Screening and expression of a gene encoding protease inhibitor protein of microorganisms associated with the sponge spheciospongia vesparium collected from mientrung sea, Vietnam

of total DNA of sponge-associated microorganisms by method of Abe et al. (2014) with some minor improvements to suit the conditions of Vietnam. 2.2.2. Sponge identification by molecular biology (Medlin et al., 1988) 2.2.3. Analysis of metagenomics data Sequencing DNA metagenome by Illumina HiSeq 2500 system (BaseClear - Netherlands). The collected data were analyzed by bioinformatic tools. 2.2.4. Synthesis of PIs gene from database metagenomics and design plasmid carrying PIs gene The PI-QT gene was screened from the metagenomic QT2 database, synthesized and cloned into the pUC57 vector, with the restriction enzymes EcoRI and NotI at both ends (GenScipt, USA). Next, the vector pUC57 (Thermo Fisher Scientifc, USA) containing the gene PI-QT and expression vector pET-32a(+) (Invitrogen, USA) were digested with restriction enzymes EcoRI and NotI (Fermentas, Lithuania). The gene PI-QT was then purifed and inserted into expression vector pET-32a(+) (expression in E. coli) and pPIC9 (expression in P. pastoris) using enzyme T4 ligase (Thermo, USA). 2.2.5. Methods used for expression and recovery of recombinant PIs in the E. coli BL21(DE3) expression system Methods of transformation, expression and optimization of gene expression in E. coli cells according to Do ThiHuyenet al., 2008; Nguyen ThiQuyet al., 2013. Western blot analysis (according to the instructions of Invitrogen, USA). Purification of recombinant PIs protein: Purification of the recombinant protein PI-QT was performed by chromatography on Ni-NTA afinity chromatography column according to the Ni-NTA 6 Purification System (Novex by life technologies) with some minor modifications. Remove the TRx-His-tag peptide from the recombinant protein (Novagen's instructions). Identification and analysis of proteins by proteomics/ bioinformatic tools Protein Biochemistry Department, Institute of Biotechnology). 2.2.6. Methods used to for expression and recovery of recombinant PIs in the Pichiapastoris expression system According to the instructions of the Pichia expression kit (Invitrogen). 2.2.7. Protease inhibitory assay (Sapna., 2013; Jiang et al., 2011) 2.2.8.Characterization of protease inhibitor PI-QT protein (Sapna., 2013). 2.2.9. Statistical Analysis The assays were performed in triplicate and expressed as the mean ± standard error of the mean (SEM). The statistical analysis was performed by t-test and one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison tests using the SPSS v.22 (SPSS Inc, Chicago, IL, USA). The results were considered to be signifcant at P < 0.05. CHAPTER III: RESULTS AND DISCUSSION 3.1. Collect of sponge samples Use SCUBA method, 8 samples of sponge were collected at Quang Tri sea. Sponges were collected at the coordinates of 107°07'06.0"E; 17°04'50.2"N. Samples were stored in 30 % glycerol, preserved in ice, transported to the laboratory and DNA extraction of sponge-associated microorganismswas carried out. 3.2. DNA metagenome extraction results 7 3.2.1. Extraction of DNA metagenome of microorganism associated withsponge collected at Quang Tri sea Total DNA of sponge-associated microorganism was extracted from 8 sponge specimens collected at the Quang Tri Sea, Vietnam. The results showed that the amount and quality of DNA extracted was different for each sample. The most isolated DNA was from QT2 sample and it had highest purity (Figure 3.1, Table 3.1). QT9 QT8 QT7 QT6 QT5 QT4 QT2 QT3 M Figure 3.1. Electrophoresis of isolated DNA from QT2 sponge- associated microorganisms (M: Marker 100 bp of Thermo) Table 3.1. The quality of total DNA extracted from spongescollected in Quang Tri sea Serial Sample DNA (ng/µL) A260/A230 A260/A280 1 QT2 202,5 2,0 1,80 2 QT3 165,1 1,8 1,71 3 QT4 160,9 1,83 1,54 4 QT5 178,7 1,82 1,32 8 5 QT6 39,8 1,9 1,62 6 QT7 73,5 1,8 1,72 7 QT8 125,8 1,77 1,41 8 QT9 36,5 1,78 1,70 The total DNA of sponge-associated microorganisms QT2 was selected (DNA concentration: 202,5ng/µl, purity A260 / A280 = 1.80) for metagenomic sequencing. 