Molecular cloning, characterization and nucleotide sequence of the gene for secreted  -amylase from Xanthomonas campestris pv. campestris

Hu, Nien-Tai; Hung, Ming-Ni; Huang, A‐Min; Tsai, Huei‐Fung; Yang, Bing; Chow, Teh-Yuan; Tseng, Yi-Hsiung

doi:10.1099/00221287-138-8-1647

Cited by 28 publications

(14 citation statements)

References 43 publications

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“…Four highly conserved regions in the a-amylase family and three invariant catalytic residues were identified through alignment with other well-known a-amylases (Hu et al 1992) based on the deduced amino acid sequences (see Fig. 3).…”

Section: Resultsmentioning

confidence: 99%

A mutant α-amylase with only part of the catalytic domain and its structural implication

Tang

Mao

et al. 2006

Biotechnol Lett

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A truncated mutant alpha-amylase, Xa-S2, was obtained from Xanthomonas campestris wild type alpha-amylases (Xa-WT) through random mutagenesis that contained 167 amino acid residues (approx 65% shorter than that of Xa-WT). Secondary structure prediction implied that Xa-S2, would be unable to form the whole (beta/alpha)(8)-barrel catalytic domain and did not have the three conserved catalytic residues of wild type alpha-amylase, but it still displays the starch-hydrolyzing activity. Xa-S2 was prepared, characterized and compared to the recombinant wild-type enzymes. The K (m) for starch was 32 mg/ml; activity was optimal at pH 6.2 and 30 degrees C. In contrast, the K (m) for starch of Xa-WT was 8 mg/ml and optimal enzyme activity was at pH 6.0-6.2 and 45-50 degrees C. Our results suggested that Xa-S2 is a new amylase with a minimal catalytic domain for hydrolyzing substrates with of alpha-1,4-glucosidic bonds.

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Section: Resultsmentioning

confidence: 99%

A mutant α-amylase with only part of the catalytic domain and its structural implication

Tang

Mao

et al. 2006

Biotechnol Lett

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“…The target protein on the membrane was visualized by incubating with a Western Lightening® Chemiluminescence Reagent Plus (PerkinElmer) mixture and detected by use of the LAS-3000 mini (Fujifilm) image reader. Rabbit antisera against α-amylase, XpsE, XpsL and XpsD were obtained from previous studies [17], [41]–[43]. Rabbit antiserum against GFP was purchased from Invitrogen.…”

Section: Methodsmentioning

confidence: 99%

Function-Related Positioning of the Type II Secretion ATPase of Xanthomonas campestris pv. campestris

Chen

2013

PLoS ONE

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Gram-negative bacteria use the type II secretion (T2S) system to secrete exoproteins for attacking animal or plant cells or to obtain nutrients from the environment. The system is unique in helping folded proteins traverse the outer membrane. The secretion machine comprises multiple proteins spanning the cell envelope and a cytoplasmic ATPase. Activity of the ATPase, when copurified with the cytoplasmic domain of an interactive ATPase partner, is stimulated by an acidic phospholipid, suggesting the membrane-associated ATPase is actively engaged in secretion. How the stimulated ATPase activity is terminated when secretion is complete is unclear. We fused the T2S ATPase of Xanthomonas campestris pv. campestris, the causal agent of black rot in the crucifers, with fluorescent protein and found that the ATPase in secretion-proficient cells was mainly diffused in cytoplasm. Focal spots at the cell periphery were detectable only in a few cells. The discrete foci were augmented in abundance and intensity when the secretion channel was depleted and the exoprotein overproduced. The foci abundance was inversely related to secretion efficiency of the secretion channel. Restored function of the secretion channel paralleled reduced ATPase foci abundance. The ATPase foci colocalized with the secretion channel. The ATPase may be transiently associated with the T2S machine by alternating between a cytoplasmic and a machine-associated state in a secretion-dependent manner. This provides a logical means for terminating the ATPase activity when secretion is completed. Function-related dynamic assembly may be the essence of the T2S machine.

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“…X. campestris is also known to produce a variety of enzymes including a periplasmic α-amylase the gene of which was cloned and expressed in Escherichia coli [2].…”

Section: Introductionmentioning

confidence: 99%

Heterologous Expression of a Hyperthermophilic α-Amylase in Xanthan Gum Producing Xanthomonas campestris Cells

Konsoula

Liakopoulou-Kyriakides

Perysinakis

et al. 2008

Appl Biochem Biotechnol

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A hyperthermophilic alpha-amylase encoding gene from Pyrococcus woesei was transferred and expressed in Xanthomonas campestris ATCC 13951. The heterologous alpha-amylase activity was detected in the intracellular fraction of X. campestris and presented similar thermostability and catalytic properties with the native P. woesei enzyme. The recombinant alpha-amylase was found to be stable at 90 degrees C for 4 h and within the same period it retained more than 50% of its initial activity at 110 degrees C. Furthermore, X. campestris transformants produced similar levels of recombinant alpha-amylase activity regardless of the carbon source present in the growth medium, whereas the native X. campestris alpha-amylase production was highly dependent on starch availability and it was suppressed in the presence of glucose or other reducing sugars. On the other hand, xanthan gum yield, which appeared to be similar for both wild type and recombinant X. campestris strains, was enhanced at higher starch or glucose concentrations. Evidence presented in this study supports that X. campestris is a promising cell factory for the co-production of recombinant hyperthermophilic alpha-amylase and xanthan gum.

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Molecular cloning, characterization and nucleotide sequence of the gene for secreted -amylase from Xanthomonas campestris pv. campestris

Cited by 28 publications

References 43 publications

A mutant α-amylase with only part of the catalytic domain and its structural implication

A mutant α-amylase with only part of the catalytic domain and its structural implication

Function-Related Positioning of the Type II Secretion ATPase of Xanthomonas campestris pv. campestris

Heterologous Expression of a Hyperthermophilic α-Amylase in Xanthan Gum Producing Xanthomonas campestris Cells

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