Cloning, mutagenesis, and structural analysis of human pancreatic α‐amylase expressed in pichia pastoris

The complex plant flavonol glycoside montbretin A is a potent (Ki = 8 nM) and specific inhibitor of human pancreatic α-amylase with potential as a therapeutic for diabetes and obesity. Controlled degradation studies on montbretin A, coupled with inhibition analyses, identified an essential high-affinity core structure comprising the myricetin and caffeic acid moieties linked via a disaccharide. X-ray structural analyses of the montbretin A-human α-amylase complex confirmed the importance of this core structure and revealed a novel mode of glycosidase inhibition wherein internal π-stacking interactions between the myricetin and caffeic acid organize their ring hydroxyls for optimal hydrogen bonding to the α-amylase catalytic residues D197 and E233. This novel inhibitory motif can be reproduced in a greatly simplified analog, offering potential for new strategies for glycosidase inhibition and therapeutic development.

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“…Methods for the construction of expression vectors for wild-type HPA have been described previously 22,24 .…”

Section: Methodsmentioning

confidence: 99%

The amylase inhibitor montbretin A reveals a new glycosidase inhibition motif

et al. 2015

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“…The methylotrophic yeast P. pastoris was chosen as the expression system for RoAmy because of its high expression level of recombinant proteins and its capacity for functional posttranslational modification of recombinant proteins. Pichia has been used to express a wide range of heterologous proteins [15], including a number of α-amylases [16][17][18]. To the best of our knowledge, this is the first report of a purified recombinant R. oryzae α-amylase that degrades starch to maltose as the main end product.…”

mentioning

confidence: 99%

Gene Cloning, Heterologous Expression, and Characterization of a High Maltose-Producing α-Amylase of Rhizopus oryzae

Song

Zuo

Niu

et al. 2011

Appl Biochem Biotechnol

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A putative α-amylase gene, designated as RoAmy, was cloned from Rhizopus oryzae. The deduced amino acid sequence showed the highest (42.8%) similarity to the α-amylase from Trichoderma viride. The RoAmy gene was successfully expressed in Pichia pastoris GS115 under the induction of methanol. The molecular weight of the purified RoAmy determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis was approximately 48 kDa. The optimal pH and temperature were 4-6 and 60 °C, respectively. The enzyme was stable at pH ranges of 4.5-6.5 and temperatures below 50 °C. Purified RoAmy had a K(m) and V(max) of 0.27 mg/ml and 0.068 mg/min, respectively, with a specific activity of 1,123 U/mg on soluble starch. Amylase activity was strongly inhibited by 5 mM Cu(2+) and 5 mM Fe(2+), whereas 5 mM Ca(2+) showed no significant effect. The RoAmy hydrolytic activity was the highest on wheat starch but showed only 55% activity on amylopectin relative to soluble corn starch, while the pullulanase activity was negligible. The main end products of the polysaccharides tested were glucose and maltose. Maltose reached a concentration of 74% (w/w) with potato starch as the substrate. The enzyme had an extremely high affinity (K(m) = 0.22 mM) to maltotriose. A high ratio of glucose/maltose of 1:4 was obtained when maltotriose was used at an initial concentration of 40 mM.

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“…Thus, we are confident that glycosylation does not strongly affect the results. This has been also found on human recombinant amylase produced in P. pastoris (Rydberg et al, 1999). Table 1 gives the kinetic parameters for the hydrolysis of the substrates tested.…”

Section: Degradation Products Produced By α-Amylase From D Melanogasmentioning

confidence: 56%

Enzymatic characterization of recombinant α-amylase in the <i>Drosophila melanogaster</i> species subgroup: is there an effect of specialization on digestive enzyme?

et al. 2013

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We performed a comparative study on the enzymological features of purified recombinant α-amylase of three species belonging to the Drosophila melanogaster species subgroup: D. melanogaster, D. erecta and D. sechellia. D. erecta and D. sechellia are specialist species, with host plant Pandanus candelabrum (Pandanaceae) and Morinda citrifolia (Rubiaceae), respectively. The temperature optima were around 57-60°C for the three species. The pH optima were 7.2 for D. melanogaster, 8.2 for D. erecta and 8.5 for D. sechellia. The k cat and K m were also estimated for each species with different substrates. The specialist species D. erecta and D. sechellia display a higher affinity for starch than D. melanogaster. α-Amylase activity is higher on starch than on glycogen in all species. α-Amylases of D. erecta and D. sechellia have a higher activity on maltooligosaccharides (G6 and G7) than on starch, contrary to D. melanogaster. Such differences in the enzymological features between the species might reflect adaptation to different ecological niches and feeding habits.

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Cloning, mutagenesis, and structural analysis of human pancreatic α‐amylase expressed in pichia pastoris

Cited by 56 publications

References 53 publications

The amylase inhibitor montbretin A reveals a new glycosidase inhibition motif

The amylase inhibitor montbretin A reveals a new glycosidase inhibition motif

Gene Cloning, Heterologous Expression, and Characterization of a High Maltose-Producing α-Amylase of Rhizopus oryzae

Enzymatic characterization of recombinant α-amylase in the <i>Drosophila melanogaster</i> species subgroup: is there an effect of specialization on digestive enzyme?

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