3.1.2. Identify the QT2 sponge by molecular technique The nearlyfull-length 18S rRNA gene fragment of the QT2 sponge sample were amplified successfully (Figure 3.2b). The obtained sequence was compared with other sequences of 18S rRNA using the BLAST (Basic Local Alignment Search Tool) on NCBI (National Center for Biotechnology Information). The result that the 18S rRNA gene sequence of QT2 sponge was similar to 99,9 % with that of Spheciospongia vesparium sponges with code AY734440. Therefore, the QT2 sample was named Spheciospongia vesparium QT2. 3.3. Establishment of a DNA metagenome database of microorganismsassociated with Spheciospongiavesparium QT2 by bioinformatics After sequencing the entire shortgunmetagenome of the QT2 sample (Base Clear, Netherlands) and purifying the data using Trimmomatic software, over 44 million paired reads were collected. The purified data were used to assemble de novo metagenome using SPAdes software. The total size of assembly is approximately 418 Mb including 102,236 contigs. The longest contig is over 855 kb, the smallest contigs is 1,000 bp, the average length is 4,089 bp. Nearly 90 % of the sequences can be mapped back to the assembly genome. The obtained results showed that the contigs mainly distributed 9 between 1,000 and 100,000 bp. The percentage of GC in the genome of QT2 samples was 61.82 %. 3.3.2. Results of gene prediction Using Prodigal and MetageneMark software, about 366 Mb (386,416 ORFs) and 361 Mb (380,886 ORFs) of the predicted genes were received, respectively. The gene prediction results of the two software are quite similar, with the largest gene being 66,639 bp, the average length is 864 bp and the GC ratio is more than 62 %. After removing all genes smaller than 250 bp, using CD-HIT software with 90 % similarity, the final genome with a total size of nearly 360 Mb was obtained, including 372,732 unified genes. Among them 262,159 complete genes (accounting for 70.33 %) (genes with both opening and ending codes); 53,162 genes lack the ending code 3’(14.26 %); 49,569 genes were missing the opening code 5' (13.3 %) and the number of genes lacking both the opening and ending codes was only 7,842 genes, accounting for 2.1 %. The length distribution shows that the prediction genes mainly ranges from about 250 bp to about 2,000 bp. 3.3.3. Results of annotation and classification of gene function Using different databases for gene annotation and the result that, for a total of 372,732 gene sequences, based on the NR database 360,564 (96.74 %) genes were annotated; 266,553 genes were annotated according Swiss-Prot, accounting for 71.51 %; 274,632 genes accounting for 73.68 % were annotated based COG database; only 11,974 (3.21 %) genes were annotated CAZy database; the number of genes annotated according the GO database is 165,552 genes accounting for 44.42 %, 244,436 genes are annotated based on the KEGG database accounting for 65.58 %; For the Pfam database, 273,826 (73.46 %) genes were annotated. 10 3.3.4. Biodiversity of microorganisms associated with Spheciospongia vesparium QT2 sponge. 10 phyla, 21 classes, 36 orders, 22 families and 16 genera were identified for bacteria associated with Spheciospongia vesparium QT2 sponge in Quang Tri sea. 3.3.5. Screening gene with protease inhibitory activity (PIs) based on metagenomic database Using bioinformatic tools, 50 complete genes related to protease inhibitors were screened from metagenomic database of QT2 (Quang Tri) (Table 3.8). Of these, 28 genes, accounting for 56 % of the annotated genes belong to the Serpin (Serine protease inhibitor) family; the remaining 22 genes (44 %) belong to Inter-alpha-trypsin inhibitor family. The shortest gene is 198 bp, coding for 66 amino acids; The longest gene is 2406 bp, which encodes for 802 amino acids. Some genes have also been identified as new genes in Vietnam. Table 