Graphene oxide-enhanced colorimetric detection of Mec A gene based on toehold-mediated strand displacement.

Mec A, as a representative gene mediating resistance to ß-lactam antibiotics in methicillin-resistant Staphylococcus aureus (MRSA), allows a new genetic analysis for the detection of MRSA. Here, a sensitive, prompt, and visual colorimetry is reported to detect the Mec A gene based on toehold-mediated strand displacement (TMSD) and the enrichment effect of graphene oxide (GO). The Mec A triggers to generate the profuse amount of signal units of single-stranded DNA (SG) composed of a long single-stranded base tail and a base head: the tail can be adsorbed and enriched on the surface of GO; the head can form a G quadruplex structure to exert catalytic function towards 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid). Therefore, through the enrichment effect of GO, the signal units SG reflects different degrees of signal amplification on different substrates (such as aqueous solution or filter membrane). This strategy demonstrates a broad linear working range from 100 pM to 1.5 nM (solution) and 1 pM to 1 nM (filter membrane), with a low detection limit of 39.53 pM (solution) and 333 fM (filter membrane). Analytical performance in real samples suggests that this developed colorimetry is endowed with immense potential for clinical detection applications.

A FRET based ultrasensitive fluorescent aptasensor for 6'-sialyllactose detection.

As a kind of human milk oligosaccharide, 6'-sialyllactose (6'-SL) plays an important role in promoting infant brain development and improving infant immunity. The content of 6'-SL in infant formula milk powder is thus one of the important nutritional indexes. Since the lacking of efficient and rapid detection methods for 6'-SL, it is of great significance to develop specific recognition elements and establish fast and sensitive detection methods for 6'-SL. Herein, using 6'-SL specific aptamer as the recognition element, catalytic hairpin assembly as the signal amplification technology and quantum dots as the signal label, a fluorescence biosensor based on fluorescence resonance energy transfer (FRET) was constructed for ultra-sensitive detection of 6'-SL. The detection limit of this FRET-based fluorescent biosensor is 0.3 nM, and it has some outstanding characteristics such as high signal-to-noise ratio, low time-consuming, simplicity and high efficiency in the actual sample detection. Therefore, it has broad application prospect in 6'-SL detection.

A lateral flow assay for miRNA-21 based on CRISPR/Cas13a and MnO2 nanosheets-mediated recognition and signal amplification.

The point-of-care testing (POCT) of miRNA has significant application in medical diagnosis, yet presents challenges due to their characteristics of high homology, low abundance, and short length, which hinders the achievement of quick detection with high specificity and sensitivity. In this study, a lateral flow assay based on the CRISPR/Cas13a system and MnO2 nanozyme was developed for highly sensitive detection of microRNA-21 (miR-21). The CRISPR/Cas13a cleavage system exhibits the ability to recognize the specific oligonucleotide sequence, where two-base mismatches significantly impact the cleavage activity of the Cas13a. Upon binding of the target to crRNA, the cleavage activity of Cas13a is activated, resulting in the unlocking of the sequence and initiating strand displacement, thereby enabling signal amplification to produce a new sequence P1. When applying the reaction solution to the lateral flow test strip, P1 mediates the capture of MnO2 nanosheets (MnO2 NSs) on the T zone, which catalyzes the oxidation of the pre-immobilized colorless substrate 3,3',5,5'-tetramethylbenzidine (TMB) on the T zone and generates the blue-green product (ox-TMB). The change in gray value is directly proportional to the concentration of miR-21, allowing for qualitative detection through visual inspection and quantitative measurement using ImageJ software. This method achieves the detection of miR-21 within a rapid 10-min timeframe, and the limit of detection (LOD) is 0.33 pM. With the advantages of high specificity, simplicity, and sensitivity, the lateral flow test strip and the design strategy hold great potential for the early diagnosis of related diseases.

A novel dual-recognition fluorescent biosensor for sialyl-Lewisx sensitive detection.

