Appl Biochem Biotechnol (2014) 172:2119–2131 DOI 10.1007/s12010-013-0674-4

Cloning, Expression, and Characterization of a Milk-Clotting Aspartic Protease Gene (Po-Asp) from Pleurotus ostreatus Chaomin Yin & Liesheng Zheng & Liguo Chen & Qi Tan & Xiaodong Shang & Aimin Ma Received: 16 September 2013 / Accepted: 3 December 2013 / Published online: 13 December 2013 # Springer Science+Business Media New York 2013

Abstract An aspartic protease gene from Pleurotus ostreatus (Po-Asp) had been cloned based on the 3′ portion of cDNA in our previous work. The Po-Asp cDNA contained 1,324 nucleotides with an open reading frame (ORF) of 1,212 bp encoding 403 amino acid residues. The putative amino acid sequence included a signal peptide, an activation peptide, two most possible N-glycosylation sites and two conserved catalytic active site. The mature polypeptide with 327 amino acid residues had a calculated molecular mass of 35.3 kDa and a theoretical isoelectric point of 4.57. Basic Local Alignment Search Tool analysis showed 68–80 % amino acid sequence identical to other basidiomycetous aspartic proteases. Sequence comparison and evolutionary analysis revealed that Po-Asp is a member of fungal aspartic protease family. The DNA sequence of Po-Asp is 1,525 bp in length without untranslated region, consisting of seven exons and six introns. The Po-Asp cDNA without signal sequence was expressed in Pichia pastoris and sodium dodecyl sulfatepolyacrylamide gel electrophoresis demonstrated the molecular mass of recombinant Po-Asp was about 43 kDa. The crude recombinant aspartic protease had milk-clotting activity. Keywords Pleurotus ostreatus . Aspartic protease (Po-Asp) . Bioinformatics analysis . Pichia pastoris . Expression . Milk clotting

Introduction Proteases are one of the most important groups of industrial enzymes which account for about 65 % of the global market [1]. Proteases with high activity and stability in acid pH range have C. Yin : A. Ma (*) Key Laboratory of Agro-Microbial Resources and Utilization, Ministry of Agriculture, College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China e-mail: [email protected] L. Zheng : L. Chen College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, China Q. Tan : X. Shang (*) National Research Center for Edible Fungi Biotechnology and Engineering, Key Laboratory of Applied Mycological Resources and Utilization, Ministry of Agriculture, Shanghai Key Laboratory of Agricultural Genetics and Breeding, Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, Shanghai 201403, China e-mail: [email protected]

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important industrial applications, especially in the production of seasonings, soy sauce, cheese, and food processing aids [2–5]. Aspartic proteases (APs, EC. 3.4.23), as a kind of acid protease, are widely distributed in a variety of organisms such as viruses, bacteria, fungi, plants, and animals [6–9]. According to the MEROPS database (http://www.merops.ac.uk), APs are now grouped into 16 different families based on their amino acid sequence homology [10]. Though most aspartic proteases share common characteristics and properties, such as sequence homology, high β-sheet content, aspartic acid active site residues, preference for hydrophobic amino acids, activity at low pH, and inhibition by pepstatin, there exist some differences in their catalytic properties, cellular localization, and biological functions [4, 8, 11]. The well-known representatives of aspartic protease include pepsin, chymosin, and cathepsin D [12]. Especially chymosin, which has a significant property of coagulating milk, is widely applied in the dairy industry to coagulate casein during cheese manufacturing. Since cheese is a popular dairy product worldwide, the demand of chymosin has rapidly increased [13]. The scarcity and high price of traditional milk coagulating agent (calf rennet) has promoted research towards alternative milk coagulants produced by the native or genetically modified microorganisms [14]. Pichia pastoris is an excellent system for production of a wide variety of recombinant proteins because it has the advantages of low cost, fast growth, high productivity, and simple manipulation [15, 16]. Recently, more and more chymosin genes have been successfully expressed in P. pastoris and the recombinant chymosins have been used in cheese production [3, 13, 17–19]. Mushrooms are macrofungi which belong to basidiomycetes and ascomycetes. Up to now, little is known about the aspartic proteases from mushrooms. Pleurotus ostreatus, or oyster mushroom, is one of the most widely cultivated edible fungi in the world. In our previous work, a 658 bp 3′ cDNA fragment of P. ostreatus aspartic protease (Po-Asp) was isolated through a differential screening method [20]. In order to gain a better understanding and utilization of aspartic protease from P. ostreatus, here we conducted molecular cloning and characterization of the Po-Asp and its recombinant expression in P. pastoris.

