Expressing antimicrobial peptide cathelicidin-BF in Bacillus subtilis using SUMO technology

Small ubiquitin-related modifier (SUMO) technology has been widely used in Escherichia coli expression systems to produce antimicrobial peptides. However, E. coli is a pathogenic bacterium that produces endotoxins and can secrete proteins into the periplasm, forming inclusion bodies. In our work, cathelicidin-BF (CBF), an antimicrobial peptide purified from Bungarus fasciatus venom, was produced in a Bacillus subtilis expression system using SUMO technology. The chimeric genes his-SUMO-CBF and his-SUMO protease 1 were ligated into vector pHT43 and expressed in B. subtilis WB800N. Approximately 22 mg of recombinant fusion protein SUMO-CBF and 1 mg of SUMO protease 1 were purified per liter of culture supernatant. Purified SUMO protease 1 was highly active and cleaved his-SUMO-CBF with an enzyme-to-substrate ratio of 1:40. Following cleavage, recombinant CBF was further purified by affinity and cation exchange chromatography. Peptide yields of ~3 mg/l endotoxin-free CBF were achieved, and the peptide demonstrated antimicrobial activity. This is the first report of the production of an endotoxin-free antimicrobial peptide, CBF, by recombinant DNA technology, as well as the first time purified SUMO protease 1 with high activity has been produced from B. subtilis. This work has expanded the application of SUMO fusion technology and may represent a safe and efficient way to generate peptides and proteins in B. subtilis.     

Novel extremely acidic lipases produced from Bacillus species using oil substrates

The extremely acidophilic microorganisms Bacillus pumilus and Bacillus subtilis were isolated from soil collected from the commercial edible oil and fish oil extraction industry. Optimization of conditions for acidic lipase production from B. pumilus and B. subtilis using palm oil and fish oil, respectively, was carried out using response surface methodology. The extremely acidic lipases, thermo-tolerant acidic lipase (TAL) and acidic lipase (AL), were produced by B. pumilus and B. subtilis, respectively. The optimum conditions for B. pumilus obtaining the maximum activity (1,100 U/mL) of TAL were fermentation time, 96 h; pH, 1; temperature, 50 °C; concentration of palm oil, 50 g/L. After purification, a 7.1-fold purity of lipase with specific activity of 5,173 U/mg protein was obtained. The molecular weight of the TAL was 55 kDa. The AL from B. subtilis activity was 214 U/mL at a fermentation time of 72 h; pH, 1; temperature, 35 °C; concentration of fish oil, 30 g/L; maltose concentration, 10 g/L. After purification, an 11.4-fold purity of lipase with specific activity of 2,189 U/mg protein was obtained. The molecular weight of the extremely acidic lipase was 22 kDa. The functional groups of lipases were determined by Fourier transform-infrared (FT-IR) spectroscopy.     

Expression, purification, and biochemical properties of arginase from Bacillus subtilis 168

The arginine-degrading and ornithine-producing enzymes arginase has been used to treat arginine-dependent cancers. This study was carried out to obtain the microbial arginase from Bacillus subtilis, one of major microorganisms found in fermented foods such as Cheonggukjang. The gene encoding arginase was isolated from B. subtilis 168 and cloned into E. coli expression plasmid pET32a. The enzyme activity was detected in the supernatant of the transformed and IPTG induced cell-extract. Arginase was purified for homogeneity from the supernatant by affinity chromatography. The specific activity of the purified arginase was 150 U/mg protein. SDS-PAGE analysis revealed the molecular size to be 49 kDa (Trix·Tag, 6×His·Tag added size). The optimum pH and temperature of the purified enzyme with arginine as the substrate were pH 8.4 and 45°C, respectively. The K_m and V_max values of arginine for the enzyme were 4.6 mM and 133.0 mM/min/mg protein respectively. These findings can contribute in the development of functional fermented foods such as Cheonggukjang with an enhanced level of ornithine and pharmaceutical products by providing the key enzyme in arginine-degradation and ornithine-production.     

