IntroductionGalactose oxidase (GalO

Introduction
Galactose oxidase (GalOx1; d-galactose:oxygen 6-oxidoreductase, EC 1.1.3.9) is a monomeric 68-kDa enzyme that contains a single copper ion [1] and an amino acid-derived cofactor [2] and [3], formed by cross-linking of a Cys and a Tyr residue in the direct vicinity of the copper [4], [5] and [6]. The thioether bond of the Tyr-Cys cross-link is post-translationally generated [4] and [7] and has been shown to affect the stability, the reduction potential [8] and the catalytic efficiency of the enzyme [9] and [10]. It has been classified as a member of the carbohydrate active-enzyme family AA5, subfamily 2 [11]. GalOx catalyzes the two-electron oxidation [3] and [12] of the C6-hydroxyl group of nonreducing d-galactose residues [13] as well as a range of primary alcohols to the corresponding aldehydes with concomitant reduction of oxygen to hydrogen peroxide [14], [15], [16] and [17]. During catalysis both the metal ion and the cysteine-modified tyrosine group undergo 1-electron redox interconversions [18]. Despite a wide substrate specificity, GalOx is strictly regioselective and no secondary alcohols are oxidized [19]. However, the enzyme accepts a wide variety of primary alcohols such as benzyl alcohol [20], and glycerol [21] as reducing substrates. GalOx displays remarkable stereospecificity in its reaction with sugars [22], being highly sensitive for the orientation of the C4-OH group, and hence it shows activity with galactose but not with glucose. Because of this specificity, various analytical techniques are based on GalOx, such as the determination of lactose in milk and dairy products [23] or the histochemical examination of mucus-secreting cells [24]. Furthermore, GalOx has been used in biosensors for the measurement of galactose and its derivatives in biological fluids [25], to label galactose residues in glycoconjugates [26], and for the induction of interferon in human lymphocyte culture [27] and [28]. GalOx is viewed as a competitive and cost-effective catalyst compared to chemical conversion for the manufacturing of fine chemicals for pharmaceutical purposes or in food industry, for example GalOx was used for conversion of sugars like d-galactose to food-grade cross-linking agents [29], [30], [31] and [32]. Another important application for GalOx is the modification of cell surface carbohydrates and has been used in cell labeling studies and histochemical staining [19]. GalOx is interesting for the use in industrial processes such as derivatization of guar gum and related polymers as well [33] and [34].


The enzyme is secreted by a number of fungal species, of which Fusarium graminearum (formerly classified as Dactylium dendroides) is the most extensively studied [35], [36], [37], [38], [39], [40], [41], [42] and [43]. The production and purification of GalOx has been reported from its natural fungal source [26], [39], [44], [45], [46], [47], [48] and [49], furthermore, various GalOx genes were cloned and successfully expressed in the filamentous fungi Aspergillus nidulans [50] and [51], Aspergillusoryzae and Fusarium venenatum [52], which have no endogenous GalOx, in the methylotrophic yeast Pichia pastoris [4], [10], [33], [36], [53], [54], [55] and [56] and in the bacterium Escherichia coli [55], [57], [58] and [59]. Typically, wild-type fungal GalOx is produced as a preproform carrying an N-terminal signal sequence, which is removed upon secretion, yielding the immature proform. The prosequence in this form was suggested to function as an intramolecular chaperone supporting copper binding and cofactor formation [42] and [50]. The maturation of GalOx requires several successive steps including cleavage of the signal sequence, which directs translocation, metal binding and cofactor processing [12] and [43]. Subsequently, the prosequence is removed and the Tyr-Cys cofactor is formed by self-processing reactions [2] and [7].