Materials and Methods Strains and Plasmids P. ostreatus Pd739 was maintained on potato dextrose agar (Difco, USA) slant at room temperature. P. pastoris GS 115 (Invitrogen, Shanghai, China) was cultured in yeast extract peptone dextrose medium (Difco) as expression host. Escherichia coli DH5α (Takara, Dalian, China) was grown in Luria-Bertani medium (LB; Difco) for standard bacterial cloning. Plasmid pMD®18-T (Takara) was used as cloning vector. pPIC9K vector (Invitrogen, Shanghai, China) was used for gene expression in P. pastoris. RNA Isolation and Full-Length cDNA Cloning Total RNA was extracted using the RNAiso™ Plus (Takara) according to the manufacturer’s protocol. The concentration and quality of the total RNA were estimated by measuring the absorbance ration of 260/280 nm and agarose gel electrophoresis, respectively. The total RNA was reverse transcribed into the first-strand cDNA using the SMART™ RACE cDNA Amplification Kit (Clontech, USA) according to the user’s manual. The Po-Asp 5′ portion cDNA was amplified using the specific primer APS5 (Table 1) based on the acquired sequence combined with the universal primer (UPM in Table 1) supplied by

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Table 1 Primers for PCR amplification in this study Names

Sequences (5′→3′)

Descriptions

APS5

CGATTTGCGTGTTCAGCATCTCCG

Primers for 5′ RACE PCR

UPM (long)

CTAATACGACTCACTATAGGGCAA GCAGTGGTATC AACGCAGAGT

The universal primer for 5′ RACE PCR

UPM (short)

CTAATACGACTCACTATAGGGC

APF

ACCTCCTTCGCTTCAACCA

APR

AACATAGGCAAAGAGTCC

Primers for amplification of full-length cDNA and genomic DNA

Asp-EF

GCTTACGTAGATGGCCTGCAC CGTCTT

Primers for expression, underlined parts indicate SnaB I and Not I restriction sites, respectively

Asp-EB

ATTTGCGGCCGCTTAATGATGA TGATGATGATGT GCAGCAAG TGCGAATCC

α-factor

TACTATTGCCAGCATTGCTG

Aox1-3

GCAAATGGCATTCTGACAT

Primers for amplification of incorporation gene

the SMART™ RACE cDNA Amplification Kit. The thermal cycling conditions were as follows: 94 °C for 10 min followed by 35 cycles at 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 1 min, with a final extension at 72 °C for 10 min. The PCR product was purified with AxyPrep™ DNA gel extraction kit (Axygene, Hangzhou, China), then cloned into pMD18-T and transformed into E. coli DH5α by heat shock. cDNA inserts isolated from positive clones were sequenced (Invitrogen). The full-length cDNA was cloned into E. coli DH5α using a pair of gene-specific primers APF and APR (Table 1), which were designed from the full-length cDNA sequence obtained by joining the fragments of 5′ and 3′ ends with DNAMAN 6.0. DNA Extraction and Genomic DNA Cloning Genomic DNA was extracted from P. ostreatus Pd739 mycelia using the CTAB method [21]. Gene-specific primers APF and APR (Table 1) were used to clone the genomic sequence. The PCR reaction conditions were as follows: 94 °C for 10 min followed by 35 cycles at 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 1 min, with a final extension at 72 °C for 10 min. The PCR products were cloned into pMD®18-T and transformed into E. coli DH5α for sequencing. Bioinformatics Analysis Sequence similarity was analyzed using Basic Local Alignment Search Tool (BLAST) from National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov/ BLAST/). The open reading frame (ORF) was found using NEW GENSCAN (http://genes. mit.edu/GENSCAN.html). The signal peptide and the cleavage site were predicted using SignalP (http://www.cbs.dtu.dk/services/SignalP/). The asparagine-linked glycosylation sites were determined by NetNGlyc 1.0 (http://www.cbs.dtu.dk/services/NetNGlyc). Protein motifs were identified using MOTIF Search (http://www.genome.jp/tools/motif/) and the Conserved Domain Database from NCBI (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). The theoretical isoelectric point and molecular weight were predicted using Compute PI/MW (http://expasy.org/tools/protparam.html). Multisequence alignment was generated using CLUSTALX, and phylogenetic analysis was conducted using MEGA 5.0. Homology modeling was performed using the Swiss-Model (http://swissmodel.expasy.org).

