Assessment of the diversity and the role of some modules in the structure of cellulolytic enzymes of bacteria in thegoat’s rumen

rification of modular enzymes encoded by selected sequence (XFn3Egc) in fusion form with SUMO partner. 4. Investigating the role of functionally unknown domains on the cellulolytic activity of enzyme. 5. Determining some characteristics of recombinant enzyme which is expressed by using selected sequence encoding modular structure. 4. New contributes of the thesis 1. Based on 816 ORFs encoding cellulases of bacteria in Vietnam goat’s rumen that were mined from in metagenomic DNA data, 243 deduced modular cellulases had FN3 or Ig domains. Among complete cellulases containing FN3, 99.2% FN3 domains were found to be accompanied with betaglucosidase catalytic domains GH3 while only a FN3 module was determined to be collocated with endoglucanase catalytic domain GH5. Besides, all Ig modules were associated with endoglucanase catalytic domains GH9. It is rather uncommon to find endoglucanase GH5 collocated with FN3 domain. 4 2. For the investigation of the FN3 function in enzyme structure, genes encoding endoglucanase GH5 (XFn3Egc) was artificial synthezised and whole gene, and different modular structures (Fn3, XFn3, Fn3Egc, Egc) were expressed in E. coli, purified and functional characterized. FN3 module was determined to have ability to increase the solubility and stability of catalytic domain as well as to loosen crystal cellulose in filter paper surface to enable enzyme access on cellulose for hydrolysis. It also was found to increase affinity of enzyme to the soluble substrate as CMC. 3. The SXFn3Egc has optimal activity at 40 o C, pH 4 and stable below 60 o C in 90 minutes. The Km and Vmax of SXFn3Egc were 1.26 mg/ml and 148.12 µmol/min/ml respectively. This enzyme showed a 2-fold increase in catalytic activity at a concentration of 40 mM Mn 2+ . In contrast, the activity decreased that caused by using metal ions (Ca 2+ , Mg 2+ , Ni 2+ , K + , Co 2+ , Cu 2+ , Zn 2+ , Fe 3+ ) or chemicals (SDS, urea, 2-mercaptoethanol, EDTA, tween 80, triton X-100). CHAPTER 1. OVERVIEW 1.1. Cellulose Cellulose is a large molecular compound composed of a linear chain of β-D-glucose units, which is the main component of the plant cell walls. The use of cellulose as a renewable resource in several industries such as food processing, manufacturing of biofuels, pure chemicals has been a sustainable development tendency in the economy and environment. 1.2. Cellulase Cellulase is a primary group of enzymes, which are able to cut the β-1,4-glycoside bond of cellulose to release the high-value final product - glucose. Cellulase is often classified into three major groups (endoglucanases, exoglucanases, β-glucosidases) with different types of hydrolysis activity. Cellulase may have only 5 catalytic module in structure or contains extra domains such as CBM or functionally unknown domains (FN3, Ig). 1.3. Metagenomics in gene mining Metagenomics is described as a group of techniques in molecular biology, bioinformatics that allows studying the genomic diversity of most microbes recovered directly from environmental samples. Metagenomics is proved as an effective method to discover new enzymes, bioactive substances for many applications. In this study, we are going to mine new cellulases, especially modular cellulases (containing FN3, Ig modules) based on 816 ORFs encoding cellulases. This data was analyzed from 164,644 ORFs and assembled from 8.46 Gb of bacterial metagenomic DNA in Vietnam goats rumen. CHAPTER 2.MATERIALS AND METHODS 2.1. Materials, chemical and equipment  Materials: The 816 ORFs encoding cellulases from bacterial metagenomic DNA in goat’s rumen.  Microorganisms, plasmids of Invitrogen (USA), PCR primers of GenScript (USA); chemicals of Bio-Lab (USA), Fermentas (USA), Sigma (USA), Merck (Germany). 2.2. Methods 2.2.1. Molecular biology techniques, microorganisms Transformation of plasmid DNA into E. coli (Froger et al., 2007); Extraction of plasmid DNA from E. coli and electrophoresis on agarose gel (Sambrook et al., 2001); DNA was purified from agarose gel by the DNA kit Qiagen - QIAquick Gel Extraction Kit; Optimizing of triplet code was carried out based on online software of Genscript (Rare Codon Analysis Tool). 2.2.2. Protein biochemical methods Recombinant proteins were purified by affinity chromatography column Ni-NTA (Invitrogen) and evaluated the 6 purity by Quantity One (Bio-Rad); protein was quantified by using Bradford method (Bradford, 1976); determination of endoglucanase activity on CMC substrate (Miller, 1959) and filter paper (Camassolaet al.,2012) with some slightly modification; determination of cellulase activity on agar-CMC agar plates (Teather et al., 1982) and Zymogram analysis (Champasri et al., 2015); the effect of the enzyme on the surface of filter paper was evaluated by taking pictures on SEM scanning electron microscopy (Kataeva et al., 2002). 