Sialyl-Lewisx (SLex) is a tetrasugar, which plays an important role in initial inflammation and cancer cell metastasis, and can be used as a marker for cancer diagnosis and prognosis or a therapeutic target. Detecting SLex from complex biological media remains a significant challenge. Herein, a single-stranded DNA aptamer of SLex was screened based on the double-stranded DNA library-modified magnetic bead (MB)-SELEX technology. After 14 rounds of screening, 12,639 sequences were obtained and divided into nine families. Three representative sequences were selected based on the number of sequence repeats and Gibbs binding free energy, and the aptamer SLex-Apt2 with 80 nt length (Kd = 23.01 nM) had the best affinity and relatively high specificity for targeting SLex. Then, a novel dual-recognition fluorescent biosensor for SLex-sensitive detection based on aptamer SLex-Apt2 bio-dots and 3-aminobenzoboric acid-modified MB was developed. This method can detect SLex as low as 32 µM and has a good linear response in the range 100 µM to 2 mM. It has the advantages of low preparation cost, good targeting, and avoiding the occurrence of false-positive and false-negative detection results, which makes the biosensor more valuable in biological detection and clinical diagnosis.

Nanosensor Based on the Dual-Entropy-Driven Modulation Strategy for Intracellular Detection of MicroRNA.

The entropy-driven strategy has been proposed as a milestone work in the development of nucleic acid amplification technology. With the characteristics of an enzyme-free, isothermal, and relatively simple design, it has been widely used in the field of biological analysis. However, it is still a challenge to apply entropy-driven amplification for intracellular target analysis. In this study, a dual-entropy-driven amplification system constructed on the surface of gold nanoparticles (AuNPs) is developed to achieve fluorescence determination and intracellular imaging of microRNA-21 (miRNA-21). The dual-entropy-driven amplification strategy internalizes the fuel chain to avoid the complexity of the extra addition in the traditional entropy-driven amplification strategy. The unique self-locked fuel chain system is established by attaching the three-stranded structure on two groups of AuNPs, where the Cy5 fluorescent label was first quenched by AuNPs. After the target miRNA-21 is identified, the fuel chain will be automatically unlocked, and the cycle reaction will be driven, leading to fluorescence recovery. The self-powered and waste-recycled fuel chain greatly improves the automation and intelligence of the reaction process. Under the optimal conditions, the linear response range of the nanosensor ranges from 5 pM to 25 nM. This nanoreaction system can be used to realize intracellular imaging of miRNA-21, and its good specificity enables it to distinguish tumor cells from healthy cells. The development of the dual-entropy-driven strategy provides an integrated and powerful way for intracellular miRNA analysis and shows great potential in the biomedical field.

Correction: A fluorescent aptasensor for ATP based on functional DNAzyme/walker and terminal deoxynucleotidyl transferase-assisted formation of DNA-AgNCs.

Correction for 'A fluorescent aptasensor for ATP based on functional DNAzyme/walker and terminal deoxynucleotidyl transferase-assisted formation of DNA-AgNCs' by Shixin Cai et al., Analyst, 2023, 148, 799-805, https://doi.org/10.1039/D2AN02006H.

A fluorescent aptasensor for ATP based on functional DNAzyme/walker and terminal deoxynucleotidyl transferase-assisted formation of DNA-AgNCs.

