Computational protocol: A novel carboxyl-terminal protease derived from Paenibacillus lautus CHN26 exhibiting high activities at multiple sites of substrates

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Protocol publication

[…] Sediment samples were collected from a few aquaculture ponds of fishery farms located in Shanghai, China. Samples were homogenized in nine volumes of a sterilized PBS solution buffer (pH 7.4) [], and microbial cells in supernatant were collected as previously described by Zhang et al. []. Serial dilutions were made and spread onto the selective skim milk agar plates, which were incubated at 30°C for 48 h. Single colonies were scored positive for protease activity using a skim milk assay in which the isolates form clear zone around their colonies resulting from skim milk hydrolysis are scored as positive in protease activity. Conventional phenotypic and biochemical characterizations were carried out for new isolates according to the Bergey’s manual of determinative bacteriology [] using the microscope (model 36XV, Shanghai Wanheng Precision Instrument Co. Ltd, Shanghai, China) and the bacterial biochemical test kits (Hangzhou Tianhe Microorganism Reagent Co. Ltd., Hangzhou, China). The 16S rRNA gene from the isolate was amplified and sequenced using the bacterial universal primers 27 F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R (5’-TACCTTGTTACGACTT-3’) [].Automated DNA sequencing was carried out using ABI3730XL sequencer (Applied Bio-systems, USA) and BigDye Terminator version 3.1 kit at the China Human Genome Center (Shanghai, China). The sequences were analyzed by the program Bioedit (Version 7.0.9, and the Basic Local Alignment Search Tool (BLAST) ( Multiple sequence alignments were performed using the ClustalW2 software ( []. The neighbor-joining method in the molecular evolutionary genetic analysis software package MEGA (version 4.0) [] was used to construct a phylogenetic tree. A bootstrap analysis with 1000 replicates was carried out to check the reliability of the tree. Signal peptide sequences were identified using the SignalP-NN Version 4.0 Server []. Oligonucleotide primers were synthesized by Shanghai Sangon Biological Engineering Technology Services Co., Ltd. (Shanghai, China). […]

Pipeline specifications

Software tools BioEdit, BLASTN, Clustal W, MEGA, SignalP
Application Phylogenetics
Organisms Escherichia coli