Cloning, sequencing, and expression of a β-1,4-endoxylanase gene from Indonesian Bacillus licheniformis strain I5 in Escherichia coli

The gene encoding an endo-β-1,4-xylanase from an Indonesian indigenous Bacillus licheniformis strain I5 was amplified using PCR, cloned, and expressed in Escherichia coli. The nucleotide sequence of a 642 bp DNA fragment was determined, revealing one open reading frame that encoded a xylanase. Based on the nucleotide sequence, calculated molecular mass of the enzyme was 23 kDa. This xylanase has a predicted typical putative signal peptide; however, in E. coli, the active protein was located mainly in intracellular form. Neither culture supernatant of recombinant E. coli nor periplasmic fraction has significantly detectable xylanase activity. The deduced amino acid of the gene has 91% identity with that of Bacillus subtilis endoxylanase. Optimal activity of the recombinant enzyme was at pH 7 and 50°C     

Design,characterization and expression of a novel hybrid peptides melittin (1–13)-LL37 (17–30)

Hybridizing of different antimicrobial peptides (AMPs) has been a common practice for obtaining novel hybrid AMPs with elevated antibacterial activity but minimized cytotoxicity. The hybrid peptides melittin (1-13)-LL37 (17-30) (M–L) combining the hydrophobic N-teriminal fragment of melittin (M) with the core antibacterial fragment of LL37 (L), was designed for the first time to explore its antibacterial activity and hemolytic activity against bacteria and sheep erythrocyte respectively. Results showed that M–L had an even more potent antibacterial activity against all indicator strains (especially gram-positive bacteria) than M and L, whereas didn’t exhibit hemolytic activity to sheep erythrocytes, implying M–L can be served as a potential therapeutic drug to substitute traditional antibiotics. However the high expense of biosynthesis limited its further research, therefore fusion expression of M–L was carried out in Escherichia coli (E. coli) for overproducing the hybrid peptide so as to solve the problem. The DNA sequence encoding M–L with preferred codons was cloned into the pET-SUMO vector for protein expression in E. coli BL21 (DE3). After IPTG induction, approximately 165 mg soluble fusion protein SUMO-M–L was recovered per liter supernatant of the fermentation ultrasonic lysate using Ni–NTA Sepharose column (92 % purity). And 23 mg recombinant M–L was obtained per liter culture after cleavage of SUMO protease and purification of Ni–NTA Sepharose column. In sum, this research not only supplied an effective approach for overproducing hybrid peptide M–L, but paved the way for its further exploration on pharmaceutical potential and medical importance.     

Overexpression and Biochemical Characterization of a Thermostable Phytase from Bacillus subtilis US417 in Pichia pastoris

The overexpression of the native gene encoding the thermostable Bacillus subtilis US417 phytase using Pichia pastoris system is described. The phytase gene, in which the sequence encoding the signal peptide was replaced by that of the α-factor of Saccharomyces cerevisiae, was placed under the control of the methanol-inducible promoter of the alcohol oxidase 1 gene and expressed in Pichia pastoris. Small-scale expression experiments and activity assays were used to screen positive colonies. A recombinant strain was selected and produces 43 and 227 U/mL of phytase activity in shake flasks and in high-cell-density fermentation, respectively. The purified phytase was glycosylated protein and varied in size (50–65 kDa). It has a molecular mass of 43 kDa when it was deglycosylated. The purified r-PHY maintains 100 % of its activity after 10 min incubation at 75 °C and pH 7.5. This thermostable phytase, which is also active over broad pH ranges, may be useful as feed additives, since it can resist the temperature used in the feed-pelleting process.     

