酶进行分离纯化。纯化的外菊粉酶P-II对菊粉的比活性为6.6 U/mg，对蔗糖的比活性为22 U/mg。酶P-III对菊粉的活性为108 U/mg，对蔗糖无活性。用Sephadex G-200凝胶过滤法测定外菊粉酶P-II和内菊粉酶P-III的M__-_rs分别为47和56 kDa。P-II酶活性的最佳pH和温度分别为pH 5.0和55℃，P-III酶活性的最佳pH和温度分别为pH 5.3和45℃。两种酶均被Mn^<2+>激活，而被Ag^+、Hg^<2+>或p__—氯汞苯甲酸酯灭活。菊粉酶P-II和P-III的表观K__-_m值分别为5.8和0.80 mM。

1. An endoinulinase gene of Penicillium sp. strain TN-88 was cloned and sequenced. An open reading frame of the gene was not interrupted by introns, and it encoded a 25 amino acid signal peptide and a 490 amino acid mature protein. The mature protein contained three Cys residues and ten potential N-linked glycosylation sites. The deduced amino acid sequence showed 72 and 85 % identities with those of Aspergillus niger and Penicillium purpurogenum endoinulinase genes, respectively. A neighbor-joining tree showed that fungal endoinulinases are closely related to bacterial levanases.2. Endoinulinase was partially purified from the culture filtrate of a filamentous fungus A. niger mutant 817. The enzyme preparation was immobilized covalently onto a porous cellulose derivative, Amino-Cellulofine. A 5 % (w/v) solution (pH 5.0) of inulin was continuously hydrolyzed in a packed-bed column reactor containing the immobilized enzyme. The majority of the hydrolysis products were inulo-oligosaccharides with a degree of polymerization of 3 to 5. In vtro studies indicated that both the F_3 and F_4 was preferentially utilized in vitro by Bifidobacterium spp.3. Intracellular exo- and endoinulinases were extracted from mycelia of A. niger strain 12 and purified by DEAE-Cellulofine A-500, Sephadex G-100, and Sephadex G-200 chromatographies. The purified exoinulinase P-II had specific activities of 6.6 U/mg toward inulin and 22 U/mg toward sucrose. The endoinulinase P-III showed 108 U/mg toward inulin, but no activity toward sucrose. M__-_rs of exoinulinase P-II and endoinulinase P-III were determined by gel filtration using Sephadex G-200 as 47 and 56 kDa, respectively. Optimal pH and temperature for enzyme activity were pH 5.0 and 55 ℃ for P-II, and pH 5.3 and 45 ℃, for P-III. Both the enzymes were activated by Mn^<2+>, and inactivated by Ag^+, Hg^<2+> or p__--chloromercuribenzoate. Inulinases P-II and P-III exhibited apparent K__-_m values of 5.8 and 0.80 mM, respectively.