3.8.Screening result for genes with protease inhibitory activity from metagenomic database of QT2 Contig Prokka A.a Uni_1 UniProtKB_ product Uni_ score Uni_ evalue 1 00016 5808 442 Q5RB37 ITIH chain H3 89.4 1.00E-17 2 000019 06418 323 O02668 ITIH chain H2 61.2 5.00E-09 3 000019 0645 398 Q61703 ITIH chain H2 71.6 4.00E-12 4 000046 10704 429 Q9D154 Serpin 184 6.00E-52 5 000046 10705 405 Q5BIR5 Serpin 214 1.00E-63 6 000127 20087 328 Q3T052 ITIH chain H4 62 3.00E-09 7 000172 24210 400 Q8BJD1 ITIH chain H5 71.6 4.00E-12 8 000213 27784 418 Q5BIR5 Serpin B8 216 5.00E-64 9 000213 27785 405 Q99574 Neuroserpin 209 2.00E-61 10 000314 34813 736 A6X935 ITIH 171 6.00E-43 11 000433 41698 419 Q5BIR5 Serpin B8 231 6.00E-70 12 000631 52340 325 Q3T052 ITIH chain H4 58.9 3.00E-08 13 000726 56621 398 Q61703 ITIH chain H2 57 2.00E-07 14 000981 67673 428 Q90935 Neuroserpin 214 1.00E-62 15 001114 72799 419 Q5BIR5 Serpin B8 219 4.00E-65 16 001390 82416 386 A6X935 ITIH 114 4.00E-26 17 001690 91580 454 Q5BIR5 Serpin B8 152 5.00E-40 18 001737 93032 412 Q5BIR5 Serpin B8 214 1.00E-63 19 001737 93033 223 Q99574 Neuroserpin 87.8 6.00E-19 11 20 001813 95168 412 Q99574 Neuroserpin 207 3.00E-60 21 002069 102253 280 Q8PTN8 Serpin 175 7.00E-50 22 002236 106102 324 A2VE29 ITIH chain H5 64.7 4.00E-10 23 002339 108516 478 Q14624 ITIH chain H4 63.5 2.00E-09 24 002592 114432 355 Q90935 Neuroserpin 197 3.00E-57 25 002838 119867 334 Q14624 ITIH chain H4 55.5 3.00E-07 26 003102 125566 401 Q8BJD1 ITIH chain H5 58.5 5.00E-08 27 003892 140659 323 Q3T052 ITIH chain H4 67 7.00E-11 28 004584 152589 631 Q61703 ITIH chain H2 66.2 6.00E-10 29 005997 173538 430 Q8PTN8 Serpin 206 2.00E-59 30 006820 184178 417 Q5BIR5 Serpin B8 237 5.00E-72 31 007047 186946 497 Q61703 ITIH chain H2 122 2.00E-28 32 007181 188591 146 Q96P15 Serpin B11 105 5.00E-26 33 007964 197295 66 Q90935 Neuroserpin 51.6 7.00E-08 34 008443 202257 443 P50453 Serpin B9 213 2.00E-62 35 010618 222724 382 Q8BJD1 ITIH chain H5 64.3 8.00E-10 36 012483 237228 378 Q9JK88 Serpin I2 57 1.00E-07 37 015758 258680 430 Q9S7T8 Serpin-ZX 143 1.00E-36 38 020504 283125 394 Q90935 Neuroserpin 73.2 8.00E-13 39 020772 284248 402 Q90935 Neuroserpin 213 1.00E-62 40 020806 284376 802 Q61703 ITIH chain H2 142 2.00E-33 41 020909 284844 362 A6X935 ITIH 104 6.00E-23 42 021896 288956 722 P56652 ITIH chain H3 170 8.00E-43 43 024785 300295 303 Q29052 ITIH chain H1 55.5 3.00E-07 44 030105 318139 717 Q9GLY5 ITIH chain H3 116 2.00E-25 45 033816 328453 391 B4USX2 Serpin B10 220 1.00E-65 46 038363 339464 376 Q9CQV3 Serpin B11 82 7.00E-16 47 040171 346561 423 Q99574 Neuroserpin 211 1.00E-61 48 044964 352966 457 Q5JJ64 Serpin 249 7.00E-76 49 060339 377096 149 Q9UIV8 Serpin B13 66.6 4.00E-12 50 067320 385523 98 Q5NBM Putative serpin 66.2 1.00E-12 Note: ITIH: Inter-alpha-trypsin inhibitor heavy; Serpin: serine protease inhibitor; I: Inhibitor 3.4. Expression of protease inhibitory gene (PIs) in Escherichia coli system Based on the PIs gene sequence from metagenomic databaseof QT2, a number of primers for cloning the protease inhibitor genes of the desired serpin family were designed, using PCR method with template was DNA metagenome of QT2. However, despite many changes of different parameters during the PCR cycle, the desired product was not received. Therefore, based on the result of gene screening (Table 3.8), the sequences of contig 12 000046, contig000433 and contig020504 were synthesized (GenScript) and inserted into cloning vector pUC57. The genes that were selected in addition to belonging to the serpin family (serine protease inhibitor), but also have some advantages over other contigs such as: new genes, complete genes less than 85 % similarity with genes related to protease inhibitors published in NCBI gene bank), predicted molecular weight of the proteins about 30-55 kDa (convenient for expression, purification and recovery of recombinant proteins). In addition, these contigs have high Uni-evalue. The contig 000046, contig000433 and contig020504 were named PI-QT, PI- QT1 and PI-QT2, respectively. During the study, the genes PI-QT, PI-QT1, PI-QT2 were successfully inserted into expression vectors pET-32a(+) and pPIC9 to form recombinant vectors. However, the PI-QT1 and PI-QT2 proteins expressed in the E.coli BL21 (DE3) cells in the form of inclusion bodies and were not expressed in P. pastoris SDM 1168. Only PI-QT protein expressed in both E. coli and P. pastoris systems in the soluble state. Therefore, in this thesis, we only report on PI-QT gene expression in the two expression systems. 