The development of sensitive adenosine triphosphate (ATP) sensors is imperative due to the tight relationship between the physiological conditions and ATP levels in vivo. Herein, a fluorescent aptasensor for ATP is presented, which adopts a strategy that combines a split aptamer and a DNAzyme/walker with terminal deoxynucleotidyl transferase (TDT)-assisted formation of DNA-AgNCs to realize fluorescence detection of ATP. A multifunctional oligonucleotide sequence is rationally designed, which integrates a split aptamer, a DNAzyme and a DNA walker. Both multifunctional oligonucleotide and its substrate strand are connected to the surface of Fe3O4@Au nanoparticles via Au-S bonds. The existence of ATP can induce the formation of the complete aptamer, and then activate the DNAzyme to circularly cleave the substrate strand, leaving 2',3'-cyclophosphate at the 3'end of the strand. This blocks the polymerization of dCTP to form poly(C) even in the presence of TDT and dCTP, due to the lack of free 3'-OH. In contrast, when ATP is absent, the DNAzyme/walker cannot work and then TDT catalyzes the formation of poly(C) at the free 3'-OH of the substrate strand, which is subsequently utilized as the template to prepare DNA-AgNCs. The fluorescence response derived from AgNCs thus reflects the ATP concentration. Under the optimum conditions, the aptasensor shows a linear response range from 5 nM to 10 000 nM, with a detection limit of 0.27 nM. The level of ATP in human serum can be effectively measured by the aptasensor with good recovery, indicating its application potential in medical samples.

Fluorescent Aptasensor for Highly Specific Detection of ATP Using a Newly Screened Aptamer.

Owing to the significant roles of adenosine triphosphate (ATP) in diverse biological processes, ATP level is used to research and evaluate the physiological processes of organisms. Aptamer-based biosensors have been widely reported to achieve this purpose, which are superior in their flexible biosensing mechanism, with a high sensitivity and good biocompatibility; however, the aptamers currently used for ATP detection have a poor ability to discriminate ATP from adenosine diphosphate (ADP) and adenosine monophosphate (AMP). Herein, an ATP-specific aptamer was screened and applied to construct a fluorescent aptasensor for ATP by using graphene oxide (GO) and strand displacement amplification (SDA). The fluorescence intensity of the sensor is linearly related to the concentration of ATP within 0.1 µM to 25 µM under optimal experimental conditions, and the detection limit is 33.85 nM. The biosensor exhibits a satisfactory specificity for ATP. Moreover, the experimental results indicate that the biosensor can be applied to determine the ATP in human serum. In conclusion, the screened aptamer and the biosensor have promising applications in the determination of the real energy charge level and ATP content in a complex biological system.

A lateral flow strip for on-site detection of tobramycin based on dual-functional platinum-decorated gold nanoparticles.

A novel lateral flow strip assay has been developed for rapid on-site detection of tobramycin. In this assay, unique dual-functional platinum-decorated gold nanoparticles (Au@Pt NPs) are synthesized by covering conventional gold nanoparticles (AuNPs) with an ultra-thin Pt film. Au@Pt NPs retain the plasmon activity of AuNPs and exhibit ultra-high catalytic activity that the Pt skin can achieve. The aptamer (Apt) specific for tobramycin and its complementary DNA (cDNA) are loaded on Au@Pt NPs as a duplex probe through the thiol group modified at the 5' end of the cDNA. When tobramycin is present, it binds specifically to the aptamer, resulting in its dehybridization from the cDNA and detachment from the surface of Au@Pt NPs. Then Au@Pt NPs can be captured by the fixed probe (DNA1) on the test zone (T zone) of the lateral flow strip through the hybridization between DNA1 and cDNA. The dual-functional Au@Pt NPs provide two different detection modes: one is based on the color of AuNPs (low sensitivity mode) and the other is based on the chromogenic reaction catalyzed by the Pt nanozyme (high sensitivity mode). The strip can complete the visual detection process of tobramycin within 10 min, and the cutoff values for the naked eye detection in the two modes are 60 nM and 5 nM, respectively. Furthermore, using a portable scanning reader and ImageJ software, quantitative detection can be achieved. The limits of detection (LOD) of the two modes are 0.09 nM and 0.02 nM, respectively. The strip has been successfully applied to detect tobramycin in different food samples. Therefore, Au@Pt NPs and the strip provide a highly sensitive, rapid and economical way for in-spot detection of tobramycin residues. The strip can be run in two modes, which can realize the on-demand adjustment of the detection performance and offer wider application prospects in diverse scenarios.

A fluorescent aptasensor for Staphylococcus aureus based on strand displacement amplification and self-assembled DNA hexagonal structure.