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Expression of the Po-Asp in P. pastoris For heterologous expression of the predicted mature Po-Asp in P. pastoris, we amplified its cDNA using primers Asp-EF and Asp-EB (Table 1). The resulting fragment was cloned into the PMD®18-T vector and used to transform E. coli DH5α. The recombinant vectors were extracted using the AxyPrep™ Plasmid miniprep Kit (Axygene, Hangzhou, China) and digested with SnaB I and Not I, respectively. Then, this fragment was ligated into pPIC9K vector which was treated with the same enzymes. The recombinant vector pPIC9K-Asp was checked for accurate insertion by sequencing and restriction enzyme analysis. The resulting plasmid was transformed in E. coli DH 5α for amplification. After linearized with Pme I and dephosphorylated with alkaline phosphatase (E. coli C75), the recombinant vector pPIC9K-Asp and the pPIC9K control were electrotransformed into P. pastoris GS115 competent cells pretreated with D-sorbitol. The incorporation of Po-Asp and P. pastoris genome was confirmed by PCR with α-factor primer and Aox1-3 primer, combining with Po-Asp specific primers Asp-EF and Asp-EB (Table 1). To express Po-Asp protein, cells from a single colony of P. pastoris containing either the empty vector pPIC9K (control) or pPIC9K-Asp were inoculated into 100 mL liquid BMGY medium (1 % (w/v) yeast extract, 2 % (w/v) peptone, 100 mM potassium phosphate, 1.34 % (w/v) yeast nitrogen base, 0.4 mg/L biotin, 0.5 % (v/v) glycerol), respectively. The cultures were grown at 28 °C in vigorous shaking (250 rpm) over night. After the cultures reached an OD 600 nm in 2.0, the cells were collected (3,000×g, 5 min) and resuspended in 150 mL BMMY medium (similar to BMGY, but containing 2 % (v/v) methanol instead of glycerol as inducer). In order to maintain the induction, absolute methanol was fed daily to 0.5 % (v/v) every 24 h, and then culture supernatants and cells were harvested by centrifugation (8,000×g, 20 min) after 4 days of induction. We monitored the protein expression level by assaying Po-Asp activity using azocasein (Sigma, USA) as a substrate and by analyzing proteins isolated from subsamples with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE and Western Blot Analysis Collected samples were analyzed by SDS-PAGE [22] using 12 % polyacrylamide gels with a Mini-Protean 3 Cell (Bio-Rad, USA). Samples were treated with 4× protein SDS-PAGE loading buffer (Takara) in boiling water for 10 min before loading. The gel was run with 1× Tris-Glycine SDS running buffer (0.025 M Tris, 0.192 M glycine, pH 8.5, and 0.1 % SDS) for 2 h in 115 V. After running the gel, it had been washed and stained with Coomassie Brilliant Blue R-250 (Sigma). For Western blot analysis, proteins separated by SDS-PAGE were transferred onto a PVDF membrane for 2 h at 11 mA. After blocking with 5 % skim milk in phosphate-buffered saline buffer containing 0.05 % Tween-20 (PBST) for 2 h at room temperature (RT), the membranes were incubated in mouse anti-His-Tag antibody (Tiangen, China) with a dilution of 1:2,000 for 2 h at RT and washed three times for 15 min in PBST. Then, the membranes were incubated in goat anti-mouse IgG conjugated with horseradish peroxidase (HRP) (Tiangen) solution (1: 2000 dilutions) for 2 h at RT. After washing, 3, 3-diaminobenzideine (DAB) western blotting detection reagent (Tiangen) was used for detection of positive reactions and the resultant complexes were processed for the detection system using an MF-ChemiBIS 3.2 (DNR, Israel). Skimmed Milk-Clotting Test Before milk-clotting test, we concentrated the supernatant using the dialysis bag (the molecular weight cutoff is 8,000 to 14,000) and PEG 20,000 at 4 °C for overnight. Milk-clotting activity

