Construction and application of recombinant strain for the production of an alkaline protease from Bacillus licheniformis

doi:10.1016/j.jbiosc.2014.08.002

Journal of Bioscience and Bioengineering

Volume 119, Issue 3, March 2015, Pages 284-288

https://doi.org/10.1016/j.jbiosc.2014.08.002 Get rights and content

The alkaline protease gene, Apr, from Bacillus licheniformis 2709 was cloned into an expression vector pET – 28b (+), to yield the recombinant plasmid pET-28b (+) – Apr. The pET-28b (+) – Apr was expressed in a high expression strain E. coli BL21. The amino acid sequence deduced from the DNA sequence analysis revealed a 98% identity to that of Bacillus licheniformis 2709. Sodium salt-Polyacrylamide gel electrophoresis (SDS-PAGE) was used to access the protein expression. SDS-PAGE analysis indicated a protein of Mr of 38.8 kDa. The medium components and condition of incubation were optimized for the growth state of a recombinant strain. The optimal composition of production medium was composed of glucose 8 g/L, peptone 8 g/L and salt solution 10 mL. The samples were incubated on a rotary shaker of 180 r/min at 37°C for 24 h.

Section snippets

Materials and instruments

B. licheniformis strain 2709 was purchased from the China General Microbiological Culture Collection Center. The genome of B. licheniformis 2709 was used as template to obtain the Apr gene using polymerase chain reaction (PCR) technology. The pET-28b (+) vectors which were modified as expression vector carry an N-terminal His·Tag/thrombin/T7·Tag configuration plus an optional C-terminal His·Tag sequence. Bacterial strains and plasmids used in the study are listed in Table 1. Escherichia coli

The result of PCR and SDS-PAGE analysis

The PCR amplification about 1110 bp fragment from the recombinant strain is shown in Fig. 1A. The construction sketch of expression vector pET – 28b (+) – Apr is shown in Fig. 1B. SDS-PAGE shows the expression level of the strain with the pET – 28b (+) – Apr plasmid while the control group only contained pET-28b (+) in the strain (Fig. 1C). It indicates that the molecular weight of the expressed recombinant was estimated to be 38.8 kDa, which corresponds with the prediction by gene sequencing.

Acknowledgments

The authors acknowledge the financial support provided by the Key Projects in the National Science & Technology Pillar Program during the Twelfth Five-Year Plan Period (2012BAD33B03) and the Youth Scientific Innovation Leading Talent and Team Building Project of Jilin Province (20140519014JH).

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Citation Excerpt :
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The biocorrosion of magnesium in the external physiological environment is still difficult to accurately evaluate the degradation behavior in vivo, particularly, in the microenvironment of the patients with hyperglycemia or diabetes. Thus, we explored the synergistic effects of glucose and protein on the biodegradation of pure magnesium, so as to have a deeper understanding the mechanism of the degradation in vivo. The surface morphology and corrosion product composition of pure magnesium were investigated using SEM, EDS, FTIR, XRD and XPS. The effect of glucose and albumin on the degradation rate of pure magnesium was investigated via electrochemical and immersion tests. The adsorption of glucose and albumin on the sample surface was observed using fluorescence microscopy. The results showed that the presence of 2 g/L glucose changed the micromorphology of corrosion products on the magnesium surface by reacting with metal cations, thus inhibiting the corrosion of pure magnesium. Protein formed a barrier layer to protect the magnesium at early stage of immersion. The chelation reaction between protein and magnesium surface might accelerate the degradation at later stage. There may be a critical glucose (albumin) content. Biodegradation of pure magnesium was inhibited at low concentrations and promoted at high concentrations. The synergistic effect of glucose and protein restrained the adsorption of aggressive chloride ions to a certain extent, and thus inhibited the degradation of pure magnesium considerably. Moreover, XPS results indicated that glucose promoted the adsorption of protein on the sample surface.
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Citation Excerpt :
The proteases are highly valuable commercial enzymes as various applications in food, pharmaceutical, detergent and dairy industries (Gupta et al., 2002; Vellard, 2003; Reddy et al., 2006; Ribitscha et al., 2010; Bhunia and Dey, 2012; Bhunia et al., 2013). Several Bacillus sp. like B. cereus (Prakash et al., 2014), Bacillus licheniformis (Lin et al., 2015), Bacillus megaterium (Asker et al., 2013), B. sterothermophilus (Sookkheo et al., 2000), Bacillus mojavensis (Haddar et al., 2009), Bacillus pumilus (Jaouadi et al., 2008) and B. subtilis (Maruthiah et al., 2013) were involved in production of a wide variety of extracellular enzymes, including proteases. The molecular docking approach can be used to model the interaction between the molecule and a protein at the atomic level, which allow us to characterize the behavior of molecules in the binding site of target proteins as well as to elucidate fundamental biochemical processes (Marrone et al., 1997; Huang and Zou, 2010; Ezat et al., 2014).
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Biotechnological applications of Bacillus licheniformis
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Section snippets

Materials and instruments

The result of PCR and SDS-PAGE analysis

Acknowledgments

References (29)

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Curr. Opin. Biotechnol.

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Enzyme Microb. Technol.

De-repression and subsequent induction of protease synthesis by Bacillus mojavensis under fedbatch operations

Process Biochem.

Expression of recombinant Thermomonospora fusca xylanase A in Pichia pastoris and xylooligosaccharides released from xylans by it

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In vitro degradation of pure magnesium―the synergetic influences of glucose and albumin

Molecular modeling and docking of protease from Bacillus sp. for the keratin degradation

Microbial proteases and their applications

Biotechnological applications of Bacillus licheniformis

Cloning, Heterologous Expression and Characterization of an Intracellular Serine Protease from Bacillus sp. LCB10