A fluorescent aptasensor for Staphylococcus aureus (S. aureus) is designed, which takes advantage of strand displacement amplification (SDA) technology and unique self-assembled DNA hexagonal structure. In the presence of S. aureus, a partially complementary strand of S. aureus aptamer (cDNA) is competitively released from cDNA/aptamer duplex immobilized on magnetic beads due to the affinity of the aptamer for S. aureus. The addition of primer starts the SDA reaction. With the catalysis of Bsm DNA polymerase and Nb.bpu10I endonuclease, a large number of single-stranded DNA (ssDNA) is produced, which induces the opening of a hairpin probe and the subsequent self-assembly to form a hexagonal structure. The staining of the DNA hexagon with SYBR Green I excites the fluorescence signal, which is used for detection. The aptasensor exhibits a broad linear range from 7 to 7 × 107 CFU/mL, with a detection limit of 1.7 CFU/mL for S. aureus. The sensor shows negligible responses to other bacteria. Moreover, the aptasensor has been applied to detect S. aureus in milk samples, and the results demonstrate the general applicability of the sensor and its prospect in systematic detection of S. aureus in food safety control and medicine-related fields. Graphical abstract The presence of S. aureus can be converted to the formation of unique DNA hexagonal structure and subsequent fluorescent signal by the combination of SDA with self-assembly technology.

Visual detection of kanamycin with DNA-functionalized gold nanoparticles probe in aptamer-based strip biosensor.

Kanamycin has been widely used to treat human and animal diseases. The excessive use of kanamycin causes its accumulation in animal-derived foods, and eventually threats human health. In the present study, we develop a lateral flow strip biosensor for fast and sensitive detection of kanamycin. The strip biosensor combines the easy separation of magnetic microspheres (MMS) with target-mediated chain displacement of single-stranded DNA and the capture of the visible DNA-functionalized gold nanoparticles (AuNPs) probe. The presence of kanamycin can competitively bind to the aptamer and release cDNA to the supernatant. The concentration of free cDNA, which is the direct target of the strip, is proportional to the concentration of kanamycin. The capture of DNA-functionalized AuNPs on the test zone of the strip through cDNA-induced hybridization provides a visual detection signal. The assay can be completed within 20 min. The visual detection limit by naked eyes of the strip is 50 nM. A linear detection range of 5-500 nM is derived for quantitative determination, with the detection limit of 4.96 nM (S/N = 3). This lateral flow strip biosensor can quickly and sensitively detect kanamycin in different food samples, which holds great application potential in medicine and daily life.

Ultrasensitive detection of the androgen receptor through the recognition of an androgen receptor response element and hybridization chain amplification.

An ultrasensitive electrochemical detection of the androgen receptor (AR) was developed based on the protection of a DNA duplex by the AR from restriction endonuclease-mediated digestion and a subsequent hybridization chain reaction (HCR). Two partially complementary DNA probes P1 and P2 were designed to form an androgen receptor binding probe (ARBP) through hybridization. The ARBP contains a duplex at one end and two single-stranded tails at the other end. The duplex part containing the recognition sites of the AR and NspI restriction endonuclease was immobilized on an Au electrode, whereas the single-stranded parts served as capture probes to activate the HCR. In the absence of the AR, NspI can cleave the duplex and release the capture probes, and thus, no HCR occurs. However, the AR can bind to the ARBP and protect the duplex from cleavage; therefore, the capture probes can trigger the HCR between four carefully designed G-quadruplex forming hairpin probes and the capture probes, resulting in the formation of numerous G-quadruplexes. Finally, differential pulse voltammetry (DPV) was carried out to quantify the AR. The assay revealed a detection limit of 7.64 fM. The verification of its high specificity and practicability in serum samples indicated its potential applications in the fields of clinical examination and disease diagnosis.

Switchable DNA tweezer and G-quadruplex nanostructures for ultrasensitive voltammetric determination of the K-ras gene fragment.

