Cloning of a Gene Encoding Raw-Starch-Digesting Amylase from a
            <i>Cytophaga</i>
            sp. and Its Expression in
            <i>Escherichia coli</i>

Jeang, Chii-Ling; Chen, Li-Shien; Chen, Mingyu; Shiau, Rong-Jen

doi:10.1128/aem.68.7.3651-3654.2002

Cited by 17 publications

(8 citation statements)

References 18 publications

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“…Thus, the raw-starch-hydrolyzing enzymes have gained much importance for effective utilization of natural resources since they also overcome viscosity problems (Jeang et al 2002). The experiments in the present study proved a strong correlation between the adsorption and hydrolysis rates.…”

Section: Discussionsupporting

confidence: 56%

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Molecular cloning, overexpression and characterization of the raw-starch-digesting α-amylase of Bacillus amyloliquefaciens

et al. 2010

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Raw starch is the most abundant source of glucose in the world. Therefore, finding enzymes capable of digesting raw starch would find high industrial demand. The α-amylase gene of Bacillus amyloliquefaciens ATCC 23842 was amplified, cloned and overexpressed in Escherichia coli BL21 (DE3) cells. The recombinant enzyme was purified to apparent homogeneity using ion exchange and gel filtration chromatography. The raw-starch digestibility of the purified enzyme was characterized by studying the hydrolysis and adsorption rate on a variety of raw starches (potato, cassava, corn, wheat and rice). The raw-starch digestion was further confirmed by scanning electron microscopy studies, which revealed an effective rate of hydrolysis. The kinetic studies revealed a relatively low Km of 2.76 mg/mL, exhibiting high affinity towards the soluble starch as the most preferred substrate and the inhibition kinetic studies revealed a high Ki value (350 mM).

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Section: Discussionsupporting

confidence: 56%

“…There was a previous report on cloning and sequencing of raw-starch-digesting amylase from a Cytophaga sp. (Jeang et al 2002), which showed high amino acid identity with the α-amylases from three Bacillus sp. (Southgate et al 1993).…”

Section: Discussionmentioning

confidence: 99%

Molecular cloning, overexpression and characterization of the raw-starch-digesting α-amylase of Bacillus amyloliquefaciens

et al. 2010

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“…The similarity between the two groups implies an evolutionary relationship, as observed previously for a cluster of bacterial enzymes with similarity to animal aamylases, suggested to be a result of horizontal gene transfer (Da Lage et al, 2004). The bacterial a-amylases belonging to subfamily GH13_5 include enzymes from Bacillus and Cytophaga species (Yuuki et al, 1985;Jeang et al, 2002;Kanai et al, 2004), some of which are thermostable (Kim et al, 2000) and used in industry (Guzman-Maldonado & Paredes-Lopez, 1995). Several of these bacterial enzymes are known to produce oligosaccharides of specific lengths from starch, including, for example, a maltohexaose-forming a-amylase from Bacillus (Kanai et al, 2004).…”

Section: Discussionmentioning

confidence: 74%

Phylogenetic and biochemical characterization of a novel cluster of intracellular fungal α-amylase enzymes

et al. 2007

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Currently known fungal a-amylases are well-characterized extracellular enzymes that are classified into glycoside hydrolase subfamily GH13_1. This study describes the identification, and phylogenetic and biochemical analysis of novel intracellular fungal a-amylases. The phylogenetic analysis shows that they cluster in the recently identified subfamily GH13_5 and display very low similarity to fungal a-amylases of family GH13_1. Homologues of these intracellular enzymes are present in the genome sequences of all filamentous fungi studied, including ascomycetes and basidiomycetes. One of the enzymes belonging to this new group, Amy1p from Histoplasma capsulatum, has recently been functionally linked to the formation of cell wall a-glucan. To study the biochemical characteristics of this novel cluster of a-amylases, we overexpressed and purified a homologue from Aspergillus niger, AmyD, and studied its activity product profile with starch and related substrates. AmyD has a relatively low hydrolysing activity on starch (2.2 U mg "1 ), producing mainly maltotriose. A possible function of these enzymes in relation to cell wall a-glucan synthesis is discussed. INTRODUCTIONa-Amylases are widely occurring enzymes which hydrolyse the a-(1,4)-glycosidic bonds in starch and glycogen, producing short maltooligosaccharides and maltose. Based on sequence similarity, most a-amylases (EC 3.2.1.1) are classified in glycoside hydrolase (GH) family 13, although some a-amylases originating from extremophilic organisms belong to family GH57 (Henrissat, 1991;Henrissat & Bairoch, 1996) (see also the CAZy website, http://www.cazy.org). Based on a phylogenetic analysis of 1691 different members of the GH13 family, the family has recently been divided into 35 subfamilies, all acting on aglycosidic bonds (Stam et al., 2006). Several of these subfamilies display a-amylase specificity, but many other enzyme reaction specificities are also represented. The tertiary structure of these enzymes is characterized by a (b/a) 8 barrel containing four highly conserved amino acid regions that form the active site (MacGregor et al., 2001), but the overall sequence similarity can be as low as 10 % and only a catalytic triad of amino acids is conserved invariantly (Machovic & Janecek, 2003). The shared (b/a) 8 barrel structure and catalytic mechanism within GH13 enzymes are believed to represent a common evolutionary origin (Kuriki & Imanaka, 1999;Janecek, 1997). The presence of the four conserved regions and a common secondary and tertiary structure allow construction of alignments and phylogenetic studies within the family. The phylogeny of a-amylases is generally in agreement with their origin, e.g. all fungal a-amylases are more related to each other than to the a-amylases originating from plants or animals. a-Amylases from bacteria, however, are scattered over several clusters, which group with animal, plant or fungal a-amylases, or form a separate branch (Janecek, 1994).Several a-amylases from yeasts and fungi have been studied previously (see e.g....