A hydrolytic ��-glutamyl transpeptidase from thermo-acidophilic archaeon Picrophilus torridus: binding pocket mutagenesis and transpeptidation

??-Glutamyl transpeptidase of a thermo-acidophilic archaeon Picrophilus torridus was cloned and expressed using E. coli Rosetta-pET 51b(+) expression system. The enzyme was expressed at 37 °C/200 rpm with ??-GT production of 1.99 U/mg protein after 3 h of IPTG induction. It was improved nearby 10-fold corresponding to 18.92 U/mg protein in the presence of 2 % hexadecane. The enzyme was purified by Ni²⁺-NTA with a purification fold of 3.6 and recovery of 61 %. It was synthesized as a precursor heterodimeric protein of 47 kDa with two subunits of 30 kDa and 17 kDa, respectively, as revealed by SDS-PAGE and western blot. The enzyme possesses hydrolase activity with optima at pH 7.0 and 55 °C. It was thermostable with a t _1/2 of 1 h at 50 °C and 30 min at 60 °C, and retained 100 % activity at 45 °C even after 24 h. It was inhibited by azaserine and DON and PMSF. Pt??-GT shared 37 % sequence identity and 53 % homology with an extremophile ??-GT from Thermoplasma acidophilum. Functional residues identified by in silico approaches were further validated by site-directed mutagenesis where Tyr327 mutated by Asn327 introduced significant transpeptidase activity.     

The simultaneous production of single-cell protein and a recombinant antibacterial peptide by expression of an antibacterial peptide gene in Yarrowia lipolytica

In this study, the DNA fragment encoding the N-terminus of scallop H2A was expressed in the marine-derived yeast Yarrowia lipolytica, which has a high protein content. After cultivation in PBB medium for 120 h, the transformant producing the highest amount of antibacterial peptide, 29a, was obtained. The supernatant from cultures of 29a had killing activity against Vibrio harveyi, V. anguillarum, and V. parahaemolyticus. After purification, the molecular mass of the recombinant antibacterial peptide was 4.5 kDa, and the purified recombinant antibacterial peptide was able to cause leakage of intracellular components from both whole and protoplast cells of V. parahaemolyticus. The results indicated that when the yeast transformant 29a was grown in YPD medium, PBB medium or hydrolysate of soybean meal containing ammonium sulfate, its cells still had a high protein content. Because this recombinant marine yeast both had a high protein content and produced the antibacterial peptide, it has high value-added applications.     

Cloning and expression in Escherichia coli of two DNA fragments from Bacillus sphaericus encoding mosquito-larvicidal activity

A library of Bacillus sphaericus 1593 DNA was constructed in Escherichia coli using pBR322 as vector and screened for clones expressing larvicidal activity against Culex mosquito larvae. Two larvicidal clones were identified and their plasmids characterized by restriction mapping. pAS233 and pAS377 contained inserts of 8.6 and 15 kb which were reduced by subcloning to 3.6 and 4.3 kb, respectively. A peptide of 29 kDa was the single product detected by maxicell expression of pAS377PT, a plasmid subcloned from pAS377. No insert-encoded peptide could be detected for pAS233HA, a subclone of pAS233, although maxicells containing this plasmid encoded larvicidal activity. The insert of pAS377PT was transcribed from a vector promoter whereas the insert of pAS233HA was transcribed from its own promoter and hence its expression in B. subtilis was possible. The insert was ligated to a shuttle vector yielding pSVI which was then used to transform B. subtilis. Recombinant E. coli and B. subtilis clones showed equivalent larvicidal activity of 1–10 μg cell protein per ml. Larvicidal activity was observed during vegetative growth for recombinant B. subtilis even though B. sphaericus 1593 synthesizes its mosquito-toxin only during sporulation.     