Voltammetric detection of the K-ras gene fragment was accomplished through the combined application of (a) a switchable DNA nanostructure, (b) the use of hairpin probe and exonuclease III (Exo III)-assisted signal amplification, (c) a split G-quadruplex, and (d) by exploiting the redox activity of DNAzyme. Three assistant oligonucleotides were designed to construct a DNA tweezer on a gold electrode. It is in "open state" in the absence of K-ras DNA. Then, a hairpin probe was introduced, whose stem-loop structure can be opened through hybridization with the K-ras DNA. Exo III is added which hydrolyzes the complementary region of the hairpin sequence to release a single-stranded rest fragment. The ssDNA hybridizes with the DNA tweezer on the electrode which thereby is switched to the "closed state". This leads to the formation of G-quadruplex due to the shortened distance of the split G-quadruplex-forming sequences in the tweezer. The voltammetric signal of the G-quadruplex-hemin complex, with a peak near -0.3 V vs. Ag/AgCl, is used as the signal output. Under the optimal conditions, the current response in differential pulse voltammetry (DPV) increases linearly with the concentration of K-ras DNA in the range of 0.01-1000 pM, and the detection limit is 2.4 fM. The assay can clearly discriminate K-ras DNA from a single-base mutation. The method has excellent selectivity and was applied to the determination of K-ras DNA in (spiked) serum samples. Graphical abstractSchematic representation of a method for the determination of the K-ras gene fragment through a combination of switchable DNA tweezer, split G-quadruplex, and exonuclease III (ExoIII)-assisted target recycling signal amplification.

A colorimetric ATP assay based on the use of a magnesium(II)-dependent DNAzyme.

A colorimetric assay for ATP is described that uses a strategy that combines the concept of split Mg(II)-dependent DNAzyme, split aptamer, and hybridization-induced aggregation of gold nanoparticles (AuNPs). Both ATP aptamer and Mg(II)-dependent DNAzyme are split into two fragments which are allocated to two well-designed DNA probes. The probes also possess mutually complementary stem sequences and spacer sequences. In the presence of ATP, the separated DNAzyme sequences in the two probes assemble via the synchronous recognition of ATP with two fragments of the aptamer. Then, the activated DNAzyme catalyzes multiple cycles of the cleavage of its substrate DNA sequence. The latter acts as a linker and induces the aggregation of two types of ssDNA-modified AuNP through the hybridization between the complementary sequences. Thus, the color of the AuNP solution remains red. However, in the absence of ATP, the detached aptamer cannot induce the assembly of DNAzyme to cleave the linker DNA. This results in the aggregation of AuNP and a concomitant color transition from red to purple. This ATP assay, performed at a wavelength of 530 nm, has a linear detection range that extends from 10 pM to 100 nM, with a detection limit of 5.3 pM. It was applied to the detection of ATP in human serum. Conceivably, the strategy has a wide scope in that it may be applied to the colorimetric detection of various other analytes through the split aptamer configuration. Graphical abstract Schematic presentation of colorimetric assay for adenosine triphosphate (ATP) based on the use of a split Mg(II)-dependent DNAzyme, a split aptamer, and by exploiting the hybridization-induced aggregation of gold nanoparticles that leads to a color change from red to purple.

Purification, characterization, and biocatalytic potential of a novel dextranase from Chaetomium globosum.

OBJECTIVE: We aimed to identify new high-yield dextranase strains and study the catalytic potential of dextranase from the strain in industrial applications. RESULTS: Dextranase-producing strains were screened from soil samples, and a potential strain was identified as Chaetomium globosum according to its phenotype, biochemical characteristics, and rDNA analysis. Crude dextranase was purified to reach 10.97-fold specific activity and 18.7% recovery. The molecular weight of the enzyme was 53 kDa with an optimum temperature and pH of 60 °C and 5.5, respectively. Enzyme activity was stable at pH 4.0-7.0 and displayed sufficient thermal stability at temperatures < 50 °C. Mn2+ (10 mM) enhanced dextranase activity by 134.44%. The enzyme was identified as an endodextranase. It displayed very high hydrolytic affinity toward high-molecular weight dextran T2000, reaching 97.9% hydrolysis within 15 min at 2 U/mL. CONCLUSION: Collectively, these results suggest that Chaetomium globosum shows higher production and specificity of dextranase than that from other reported strains. These findings may offer new insights into the potential of dextranase in the sugar, medical, and food industries.