3.4.1. Amino acid sequence and phylogeny analy- sis of protein PI-QT The gene PI-QT was 1,287 bp in length and had an open reading frame of 429 amino acid with a calcu- lated molecular mass of about 50 kDa and a theo- retical isoelectric point of 4.56. Comparison of the deduced amino acid sequence of PI-QT with the se- quences in the NCBI database showed that protein PI-QT was most similar with serpins with similari- ties <55 %. Multiple alignments of the deduced amino acids of PI-QT with the most homologous serpins in NCBI database showed that the deduced peptide PI- QT shared conserved active site residues with micro- bial serpin members. 13 The phylogenetic tree based on the neighbor-joining method located the protein PI-QT between two microbial ser- pin clades: one serpin clade from a candidate phy- lum of bacteria (Poribacteria) originally identified in the microbiome of marine sponges and another serpin clade from bacterial phyla Firmicutes and Cyanobac- teria. Based on the database comparison, the protein PI-QT was considered as a new microbial serpin. The protein structure of PI-QT was predicted by SWISS- MODEL and (PS)2-v2 model. The sequence of the gene PI-QT was deposited in the National Center for Biotechnology Information (NCBI) database with the accession number MK359987. 3.4.2. Construction of the expression vector pET-32a(+)/PI-QT The PI-QT gene was successfully inserted into expression vector pET-32a(+) to form the recombinant vector pET-32a(+)/PI- QT 3.4.3. Expression of recombinant protein in E.coli strain BL21(DE3) The recombinant vector pET-32a(+)/PI-QT was transformed into E. coli BL21(DE3) by heat shock and incubated on LB/amp medium overnight. After that, some colonies were incubated in LB/amp broth until OD600 was about 0.8 - 1.0. IPTG was then added in culture and incubated at 37 o C for 4 hours. SDS-PAGE gel analysis of expressed protein profile showed the presence of an overexpressed protein band of 64 kDa, corresponding to the size of recombinant protein PI-QT with TRx-his-tag fusion (Figure 3.15, lane 3, 5, 7), whereas this foreign protein band was not present in negative control pET32a(+) (Figure 3.15, lane 1) with out PI-QT gene and samples containing the recombinant vectors but not induced with IPTG (Figure 3.15, lane 2, 4 and 6). 14 Figure 3.15: SDS-PAGE gel of expressed protein Lane M: marker protein (Novagen); lane 1: E. coli BL21(DE3) containing vector pET-32a(+); lane 2, 4, 6: E. coli BL21(DE3) containing the recombinant vector pET-32a(+)/PI-QT was not induced by IPTG; lane 3, 5, 7: E. coli BL21(DE3) containing the recombinant vector pET-32a(+)/PI-QT was induced by IPTG 1 mM at 37 o C. However, the recombinant protein was expressed mainly in the insoluble fraction. 3.4.4. Investigation of most suitable conditions for recovery highest soluble PI-QT recombinant protein Determination of suitable IPTG concentration for recombinant protein expression Investigating the expression of PI-QT protein at different IPTG concentrations (0; 0.5; 1.0 and 1.5 mM); OD600 before induction about 0.8-1; induction at 37 o C; for 4 hours. The SDS-PAGE gel analysis showed that recombinant protein was expressed at 1 mM IPTG, but not at lower or higher concentration of IPTG (0, 0.5 and 1.5 mM). However, the expressed protein still was present mainlyin the insoluble fraction. Determination of suitable temperature for formation ofrecombinant protein in the soluble state The expression of the recombinant protein was also investigated at different