Purification and characterization of recombinant Bacillus subtilis 168 catalase using a basic polypeptide from ribosomal protein L2

An efficient purification system for purifying recombinant Bacillus subtilis 168 catalase (KatA) expressed in Escherichia coli was developed. The basic region containing 252–273 amino acids derived from E. coli ribosomal protein L2 was used as an affinity tag while the small ubiquitin-like modifier (SUMO) was introduced as one specific protease cleavage site between the target protein and the purification tags. L2 (252–273)–SUMO fusion protein purification method can be effectively applied to purify the recombinant catalase using cation exchange resin. This purification procedure was used to purify the KatA and achieved a purification fold of 30.5, a specific activity of 48,227.2 U/mg and an activity recovery of 74.5%. The enzyme showed a Soret peak at 407 nm. The enzyme kept its activity between pH 5 and 10 and between 30 °C and 60 °C, with the highest activity at pH 8.0 and 37 °C. The enzyme displayed an apparent Km of 39.08 mM for hydrogen peroxide. These results agree well with the previous reports about B. subtilis catalase. L2 (252–273)–SUMO fusion protein purification technique provides a novel and effective fusion expression system for the production of recombinant proteins.     

Cloning, Expression, and Characterization of Iron Superoxide Dismutase in Sonneratia alba, a Highly Salt Tolerant Mangrove Tree

In this study, a novel iron superoxide dismutase (FeSOD) gene from Sonneratia alba was cloned and then expressed in Escherichia coli Rosetta-gami, designated as SaFeSOD. The DNA sequence of SaFeSOD contained a 786-bp open reading frame which encodes a 261 amino-acid protein of 30.0 kDa. The 651-bp fragment coding for putative mature SaFeSOD was amplified and inserted into pET15b for expression. This recombinant SaFeSOD was subsequently isolated by Ni-trap column protein purification system. The apparent molecular mass of the purified enzyme was 25 kDa on SDS-PAGE. In comparison with FeSODs from other plant species, all iron-binding sites (His 27, His 80, Asp 164 and His 168) of SaFeSOD were conserved. SaFeSOD was found to have good pH stability in the pH range of 3.5–9.5 at 25 °C after 1 h incubation and was relatively stable and showed 78 % activity when incubated in 50 °C for 1 h. Quantitative real-time PCR experiments demonstrated that SaFeSOD was expressed in leaf, stem, flower, fruit and root tissues with the highest expression in leaf tissues.     

Cloning, Expression, and Characterization of an GHF 11 Xylanase from Aspergillus niger XZ-3S

A xylanase gene (xynZF-2) from the Aspergillus niger XZ-3S was cloned and expressed in Escherichia coli. The coding region of the gene was separated by only one intron with the 68 bp in length. It encoded 225 amino acid residues of a protein with a calculated molecular weight of 24.04 kDa plus a signal peptide of 18 amino acids. The amino acid sequence of the xynZF-2 gene had a high similarity with those of family 11 of glycosyl hydrolases reported from other microorganisms. The mature peptide encoding cDNA was subcloned into pET-28a(+) expression vector. The resultant recombinant plasmid pET-28a-xynZF-2 was transformed into E. coli BL21(DE3), and finally the recombinant strain BL21/xynZF-2 was obtained. A maximum activity of 42.33 U/mg was gained from cellular of E. coli BL21/xynZF-2 induced by IPTG. The optimum temperature and pH for recombinant enzyme which has a good stability in alkaline conditions were 40 °C and 5.0, respectively. Fe³⁺ had an active effect on the enzyme obviously.     

Trypsin Hydrolysed Protein Fractions as Radical Scavengers and Anti-bacterial Agents from <Emphasis Type="Italic">Ficus deltoidea</Emphasis>