An aptamer and functionalized nanoparticle-based strip biosensor for on-site detection of kanamycin in food samples.

A lateral flow strip biosensor for fast, sensitive, low-cost and on-site detection of kanamycin was developed by using kanamycin-specific aptamer-modified gold nanoparticles (AuNPs-apt) as a probe and oligonucleotide DNA1-modified silver nanoparticles (AgNPs-DNA1) as a signal amplification element. Through the complementary sequences of DNA1 and the aptamer, the AgNP-DNA1-apt-AuNPs complex can be formed and further captured on the test zone of the strip, where a capture probe DNA2 complementary to the 3'-terminal of DNA1 was immobilized. In the presence of kanamycin, it can competitively bind to the aptamer, and then inhibit the formation of the complex and the accumulation of AuNPs on the test zone. AuNPs-apt can finally be captured on the control zone via the specific binding between biotin and streptavidin. The assay avoids multiple incubation and washing steps and can be completed within 10 min. By observing the color change of the test zone, a qualitative detection for kanamycin can be achieved by the naked eye, with the visual limit of 35 nM. Meanwhile, a linear detection range of 1-30 nM with a low detection limit of 0.0778 nM for quantitative analysis can be achieved by using a scanning reader. The lateral flow strip biosensor exhibited high specificity and stability. Moreover, it was applied to detect kanamycin in various food samples, indicating its great potential in field testing.

Gold nanoparticle based photometric determination of tobramycin by using new specific DNA aptamers.

A magnetic bead-based SELEX was applied to identify 37 single-stranded DNA aptamers specific for tobramycin after ten rounds of selection. The aptamers were classified into nine families according to sequence analysis. Among them, several aptamers with typical sequences were selected and their dissociation constants (Kds) were determined by a fluorescent method. An aptamer termed "Ap 32", with a Kd value of 56.8 ± 4.6 nM, possesses the highest affinity and satisfactory specificity. Theoretical modeling showed that nucleotides 14-18 and 26-29 play a most significant role in the interaction between aptamer and tobramycin. Subsequently, the sequence of Ap 32 was optimized through rationally designed truncation. The truncated aptamer Ap 32-2 consists of 34 nucleotides and has a Kd that is similar to the original one. It was chosen as the optimal aptamer for use in the assay and was immobilized on gold nanoparticles. On addition of tobramycin, the color turns from red to purple. The findings were used to design a photometric assay (best performed at 520 nm) that has a linear response in the 100 nM to 1.4 µM concentration range, with a 37.9 nM detection limit. The method was successfully applied to the determination of tobramycin in (spiked) honey samples. Graphical abstract A 34-nucleotide aptamer specific for tobramycin was obtained through magnetic beads-based systematic evolution of ligands by exponential enrichment (SELEX) and structural analysis-based rational post-SELEX truncation, and then applied to the determination of tobramycin using a gold nanoparticle-based photometric assay.

Preparation of crosslinked enzyme aggregates (CLEAs) of acid urease with urethanase activity and their application.

