The α-amylase activity was measured using an α-amylase assay kit (Megazyme) with a commercial α-amylase from Aspergillus oryzae (Sigma-Aldrich) as the standard. Samples were centrifuged for 10 min at 15,000 × g at 4°C, and the supernatant was used for extracellular α-amylase quantification. For intracellular α-amylase quantification, the cell pellet was washed with distilled water and resuspended in 500 μl of phosphate-buffered saline (PBS) with 5 μl of Halt protease inhibitor cocktail (Thermo Fisher). The cell suspension was added to a lysing matrix tube, and cell lysis was performed using a FastPrep-24 tissue and cell homogenizer (MP Biomedicals) by two 60-s cycles at a speed of 6.5 m/s (samples were put on ice for 5 min between the two cycles). Cell debris was removed by centrifugation, and the supernatant fraction was used for amylase quantification.
α amylase assay kit
Manufactured by Megazyme
Sourced in Ireland
The α-amylase assay kit is a laboratory equipment designed for the quantitative determination of α-amylase activity. It provides the necessary reagents and protocols to measure the enzymatic activity of α-amylase in various sample types.
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6 protocols using α amylase assay kit
1
Quantification of Extracellular and Intracellular α-Amylase
2
Enzymatic Activity Assay of AtAMY3
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The enzymatic activity of AtAMY3 was measured using the α-Amylase Assay Kit (Ceralpha Method) from Megazyme (Megazyme, Ireland) according to the manufacturer’s instructions. Briefly, a mixture composed by an equal volume of AtAMY3 in 100 mM Tricine-NaOH, pH 7.9 and the artificial substrate blocked p-nitrophenyl maltoheptaoside (B-PNPG7) plus α-glucosidase was incubated at 40°C. After incubation, the reaction was blocked by adding 20-volume of Stopping Reagent (1% Tris, pH 11.0). The absorbance of the samples was evaluated at 400 nm using a spectrophotometer and subtracting the absorbance of blank sample treated under the same condition but without AtAMY3. An extinction coefficient at 400 nm for p-nitrophenol of 18.1 mM–1 cm–1 was used.
3
Yeast-based α-Amylase Production Protocol
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All strains and plasmids used in this study are listed in Supplementary Table 2 . Plasmids for gene overexpression were constructed by insertion of the gene fragment, which was amplified from the yeast genome then assembled with the expression vector pSPGM1 through Gibson assembly method. The standard LiAc/SS DNA/PEG method was used for yeast transformation.
For strain constructions, yeast strains were grown in SD-URA medium at 30 °C according to the auxotrophy of the cells. For α-amylase production in shake flasks, yeast strains were cultured for 96 h at 200 rpm, 30 °C with an initial OD600 of 0.05 in the SD-2×SCAA medium containing 20 g L−1 glucose, 6.9 g L−1 yeast nitrogen base without amino acids, 190 mg L−1 Arg, 400 mg L−1 Asp, 1,260 mg L−1 Glu, 130 mg L−1 Gly, 140 mg L−1 His, 290 mg L−1 Ile, 400 mg L−1 Leu, 440 mg L−1 Lys, 108 mg L−1 Met, 200 mg L−1 Phe, 220 mg L−1 Thr, 40 mg L−1 Trp, 52 mg L−1 Tyr, 380 mg L−1 Val, 1 g L−1 BSA, 5.4 g L−1 Na2HPO4 and 8.56 g L−1 NaH2PO4·H2O (pH = 6.0)42 (link).
The α-amylase activity was measured using the α-amylase assay kit (Megazyme) with a commercial α-amylase from Aspergillus oryzae (Sigma-Aldrich) as the standard. Samples were centrifuged for 10 min at 15,000 g, 4 °C and the supernatant was used for extracellular α-amylase quantification.
For strain constructions, yeast strains were grown in SD-URA medium at 30 °C according to the auxotrophy of the cells. For α-amylase production in shake flasks, yeast strains were cultured for 96 h at 200 rpm, 30 °C with an initial OD600 of 0.05 in the SD-2×SCAA medium containing 20 g L−1 glucose, 6.9 g L−1 yeast nitrogen base without amino acids, 190 mg L−1 Arg, 400 mg L−1 Asp, 1,260 mg L−1 Glu, 130 mg L−1 Gly, 140 mg L−1 His, 290 mg L−1 Ile, 400 mg L−1 Leu, 440 mg L−1 Lys, 108 mg L−1 Met, 200 mg L−1 Phe, 220 mg L−1 Thr, 40 mg L−1 Trp, 52 mg L−1 Tyr, 380 mg L−1 Val, 1 g L−1 BSA, 5.4 g L−1 Na2HPO4 and 8.56 g L−1 NaH2PO4·H2O (pH = 6.0)42 (link).
The α-amylase activity was measured using the α-amylase assay kit (Megazyme) with a commercial α-amylase from Aspergillus oryzae (Sigma-Aldrich) as the standard. Samples were centrifuged for 10 min at 15,000 g, 4 °C and the supernatant was used for extracellular α-amylase quantification.
4
Quantification of Intracellular and Extracellular Proteins
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For protein quantification, 500 µL cell cultures were centrifuged at 12 000 × g for 1 min for separation of the supernatant and cell pellet. The supernatant was used for extracellular protein measurement. For intracellular protein quantification, the cell pellet was washed with distilled water and resuspended in equal phosphate‐buffered saline (PBS). The cell resuspension was added to a lysing matrix tube for cell lysis and run on a cell homogenizer (Allsheng, Bioprep‐24R) at 6.5 m s−1 for 2 min, then cell debris was removed by centrifugation.[46 ]α‐Amylase activity was measured by a α‐amylase assay kit (Megazyme) and commercial α‐amylase (Sigma‐Aldrich, 69.6 U mg−1) from Aspergillus oryzae was used for quantification as a standard.[60 ] Lipase activity was measured based on a pNP‐palmitate hydrolysis method.[61 ]
5
Plasmid-based overexpression and CRISPR-mediated gene deletion
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PCR-amplified native candidate genes were cloned into plasmid p427-TEF between SpeI and SalI and transformed into the background strain for plasmid-based over-expression. Deletion strains were constructed by golden gate assembly of annealed oligos with gRNA sequences targeting the start and end position of the target gene, into sgRNA expression vector pWS082. The assembled plasmid and Cas9 expression vector pWS173 were linearized using EcoRV or BsmBI and co-transformed with annealed repair fragments, consisting of the joined 60 bp flanking regions of each target gene, which upon successful homology-directed repair, resulted in the deletion of the target gene. The insertion or deletion of the target gene was confirmed by Sanger sequencing. The amount of secreted amylase was measured in cultures grown in YPAD medium after 4, 24 and 48 hours using the Ceralpha method using α-amylase assay kits from Megazyme, Ireland. The relative amount of secreted α-amylase per cell was calculated by dividing the measured amount of secreted α-amylase by the optical density (OD600) as measured during the point of sampling. Baseline expression was measured using the transformed empty vector, or the background strain without gene deletion, for overexpression and deletion strains respectively.
6
Plasmid-based overexpression and CRISPR-mediated gene deletion
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