Different molecular sizes of protein hydrolysates were prepared from the crude protein extract of Ficus deltoidea using the technique of membrane ultrafiltration after trypsin hydrolysis. Gel electrophoretic images shows the presence of 12, 8, 7 and 7 protein bands for the protein fractions prepared from the molecular weight cut-off of 3, 10, 30 and 100 kDa, respectively. The protein hydrolysates were found to have higher radical scavenging activity than those unhydrolysed fractions at the similar molecular size. They exhibited significant differences in the radical scavenging activities based on one-way analysis of variance, except for the protein hydrolysates of 30 and 100 kDa. The smallest protein hydrolysates, 3 kDa appeared to have the comparable activity (30%) with bovine serum albumin as a positive control in this study. Similarly, the 3 kDa protein hydrolysates achieved the highest inhibitory activity (87.5%) against Pseudomonas aeruginosa at the concentration of 128 µg/mL. The protein hydrolysates were found to be more effective against gram negative bacteria (P. aeruginosa and Escherichia coli) because of lower minimum inhibitory concentration (MIC) and effective inhibitory concentration at 50% (EC₅₀) than gram positive bacterium (Staphylococcus aureus). Trypsin catalysed hydrolysis seemed to improve the anti-bacterial activity of protein hydrolysates in a bacterial strain dependent manner. The MIC could achieve 1–55 µg/mL at different molecular sizes of protein fractions. Mass spectra matching revealed that 26% of 226 identified proteins belonged to the category of plant defensive proteins in stress management and metal handling.     

Surface Display of Bacterial Laccase CotA on <Emphasis Type="Italic">Escherichia coli</Emphasis> Cells and its Application in Industrial Dye Decolorization

Laccase CotA from Bacillus subtilis 168 was successfully displayed on the membrane of Escherichia coli cells using poly-γ-glutamate synthetase A protein (PgsA) from B. subtilis as an anchoring matrix. Further analyses demonstrated that the fusion protein PgsA/CotA efficiently translocates to the cell surface of E. coli with an enzymatic activity of 65 U/10⁸ cells. Surface-displayed CotA was shown to possess improved enzymatic properties compared with those of the wild-type CotA, including higher thermal stability (above 90% activity at 70 °C and nearly 40% activity at 90 °C after 5-h incubation) and stronger inhibitor tolerance (approximately 80 and 65% activity when incubated with 200 and 400 mM NaCl, respectively). Furthermore, the whole-cell system was demonstrated to have high enzymatic activity against anthraquinone dye, Acid Blue 62, triphenylmethane dye, Malachite Green, and azo dye, Methyl Orange with the decolorization percentages of 91, 45, and 75%, after 5-h incubation, respectively.     

Purification and characterization of YxeI, a penicillin acylase from Bacillus subtilis

The paper reports the purification and characterization of the first penicillin acylase from Bacillus subtilis. YxeI, the protein annotated as hypothetical, coded by the gene yxeI in the open reading frame between iol and hut operons in B. subtilis was cloned and expressed in Eshcherichia coli, purified and characterized. The purified protein showed measurable penicillin acylase activity with penicillin V. The enzyme was a homotetramer of 148 kDa. The apparent K_m of the enzyme for penicillin V and the synthetic substrate 2-nitro-5-(phenoxyacetamido)-benzoic acid was 40 mM and 0.63 mM, respectively, and the association constants were 8.93 × 10² M⁻¹ and 2.51 × 10⁵ M⁻¹, respectively. It was inhibited by cephalosporins and conjugated bile salts, substrates of the closely related bile acid hydrolases. It had good sequence homology with other penicillin V acylases and conjugated bile acid hydrolases, members of the Ntn hydrolase family. The N-terminal nucleophile was a cysteine which is revealed by a simple removal of N-formyl-methionine. The activity of the protein was affected by high temperature, acidic pH and the presence of the denaturant guanidine hydrochloride.     

Cloning of a Family 11 Xylanase Gene from Bacillus amyloliquefaciens CH51 Isolated from Cheonggukjang

Bacillus amyloliquefaciens CH51, an isolate from cheonggukjang, Korean fermented soyfood, secretes several enzymes into culture medium. A gene encoding 19 kDa xylanase was cloned by PCR. Sequencing showed that the gene encoded a glycohydrolase family 11 xylanase and named xynA. xynAHis, xynA with additional codons for his-tag, was overexpressed in Escherichia coli BL21(DE3) using pET-26b(+). XynAHis was purified using HisTrap affinity column. K_m and V_max of XynAHis were 0.363 mg/ml and 701.1 μmol/min/mg, respectively with birchwood xylan as a substrate. The optimum pH and temperature were pH 4 and 25 °C, respectively. When xynA was introduced into Bacillus subtilis WB600, active XynA was secreted into culture medium.     