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was determined by adding recombinant aspartic protease to skim milk powder reconstituted (12 % w/v) in 10 mM CaCl2, pH 5.8. An enzyme/milk mixture (1:5, v/v) was incubated at 37 °C about 5 h to observe the curd formation. Controls without enzyme and with enzyme preincubated 10 min using pepstatin A (0.001 mM) were also performed. This experiment was carried out in three replicates [12].

Results Sequence Analysis of Aspartic Protease In our previous work, a 658 bp 3′ cDNA fragment of P. ostreatus aspartic protease was isolated [20]. By 5′ RACE PCR reaction, we obtained a 936 bp fragment with the gene-specific primer APS5 and universal primer UPM after trimming the adaptors. Finally, the full-length cDNA of aspartic protease Po-Asp (GenBank Accession no. KF471121) consists of 1,332 nucleotides with an ORF of 1,212 bp, a 5′ untranslated region (UTR) region of 24 bp and a 3′ UTR region of 96 bp containing the poly A tail. The ORF encodes a protein of 403 amino acid residues including a signal peptide (aa 1–16), an activation peptide (aa 17–76), and two most possible N-glycosylation sites located at the amino acid residues 33–35 (–N–P–S–) and 145–147 (–N–G–T–). After removal of signal peptides and activation peptides, we obtained a mature polypeptide of 327 amino acid residues with a theoretical isoelectric point and molecular weight (Mw) of 4.57 and 35.3 kDa, respectively. The deduced amino acid sequence displays the characteristic primary structure of typical fungal aspartic proteases with two conserved catalytic triads (DTG 110–112 and DTG 292–294) and two conserved active sites—“VILDTGSSNLWV” (107–118) and “AAIDTGTSLIAL” (289–300) (Figs. 1 and 2). Multiple Sequence Alignment and Phylogenetic Analysis The results of similarity analysis using the NCBI BLAST program showed that the deduced amino acid sequence of Po-Asp possessed homology with aspartic proteases from Trametes versicolor (76 %, accession no. EIW61956), Dichomitus squalens (80 %, EJF61236), Punctularia strigosozonata (77 %, EIN10642), Coprinopsis cinerea (77 %, EAU84813), Laccaria bicolor (77 %, EDR12869), Stereum hirsutum (76 %, EIM88453), Fomitiporia mediterranea (78 %, EJD01470), Coniophora puteana (76 %, EIW76137), and Auricularia delicata (68 %, EJD53628; Fig. 3). To examine the phylogenetic relationship of Po-Asp with other homologous aspartic proteases, a phylogenetic tree was constructed based on the amino acid sequence from 23 fungal aspartic proteases. Protein distance-based phylogenetic analysis of the fungal aspartic protease amino acid sequences resulted in the creation of a dendrogram with two clusters, and Po-Asp branched between D. squalens and P. strigosozonata aspartic protease (Fig. 4). The Gene Structure of Aspartic Protease The Po-Asp gene (GenBank Accession no. KF498707) contains 1,525 bp without UTR. It consists of seven exons and six introns (257–307, 51 bp; 364–414, 51 bp; 438–486, 49 bp; 735–793, 59 bp; 1,092–1,138, 47 bp; and 1,396–1,451, 56 bp). In Po-Asp gene, the first to the third intron (TA–GG) and the fifth intron (GT–AG) splice motifs have been identified to be common in organisms, while the special intron splice motifs of the fourth (AC–GT) and the sixth (TG–TG) also exist.