In the present paper we describe cloning and recombinant expression of a new gao gene without its prepro sequence from Fusarium sambucinum in E. coli. Furthermore, the purification and biochemical characterization of the enzyme are reported. Alternative electron acceptors, and possible activators as well as inhibitors were tested for their effect on GalOx activity

Kết quả (Việt) 1:

Giới thiệuGalactose oxidase (GalOx1; d-galactose:oxygen 6-oxidoreductase, EC 1.1.3.9) is a monomeric 68-kDa enzyme that contains a single copper ion [1] and an amino acid-derived cofactor [2] and [3], formed by cross-linking of a Cys and a Tyr residue in the direct vicinity of the copper [4], [5] and [6]. The thioether bond of the Tyr-Cys cross-link is post-translationally generated [4] and [7] and has been shown to affect the stability, the reduction potential [8] and the catalytic efficiency of the enzyme [9] and [10]. It has been classified as a member of the carbohydrate active-enzyme family AA5, subfamily 2 [11]. GalOx catalyzes the two-electron oxidation [3] and [12] of the C6-hydroxyl group of nonreducing d-galactose residues [13] as well as a range of primary alcohols to the corresponding aldehydes with concomitant reduction of oxygen to hydrogen peroxide [14], [15], [16] and [17]. During catalysis both the metal ion and the cysteine-modified tyrosine group undergo 1-electron redox interconversions [18]. Despite a wide substrate specificity, GalOx is strictly regioselective and no secondary alcohols are oxidized [19]. However, the enzyme accepts a wide variety of primary alcohols such as benzyl alcohol [20], and glycerol [21] as reducing substrates. GalOx displays remarkable stereospecificity in its reaction with sugars [22], being highly sensitive for the orientation of the C4-OH group, and hence it shows activity with galactose but not with glucose. Because of this specificity, various analytical techniques are based on GalOx, such as the determination of lactose in milk and dairy products [23] or the histochemical examination of mucus-secreting cells [24]. Furthermore, GalOx has been used in biosensors for the measurement of galactose and its derivatives in biological fluids [25], to label galactose residues in glycoconjugates [26], and for the induction of interferon in human lymphocyte culture [27] and [28]. GalOx is viewed as a competitive and cost-effective catalyst compared to chemical conversion for the manufacturing of fine chemicals for pharmaceutical purposes or in food industry, for example GalOx was used for conversion of sugars like d-galactose to food-grade cross-linking agents [29], [30], [31] and [32]. Another important application for GalOx is the modification of cell surface carbohydrates and has been used in cell labeling studies and histochemical staining [19]. GalOx is interesting for the use in industrial processes such as derivatization of guar gum and related polymers as well [33] and [34].Men tiêu hóa được bài tiết bởi một số loài nấm, trong đó Fusarium graminearum (trước đây được phân loại là Dactylium dendroides) là nghiên cứu rộng rãi nhất [35], [36], [37], [38], [39], [40], [41], [42] và [43]. Sản xuất và thanh lọc của GalOx đã được báo cáo từ nguồn tự nhiên nấm [26], [39], [44], [45], [46], [47], [48] và [49], hơn nữa, nhiều GalOx gen đã được nhân bản và thành công thể hiện trong sợi nấm Aspergillus nidulans [50] và [51], Aspergillusoryzae và Fusarium venenatum [52], mà đã không có GalOx nội sinh, trong nấm men methylotrophic Pichia pastoris [4], [10], [33], [36], [53], [54], [55] và [56] và trong các vi khuẩn Escherichia coli [55], [57], [58] và [59]. Thông thường, loại hoang nấm GalOx được sản xuất như là một preproform thực hiện một chuỗi tín hiệu N thiết bị đầu cuối, mà được lấy ra sau khi tiết, năng suất các non proform. Prosequence trong hình thức này đã được đề xuất để hoạt động như một đi kèm intramolecular hỗ trợ đồng ràng buộc và hình thành cofactor [42] và [50]. Sự trưởng thành của GalOx đòi hỏi một số bước kế tiếp trong đó có cát khai của chuỗi tín hiệu, mà chỉ đạo translocation, kim loại ràng buộc và cofactor xử lý [12] và [43]. Sau đó, các prosequence được lấy ra và cofactor Tyr-Cys được hình thành bằng cách tự xử lý phản ứng [2] và [7].Trong giấy hiện nay chúng tôi mô tả thể hiện nhân bản và tái tổ hợp của một gen gao mới mà không có của nó tự prepro từ Fusarium sambucinum trong E. coli. Hơn nữa, làm sạch và các đặc tính sinh hóa của enzyme được báo cáo. Chất nhận khác ở điện tử, và có thể tính cũng như các chất ức chế đã được thử nghiệm cho hiệu quả của họ trên GalOx hoạt động