Cloning, Large Scale Over-Expression in E. coli and Purification of the Components of the Human LAT 1 (SLC7A5) Amino Acid Transporter

The high yield expression of the human LAT1 transporter has been obtained for the first time using E. coli. The hLAT1 cDNA was amplified from HEK293 cells and cloned in pH6EX3 vector. The construct pH6EX3-6His-hLAT1 was used to express the 6His-hLAT1 protein in the Rosetta(DE3)pLysS strain of E. coli. The highest level of expression was detected 8 h after induction by IPTG at 28 °C. The expressed protein was collected in the insoluble fraction of cell lysate. On SDS-PAGE the apparent molecular mass of the polypeptide was 40 kDa. After solubilization with sarkosyl and denaturation with urea the protein carrying a 6His N-terminal tag was purified by Ni²⁺-chelating affinity chromatography and identified by anti-His antibody. The yield of the over-expressed protein after purification was 3.5 mg/L (cell culture). The human CD98 cDNA amplified from Imagene plasmid was cloned in pGEX-4T1. The construct pGEX-4T1-hCD98 was used to express the GST-hCD98 protein in the Rosetta(DE3)pLysS strain of E. coli. The highest level of expression was detected in this case 4 h after induction by IPTG at 28 °C. The expressed protein was accumulated in the soluble fraction of cell lysate. The molecular mass was determined on the basis of marker proteins on SDS-PAGE; it was about 110 kDa. GST was cleaved from the protein construct by incubation with thrombin for 12 h and the hCD98 was separated by Sephadex G-200 chromatography (size exclusion). hCD98 showed a 62 kDa apparent molecular mass, as determined on the basis of molecular mass markers using SDS-PAGE. The yield of CD98 was 2 mg/L of cell culture.     

Molecular characterization and primary functional analysis of PeVDE, a violaxanthin de-epoxidase gene from bamboo (Phyllostachys edulis)

Key message

PeVDE was expressed primarily in bamboo leaves, which was up-regulated under high light. The protein encoded by PeVDE had enzyme activity of catalyzing violaxanthin (V) to zeaxanthin (Z) through antheraxanthin (A) as assay shown in vitro.

Abstract

Violaxanthin de-epoxidase (VDE), a key enzyme of xanthophyll cycle, catalyzes conversion from violaxanthin (V) to zeaxanthin (Z) through antheraxanthin (A) to protect photosynthesis apparatus. A cDNA, PeVDE, encoding a VDE was isolated from bamboo (Phyllostachys edulis) by RT-PCR and RACE methods. PeVDE is 1,723 bp and contains an ORF encoding 451 amino acids, with a transit peptide of 103 amino acids. The mature protein is deduced to have 348 amino acids with a calculated molecular weight of 39.6 kDa and a theoretic isoelectric point of 4.5. Semi-quantitative RT-PCR assay indicated that the highest expression level of PeVDE was in leaf, which agreed with the accumulation pattern of PeVDE protein. Real time PCR results showed that PeVDE was up-regulated and reached the highest level after the treatment (1,200 μmo1 m^?2 s^?1) for 2 h, then decreased and kept at the level similar to that of 0.5 h after treatment for 8 h. To investigate the function of PeVDE, mature protein was heterologously expressed in Escherichia coli and the enzymatic activity assay was carried out using V as substrate. The pigments that formed in the reaction mixture were extracted and analyzed by HPLC method. Besides V, A and Z were detected in the reaction mixture, which indicated that the recombinant protein exhibited enzymatic activity of catalyzing V into Z through A. This study indicates that PeVDE functions through regulating the components of xanthophyll cycle, which might be one of the critical factors that contribute to the growth of bamboo under naturally varying light conditions.