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Fig. 1 The full-length cDNA and deduced amino acid sequence of the P. ostreatus aspartic protease. The number of nucleotide and amino acid sequence are located on the left. The translation initial and stop codons are marked with a frame. The putative signal peptide is indicated in bold face and underlined. The potential glycosylation residue (N) is marked by diamond shapes. The consensus motifs and conserved catalytic sites (DTG) are indicated in bold face with light gray shadow and a frame, respectively

Homology Modeling The three-dimensional structure modeling of the Po-Asp was conducted using the automated mode of Swiss-Model and displayed with DeepView software. According to the homology search in Worldwide Protein Data Bank, the aspartic protease structure of Saccharomyces cerevisiae

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Fig. 2 Conserved domains analysis of the Po-Asp

(PDB ID: 1DP5) was identified as the best template for homology modeling. The protein model consists of 11 helices and 26 beta strands (Fig. 5) which showed the Po-Asp is a highly strand protein. Based on the homology modeling, the Asp110 and Asp295 could be an active site of the acid protease and these catalytic residues are located in the deep cleft between the subdomains. These locations of catalytic residues are highly conserved in the active catalytic center of other aspartic protease. Expression of the Recombinant Po-Asp in P. pastoris The cDNA of the Po-Asp had been cloned into the expression vector pPIC9K and electrotransformed into P. pastoris GS115. The recombinant protein of pPIC9K-Asp-6His induced by the methanol had been secreted into the extracellular. SDS-PAGE showed the supernatants from the uninduced and induced cells exhibited a unique band with a molecular mass of 43 kDa (lanes 1 and 3 in Fig. 6a). On the contrary, no target band had been found in supernatant from the control (lane 2 in Fig. 6a). The Western blotting result (Fig. 6b) confirmed the unique band was indeed the His-tagged fusion protein. The Milk-Clotting Activity of Po-Asp Skimmed milk-clotting test of the recombinant protein of pPIC9K-Asp-6His was carried out. As illustrated in Fig. 7, the recombinant protein of pPIC9K-Asp-6His was able to coagulate milk (Fig. 7c), and this activity was totally inhibited in the presence of pepstatin A (Fig. 7d), while no such coagulability was observed with the addition of water (Fig. 7a) or supernatant from blank control (Fig. 7b).

Discussion In this paper, we describe the cloning, characterization, and expression analysis of a P. ostreatus aspartic protease. The Po-Asp contains two catalytic site motifs, Asp110-Thr111-Gly112 and Asp292Thr293-Gly294, which are highly conserved among fungal aspartic proteases (Figs. 1 and 2). According to the structure analysis, we confirm Po-Asp belongs to clan A of aspartic protease. Based on the alignment and comparison of the Po-Asp protein sequence using the protein BLAST program, the Po-Asp shared substantial identity (above 65 %) with aspartic proteases of other homobasidiomycetes (Fig. 3). These high homologies showed that Po-Asp had a conserved catalytic domain with catalytic aspartic acid residue in the active site, which is a common characteristic of aspartic proteases [8]. To conduct comparative evolutionary analysis of gene families across multiple fungal species, a special unrooted tree was constructed based on the deduced amino acid sequences [23]. As Fig. 4 showed, Po-Asp branched between D. squalens and P. strigosozonata. And the aspartic proteases coming from 23 species of fungi could be divided into two categories: clusters I and II. The species of cluster I belong to