temperature (20, 25, 28, and 30 oC), OD600 before induction Protein PI-QT qt 15 was about 0.8-1; IPTG concentration for 1 mM. The obtained results showed that the amount of recombinant protein in soluble fractionreached the highest value (about 45 % of total protein) when the expression process carried out at 25 o C, where as the expression of recombinant protein was not observed at 20 o C. At 28 °C and 30 °C, the amount of recombinant protein is produced higher than when expressed at 20 and 25 °C; however, only a small amount of the recombinant protein was detected in the soluble fraction (only about 20 % of total protein). Determination of suitable cell density before induction for formation of recombinant protein in the soluble state The expression of the recombinant protein at different induction cell densities (OD600 = 0.4, 0.5, 0.6, and 0.7) was investigated. The results showed that expression of the protein was not observed at OD600 = 0.4. The total amount of produced protein and protein content in the soluble fraction were increased with increasing pre-induction cell density and reached the highest values at OD600 = 0.6 - 0.7 (409 mg/L with 90 % in the soluble fraction) (Figure 3.19). Figure 3.19: Influence of induction cell density on expressionof the recombinant protein. (A) SDS - PAGE gel of protein, lane M:marker protein (Thermo, USA); lane 1: total protein of E. coli BL21 (DE3) [pET-32a (+)/PI- 16 QT]; lane 2: total protein of E. coli BL21 (DE3) [pET-32a (+)/PI- QT] in insoluble fraction; lane 3: total protein of E. coli BL21 (DE3) [pET-32a (+)/PI-QT] in soluble fraction. (B) Amount of total protein and percentage of protein in soluble fraction at different pre-induction cell density. 3.4.5. Purifcation of the recombinant protein in E. coli and identification by mass spectrometry The recombinant protein was purified by Ni-NTA afnity chromatography column using imidazole as eluent. At 100 and 300 mM imidazole,the recombinant protein was recovered higher than when using 500 mM; however, at these concentrations, protein purity was low. In the case of imidazole concentration of 500 mM, the amount of the obtained protein was lower but had higher purity than those of the two above cases (Figure 3.20). Figure 3.20: Influence of pre-induction cell density on expression of the recombinant protein. (A) SDS - PAGE gel of protein, lane M:marker protein (Thermo); lane 1: total protein of E. coli BL21(DE3) [pET-32a(+)/PI-QT]; lane 2: total protein of E. coli BL21(DE3) [pET-32a(+)/PI-QT] in insoluble fraction; lane 3: total protein of E. coli BL21(DE3) [pET- 32a(+)/PI-QT] in soluble fraction. 17 (B) Amount of total protein and percentage of protein in soluble fraction at different pre- induction cell density. Figure 3.21: (A) Western blot analysis Lane M: marker protein (Bio Basic); lane 1: the recombinant protein containing Trx-his-tag tail was hybrid to antibody anti-Trx. (B) SDS- PAGE gel of recombinant protein was removed Trx-his-tag by thrombin, lane M: marker protein (Bio-Basic); lane 1: the recombinant protein was not removed Trx-his-tag; lane 2: the recombinant protein was removed Trx-his-tag. To confirm that the purified protein was the protein PI-QT, the Western blot assay was performed using anti-Trx antibody. The Western blot analysis showed the presence of a protein band of 64 kDa on the hybrid membrane that was similar to the size of protein PI-QT with Trx-his-tag fusion (Figure 3.21A). In addition, in order to remove the Trx-his-tag from the recombinant protein PI-QT, the protein was cut off from Trx-tag by thrombin. SDS-PAGE gel analysis of the protein treated with thrombin showed 2 bands of 50 kDa and 14 kDa, which were corresponded to the sizes of protein PI-QT andTrx-his-tag, respectively (Figure 3.21 B). This revealed that the Trx-his-tag was successfully removed from the recombinant protein. 