Phylogenetic analysis of inuin-degrading enzymes and efficient expression

1.青霉内切菊粉酶基因对菌株TN-88进行了克隆和测序。该基因的开放阅读框不被内含子打断，编码一个25个氨基酸的信号肽和一个490个氨基酸的成熟蛋白。成熟蛋白含有3个半胱氨酸残基和10个潜在的N-连接糖基化位点。推导的氨基酸序列与黑曲霉和紫青霉内切菊粉酶基因的同源性分别为72%和85%。一棵相邻的树表明真菌内切菊粉酶与细菌左旋酶密切相关。从丝状真菌黑曲霉817的培养滤液中分离纯化了部分菊粉内切酶。酶制剂被共价固定在多孔性纤维素衍生物氨基纤维素钠上。在含有固定化酶的填充床柱反应器中，用5%(w/v)的菊粉溶液(pH 5.0)连续水解菊粉。水解物主要是聚合度在3~5之间的低聚糖。体外研究表明，双歧杆菌优先利用F_3和F_4。从黑曲霉12号菌丝体中提取胞外和胞内菊粉酶，经DEAE-Cellulofine A-500、Sephadex G-100和Sephadex G-200柱层析纯化。纯化的外切菊粉酶P-II对菊粉的比活为6.6U/mg，对蔗糖的比活为22U/mg。内切菊粉酶P-III对菊粉的活力为108U/mg，对蔗糖的活力为零。用Sephadex G-200凝胶过滤法测定外切菊粉酶P-II和内切菊粉酶P-III的分子质量比分别为47和56 kDa。P-Ⅱ的最适pH为5.0，最适温度为55℃，P-Ⅲ的最适pH为5.3，最适温度为45℃。这两种酶均被Mn~(2+)和Gt~(2+)激活，而被Ag~(2+)、Hg~(2+)和p_(-)-氯汞苯甲酸失活。菊粉酶P-II和P-III的表观Km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.