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Fig. 3 Multiple amino acid sequences alignment of Po-Asp with other homologues. Conservative residues are shown in dark blue boxes, identical residues in light blue boxes, and unrelated residues have a white background. Amino acid numbers are shown on the right. Accession numbers of amino acid sequences were as follows: Trametes versicolor aspartic peptidase A1 (EIW61956), Dichomitus squalens endopeptidase (EJF61236), Punctularia strigosozonata endopeptidase (EIN10642), Coprinopsis cinerea endopeptidase (EAU84813), Laccaria bicolor aspartic peptidase A1 (EDR12869), Stereum hirsutum Asp-domain-containing protein (EIM88453), Fomitiporia mediterranea aspartic peptidase A1 (EJD01470), Coniophora puteana Asp-domaincontaining protein (EIW76137), and Auricularia delicate aspartic peptidase A1 (EJD53628)

basidiomycota as well as the species of cluster II to ascomycota. Po-Asp was classified into cluster I with other basidiomycetes. Although Cryptococcus gattii, Cryptococcus neoformans, and Ustilago maydis belong to basidiomycota, significance differences had existed in morphological and physiological aspects when compared to mushroom-forming fungi.

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Fig. 4 A molecular phylogenetic tree of Po-Asp generated by the neighbor-joining (NJ) method using MEGA 5. An unrooted phylogenetic tree was generated based on the alignment of the amino acid sequences from 23 fungal species. One thousand bootstrap replicates were calculated, and bootstrap values are shown at each node. Nodes were collapsed to a single horizontal line whenever statistical support was less than 60 %. The scale bar indicates an evolutionary distance of amino acid substitutions per position. Accession numbers of amino acid sequences were as follows: C. cinerea (EAU84813), L. bicolor (EDR12869), S. hirsutum (EIM88453), D. squalens (EJF61236), P. strigosozonata (EIN10642), T. versicolor (EIW61956), F. mediterranea (EJD01470), C. puteana (IW76137), A. delicate (EJD53628), Cryptococcus gattii (XP_003191823), Cryptococcus neoformans (AFR92711), Ustilago hordei (XP_761073), Neosartorya fischeri (XP_001263323), Aspergillus fumigatus (EAL92441), Aspergillus clavatus (EAW09715), Aspergillus niger (EHA23047), Aspergillus kawachii (GAA88863), Talaromyces stipitatus (EED19431), Penicillium marneffei (EEA27026), Coccidioides posadasii (AAZ92541), Paracoccidioides brasiliensis (EEH19972), and Neurospora crassa (CAF05874). On the basis of the AP family tree, it is possible to divide typical fungal APs into two groups (clusters I and II)

In view of sequence alignment between DNA and cDNA, six introns were found in Po-Asp. One of them had the splice motif of (GT–AG) and three had the splice motif of (TA–GG); these introns were typically based on the consensus splice site and internal sequence for lariate formation [24]. At the same time, the uncommon splice motifs (AC–GT) and (TG–TG) had also been found. It has been reported that a (GG–AG) splice motif existed in the S. mansoni aspartic protease gene [25], but we did not find this splice motif. Besides, the polyadenylation signal (AATAAA) had not been found in gene Po-Asp. This is similar to the result of Schuren’s research in which no conserved sequence for the addition of a polyA tail had been found in 3′ noncoding sequences of 17 basidiomycetous genes [26]. We supposed no polyadenylation signal in Po-Asp may be caused by the analogous T- or TG-rich motif or other unknown mechanisms.