An acid urease from Providencia rettgeri JN-B815 was purified via ultrasonication, ethanol precipitation, and DEAE ion-exchange column chromatography. It was found that the enzyme exhibits not only urease activity, but also urethanase activity, which made it possible to reduce EC already existed or would produce and its precursor urea at the same time. Then, crosslinked enzyme aggregates of P. rettgeri urease (PRU-CLEAs) were prepared using genipin as crosslinking agent. The purification process of acid urease, the effects of genipin concentration, and crosslinking time on PRU-CLEAs activity were investigated. The crosslinking was performed at pH 4.5 for 2.5 h, using 0.3% genipin as crosslinking agent, and 0.3 g · L(-1) bovine serum albumin as protein feeder. Using the obtained PRU-CLEAs, the removal rate of urea was up to 9.31 mg · L(-1) · h(-1). The removal rate of urea was still up to 7.56 mg · L(-1) · h(-1) after PRU-CLEAs was re-used for 6 times. When PRU-CLEAs were applied in a batch stirred and membrane reactor, the removal rate of urea in rice wine reached 5.16 mg · L(-1) · h(-1) and the removal rate of EC was 9.21 µg · L(-1) · h(-1). Furthermore, the treatment with PRU-CLEAs revealed no significant change of volatile flavor substances in Chinese rice wine. Thus PRU-CLEAs have great potential in the elimination of EC in Chinese rice wine.

Optimized expression of prolyl aminopeptidase in Pichia pastoris and its characteristics after glycosylation.

Prolyl aminopeptidases are specific exopeptidases that catalyze the hydrolysis of the N-terminus proline residue of peptides and proteins. In the present study, the prolyl aminopeptidase gene (pap) from Aspergillus oryzae JN-412 was optimized through the codon usage of Pichia pastoris. Both the native and optimized pap genes were inserted into the expression vector pPIC9 K and were successfully expressed in P. pastoris. Additionally, the activity of the intracellular enzyme expressed by the recombinant optimized pap gene reached 61.26 U mL(-1), an activity that is 2.1-fold higher than that of the native gene. The recombinant enzyme was purified by one-step elution through Ni-affinity chromatography. The optimal temperature and pH of the purified PAP were 60 °C and 7.5, respectively. Additionally, the recombinant PAP was recovered at a yield greater than 65 % at an extremely broad range of pH values from 6 to 10 after treatment at 50 °C for 6 h. The molecular weight of the recombinant PAP decreased from 50 kDa to 48 kDa after treatment with a deglycosylation enzyme, indicating that the recombinant PAP was completely glycosylated. The glycosylated PAP exhibited high thermo-stability. Half of the activity remained after incubation at 50 °C for 50 h, whereas the remaining activity of PAP expressed in E. coli was only 10 % after incubation at 50 °C for 1 h. PAP could be activated by the appropriate salt concentration and exhibited salt tolerance against NaCl at a concentration up to 5 mol L(-1).

Characterization of an N-glycosylated Bacillus subtilis leucine aminopeptidase expressed in Pichia pastoris.

Aminopeptidase is an important flavorsome especially in protein hydrolysate debittering by removing hydrophobic amino acid residue at the N-terminal end. Besides, it is also applied to preparation of active peptides and analysis of protein sequence. In this study, leucine aminopeptidase from Bacillus subtilis was cloned and expressed in Pichia pastoris, a widely used heterologous protein expression host. Then it was purified and characterized. After methanol induction for 96 h, the aminopeptidase activity in culture supernatant reached 28.4 U ml(À1) , which was 7.1 times that of wild strain B. subtilis Zj016. The optimal temperature and pH of the purified recombinant enzyme were 60 °C and 8.5, respectively. The purified aminopeptidase was stable within 30-60 °C and pH 8.0-9.0. It was intensively inhibited by Ni(2ß) , Ca(2ß) , DL-dithiothreitol (DTT) and ethylene diamine tetraacetic acid (EDTA), but activated by Co(2ß) . The Km toward leucine-p-nitroanilines (Leu-pNA) of the enzyme was 0.97 mM. The sequence analysis of aminopeptidase indicated three potential N-glycosylation sites and it was further verified via MALDI-TOF-MS analysis. Consequently, the N-glycosylated aminopeptidase exhibited higher thermostability and catalytic efficiency. The purified enzyme exhibited two bands through sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) while a single band can be identified when the enzyme was deglycosylated. Circular dichroism spectroscopy indicated that the secondary structure of recombinant aminopeptidase was similar to the wild-type.