20 kDa 50 kDa 64 kDa 85 kDa 85 kDa M 1 50 kDa (a) (b) 1 2 M PI-QT 18 Identifion of the protein of interest by mass spectrometry MS, shows that the recombinant protein was designed having the same sequences as the theory of gene coding. PI-QT amino acid sequence after identification: MTKQLINATIIACVGLIYLLGCSGEEDVLHEDELLEMSLEEGAPTAPDGCAILAKPAGF TDNALSEANAKFGFKLLAELYNQKPNKNVFISPLSISIALTMTYNGARGATKQAMAK TLEIEGMDLGAVNQANAELREILKSADPQIELAIANSIWLRNTFDEVNPDFLDRNDRF FGAEIASLDFDDPQTVETINQWVNTNTQGKIKEILGEIEEHEVMFLINAIYFKGGWKG KFDASKTQDGVFHLLDGGEKQVPMMSHTCNYSYLESGDFRAVGLPYGEGRVSMY IFLPEHSSNLDEFLADLNAENWENWLSRFWNERDLRFIMPRFRLEYGMLLNDALKA 3.4.6. Activity of the recombinant protease inhibitor The inhibitory activity of PI-QT recombinant protein for proteases: trypsin, ɑ-chymotrypsin, thermolysin (Sigma) was determined by testing on agar plate with 1%skimmed milked, determined inhibition rate using BAPNA substrate and quantification of inhibition activity by the method of Jiang et al. (2011). As a result, the PI-QT protein at concentration 468 µg strongly inhibited trypsin (87 %; Ki = 1.33×10 -8 M), weak inhibiting ɑ-chymotrypsin (51 %; Ki = 2.87×10 -8 M) and does not inhibit thermolysin. Specific activity of the protein against trypsin and a-chymotrypsin was 975 ± 26 U/mg and 417 ± 14 U/mg, respectively. Compared to protein PI-QT, BBI (positive control) showed better inhibitory effect against trypsin and a- chymotrypsin with specifc activities of 3303 ± 66 U/mg and 1340 ± 58 U/mg, respectively. 3.5. Expression of recombinant protein in Pichiapastoris SMD1168 3.5.1. Construction of the expression vector pPIC9/PI-QT PI-QT gene was successfully inserted into expression vector pPIC9 to form the recombinant vector pPIC9/PI-QT 3.5.2. Create Pichiapastoris SMD1168 cell line containing PI-QT gene 19 Successfully created P. pastoris SMD 1168 cell line containing recombinant vector [pPIC9/PI-QT]. Test results on MD, MM and colony PCR with primers AOX1 (Pichia expression Kit, Invitrogen) showed that the recombinant P. pastoris lines had Mut s phenotype. 3.5.3. Expression of PI-QT gene in P. pastoris SMD1168 Expression of P. pastoris SMD 1168 [pPIC9-PI-QT] lines according to the manufacturer's instructions (Pichia expression kit, Invitrogen) for Mut s phenotypes, induction with 0.5 % methanol, shaking 200 cycles/minute at 28 o C. Results on 12.6 % SDS-PAGE gel and coomassie staining showed that the PI-QT protein was synthesized with M.W about 50 kDa in size, corresponding to the theoretical size of PI-QT protein at 24, 48, 72, 96 and 120 hours of culture. The highest protein content was obtained at 72 hours after induction and decreased after 96 hours induction (Figure 3.28). Figure 3.28. SDS-PAGE of the PI-QT protein expressed in P. pastoris SMD1168. M: Marker protein (Affymetric) 1: Non-induction; 2: 0 h after induction. 3-8: Induced (3: 24 h; 4: 48 h; 5: 120 h; 6:72 h; 7, 8: 96 h after induction) Protein PI-QT 20 3.5.4. Assessment of protease inhibitory activity of PI-QT protein expressed in yeast The extracellular protein of P. pastoris SMD1168 containing PI-QT and without PI-QT genes after 72 hours induction by methanol was tested for protease inhibitory activity against trypsin, ɑ- chymotrypsin and thermolysin. The results showed that, the extracellular secretion of yeast line without the PI-QT gene, there was no inhibitory activity against all three proteases tested was observed. Mean while, the extracellular secretion of P. pastoris SMD1168 containing the PI-QT gene exhibited inhibitory activity against all three protease enzymes. Among them, inhibition was strongest for trypsin, weaker for ɑ-chymotrypsin and very weak for thermolysin (Figure 3.30). Figure 3.30. Qualification of proteases inhibition ability of the expression proteins 21 a, b, c corresponding to inhibition tests with trypsin (Try), ɑ- chymotrypsin (ɑ-chy) and thermolysin (Ther). The protease concentration in each well was 0.01 mg); PI: recombinant protein PI-QT (PI 1: 0.4 mg PI-QT protein; PI 2: 1.0 mg PI-QT protein; PI 3: 2.0 mg PI-QT protein) d. Protease inhibition activity of the raw secretion of P. pastoris SMD1168 without PI-QT gene (N: Water; 1: Trypsin;