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“…It was reported that, the rsda gene of a Cytophaga sp. was cloned and expressed in E. coli DH5α and the enzyme was produced intracellularly by the transformant 39 . Similarly, the Bacillus subtilis β-1,4-glucanase gene (a similar enzyme hydrolyzing β-1,4-linkages present in noncrystalline cellulosic substrates such as carboxymethyl cellulose and trinitrophenyl carboxymethyl cellulose) was cloned in E. coli expressing the intracellularly 40 .…”

Section: Discussionmentioning

confidence: 99%

Cellulosimicrobium cellulans’dan β-1,3-Glukanaz Geninin Escherichia coli DH5α’da Klonlanması ve Ekspresyonu

Özcan¹,

Özcan²,

Baylan³

et al. 2013

Kafkas Univ Vet Fak Derg

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SummaryIn this study, β-1,3-glucanase gene of Cellulosimicrobium cellulans was amplified by PCR and cloned in pUC18 cloning vector to construct the recombinant plasmids pTEG5 and pTEG11. The recombinant plasmids pTEG5 and pTEG11 were transformed into competent Escherichia coli cells. Digestion of recombinant plasmids with SacI produced 1.9 kbp β-1,3-glucanase gene band on agarose gel which indicated the gene integration. β-1,3-Glucanase gene amplification on the recombinant vectors also indicated 1.9 kbp gene insert. Recombinant enzyme was produced by E. coli intracellularly. Intracellular components of recombinant E. coli strains with pTEG5 or pTEG11 dropped on LB-laminarin-agar plate, showed clear positive zones by Congo-red staining revealing the activity of secreted protein. Based on the zymogram analysis, the intracellular produced recombinant β-1,3-glucanase enzymes exhibited the same activity bands with C. cellulans enzyme with respect to molecular weight. Keywords: β-1,3-Glucanase, Cellulosimicrobium cellulans, Cloning, Escherichia coliCellulosimicrobium cellulans'dan β-1,3-Glukanaz Geninin Escherichia coli DH5α'da Klonlanması ve Ekspresyonu ÖzetBu çalışmada Cellulosimicrobium cellulans bakterisine ait β-1,3-glukanaz geni PCR ile amplifiye edilerek pUC18 klonlama vektörüne klonlanmış, böylece pTEG5 ve pTEG11 rekombinant plazmit DNA'lar elde edilmiştir. Rekombinant plazmit DNA'lar pTEG5 ve pTEG11 kompetent Escherichia coli hücrelerine transfer edilmişlerdir. SacI enzimi ile kesilmiş rekombinant plazmitlerin agaroz jelde elektroforezi sonucu 1.9 kbç büyüklüğündeki β-1,3-glukanaz gen bandının görünmesi, gen entegrasyonunun tamam olduğunu göstermiştir. β-1,3-glukanaz geninin rekombinant vektörlerden amplifikasyonu da 1.9 kbç büyüklüğündeki geni göstermiştir. Rekombinant enzim E. coli tarafından hücre içi olarak üretilmiştir. LB-laminarin-agar plağına damlatılan rekombinant E. coli suşlarının intraselüler içerikleri Congo-red boyaması ile pozitif zonlar üretmiş, böylece rekombinant bakterilerce üretilen proteinin aktif olduğu anlaşılmıştır. Zimogram analizinde, hücre içi üretilen rekombinant β-1,3-glukanaz enzimleri C. cellulans enzimi ile aynı moleküler ağırlığa ait aktivite bantları sergilemişlerdir.

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Cloning of a Gene Encoding Raw-Starch-Digesting Amylase from a Cytophaga sp. and Its Expression in Escherichia coli

Cited by 17 publications

References 18 publications

Molecular cloning, overexpression and characterization of the raw-starch-digesting α-amylase of Bacillus amyloliquefaciens

Molecular cloning, overexpression and characterization of the raw-starch-digesting α-amylase of Bacillus amyloliquefaciens

Phylogenetic and biochemical characterization of a novel cluster of intracellular fungal α-amylase enzymes

Cellulosimicrobium cellulans’dan β-1,3-Glukanaz Geninin Escherichia coli DH5α’da Klonlanması ve Ekspresyonu

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