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Fig. 5 Modeling structure of Po-Asp. D110 and D295 represent the Asp110 and Asp295 residues in active site, respectively. N and C terminal had also been showed

In the homology modeling, we found the mature Po-Asp was bilobed molecule, and two homologous Asp-Thr-Gly (DTG) catalytic site motifs were located in the center of the substrate-binding groove, which are conserved and essential for the catalytic activity [27]. Most of the fungal aspartic proteases were synthesized as zymogens, the similar situation had occurred in Po-Asp which has a 60 amino acids activation peptide. Some researchers guessed that this mechanism likely provides protection from proteolysis [4]. Now, scientists have found acid-triggered and autocatalytic proteolysis can make zymogen convert into active enzyme [28, 29]. Po-Asp activation peptide shares lower similarity with other fungal activation

Fig. 6 SDS-PAGE and Western blot analysis of the recombinant Po-Asp. a The SDS-PAGE analysis of the recombinant Po-Asp. M marker proteins, 1 supernatant from uninduced pPIC9K-Asp-6His, 2 supernatant from blank control, 3 supernatant from induced pPIC9K-Asp-6His. b Western blot analysis of the recombinant PoAsp. M marker proteins, 1 supernatant from blank control, 2 supernatant from induced pPIC9K-Asp-6His. The marker proteins comprised phosphorylase B (97 kDa), albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (29 kDa), and trypsin inhibitor (20 kDa)

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Fig. 7 The milk clotting test of the recombinant Po-Asp. a Milk added an equal amount of water, b milk added an equal amount of supernatant from blank control, c milk added an equal amount of supernatant from induced pPIC9K-Asp-6His, d milk added an equal amount of supernatant from induced pPIC9K-Asp-6His which has been inhibited by the pepstatin A

peptides (Fig. 3), the detailed activation mechanism is still unclear due to lack of further experimental evidence. We expressed the Po-Asp cDNA without signal sequence in P. pastoris. The target band observed in Fig. 6b suggests the Po-Asp cDNA has been successfully expressed in P. pastoris with no frame shift and non-enzymatic cleavage during expression. SDS-PAGE showed the recombinant aspartic protease has a molecular mass of about 43 kDa, which is consistent with the estimated size of the deduced amino acids (41.7 kDa). When the activation peptide is removed, the estimated molecular weight of mature polypeptides is 35.3 kDa. This result is similar to the milk-clotting aspartic peptidase PsoP1 from basidiomycete Piptoporus soloniensis [30] and Cap1 from basidiomycetous yeast Cryptococcus sp. S-2 [31]. Milk-clotting occurrence is essentially the action result of proteases that destabilize casein micelles, which are particles present in fresh milk and dispersed in a continuous phase [32]. As a result, the repulsive forces between casein micelles decrease, thereby causing aggregation of caseins [33]. At present, some aspartic proteases with milk-clotting activity have been isolated and characterized from several fungi including P. soloniensis [30], Cryptococcus sp. S-2 [31], Mucor spp. [34–37], Irpex lacteus [38], and Laetiporus sulphure [39]. In this study, we found that the recombinant Po-Asp exhibited a higher milk-clotting activity (Fig. 7). Therefore, Po-Asp could become an excellent candidate of milk-clotting enzyme in the cheese-making industry.

Conclusion In this study, we cloned a cDNA encoding Po-Asp which contained 1,324 nucleotides with an ORF of 1,212 bp. The putative 403 amino acid residues of Po-Asp have a signal peptide, an activation peptide and two conserved catalytic active sites. The DNA of Po-Asp is 1,525 bp in length consisting of seven exons and six introns. The recombinant protein of Po-Asp has been successfully expressed in P. pastoris and displayed milk-clotting activity. In order to utilize the

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recombinant Po-Asp in the cheese-making industry, the works on purification and characterization of this enzyme are ongoing. Acknowledgments We are grateful to Qi Tang for assistance in Western blot experiment, Lifen Huang for assistance in skimmed milk clotting test, and Jihong Zhu for critical reading of the manuscript. This work was supported by grants from the National Natural Science Foundation of China (31172011) and the Chinese National Science and Technology Support Program (2013BAD16B02).

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Cloning, expression, and characterization of a milk-clotting aspartic protease gene (Po-Asp) from Pleurotus ostreatus.

An aspartic protease gene from Pleurotus ostreatus (Po-Asp) had been cloned based on the 3' portion of cDNA in our previous work. The Po-Asp cDNA cont...
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