2.2.3. Bioinformatics methods The sequences containing Pfam and conservative regions were studied using Pfam database ( and BLASTP ( respectively. For the prediction of tertiary structure of enzymes, two distinct online software Phyre2 and Swiss model were used; AcalPred software was applied to predict of acidic and alkaline enzymes; the thermostability of a protein was estimated by TBI software. 2.2.4. Data processing Statistical methods, Microsoft Excel were used to calculate and show the results as ± SE (Standard Error) CHAPTER 3.RESULTS AND DISCUSSION 3.1. The GH diversity and modular structure of cellulases deduced from 816 open reading frames 3.1.1. Evaluation of the diversity and structure of GH cellulase families The 816 ORFs encoding cellulases were functionally annotated that belonged to 11 distinct GH families (Table 3.1). In particular, GH3 (400 ORFs) and GH5 (192 ORFs) were the most popular families which accounted for 49% and 23.5%, respectively. 297 complete sequences were found to exist in the form that has unique domain for the catalytic function or contains additional 7 functionally unknown modules (FN3, Ig). Specifically, 90.9% GH3 and 100% GH9 contained FN3 and Ig respectively. Besides, only one FN3 module was collocated with GH5 domain. Therefore, FN3 and Ig modules were not only basically linkers but also shown some biological functions that have not been clearly defined. Table 3.1. Summary of sequences encoding cellulases based on COG và KEGG databank GH Module ORFs GH Module ORFs GH1 GH1 16 GH16 GH16 33 GH3 GH3 198 GH16-CBM4 2 Fn3-GH3 202 GH44 GH44 2 GH5 GH5 189 GH48 GH48 1 Fn3-GH5 1 GH64 GH64-CBM6 1 GH5-CBM2 1 GH74 GH74 1 GH5-CBM37 1 GH94 GH94 50 GH8 GH8 48 CBM63 CBM63 1 GH9 GH9 11 FN3 FN3 10 GH9-Ig 30 - - 14 GH9-CBM3 2 GH9-CBM37 1 GH9-dockerin 1 3.1.2. Evaluating the diversity of structures of completed modular cellulase The investigation of 243 ORFs encoding modular cellulases showed that 148 ORFs had a completed structure (131 ORFs encoding enzymes contain FN3 domain, 17 ORFs encoding enzymes have Ig domain). Specifically, 17 ORFs encoding endoglucanases GH9 contained Ig module (Ig-GH9); 130 ORFs (in total 131 ORFs encoding enzymes contain FN3 domain) are responsible for encoding beta-glucosidase GH3 (GH3-Fn3) and only one endoglucanase GH5 domain was accompanied with Fn3 domain (Fn3-GH5). The FN3 module situating in front of catalytic domain of endoglucanase GH5 at N-terminal is known as an uncommon structure. Therefore, it is 8 necessary to study the role of this module in the efficiency of enzymes hydrolysis. 3.1.3. Evaluation of diversity of the ORFs encoding for cellulase For clearly understanding about the bacterial community and their role in the digestion of cellulose in Vietnam goat's rumen, we have identified the origin of 816 ORFs encoding cellulase. In particular, 221 ORFs encoding cellulase mainly belonged to Bacteroidetes (153 ORFs) and Firmicutes (53 ORFs) accounted for 69.2% and 24.0%, respectively. Bacteroidesuniformis (29 ORFs), Prevotellabuccal (25 ORFs) were the most dominant species that containing genes coding cellulase; Ruminococcus flavefaciens (7 ORF) was determined as a typical species belonged to the cellulolytic bacterial group with high efficiency of cellulose biomass hydrolysis. 3.1.4. Evaluation of the similarity of amino acid sequences deduced from annotated ORFs encoding cellulase Based on two NR and CAZy, 297 completed ORFs encoding cellulases were demonstrated the similarity below 85% (new sequence) accounted for 80.1% and 77.4% respectively. By investigation of 148 completed ORFs encoding cellulase with modular structure, 17 completed ORFs encoding endoglucanase containing Ig module were firstly reported; 131 completed ORF encoding cellulase having FN3 module, in which 90 sequences were initially studied accounting for over 68% (89 ORFs encoding beta- glucosidase, 01 ORF encoding endoglucanase). Thus, this data is expected to exploit numerous new genes, especially the completed sequences encoding cellulases containing modules such as FN3, Ig. 3.1.5. Prediction of properties of enzymes based on sequences Rapid prediction of some optimal conditions for enzyme activity such as pH range, temperature, pI value is necessary to 9 initially screen the prominent genes and study their application. The investigation of some properties of modular cellulases based on 243 ORFs showed that most enzymes (from 130 ORFs) were stable at 55-65 o C, meanwhile, cellulases encoded by 139 ORFs maintain activity at alkaline pH and 146 enzymes have pI above 5-6. By the survey of the pI values of 148 completed sequences containing FN3 and Ig-like domains, two sequences (an Ig-GH9 and a FN3-GH5) were determined to have pI higher than 9. 3.2. Selection of sequences of the typical modular enzymes to investigate the role of modules 3.2.1. Investigation of the three-dimension structure of enzyme containing FN3 modules The existence of FN3 module in GH5 endoglucanase was found to be a rare structure compared to common Fn3-GH3 structure, which was selected for the study. The gene sequence encoding for mature endoglucanase had a length of 1545 nucleotides. The results of homologous comparative analysis by BLASTN and classification by MEGAN software showed that this gene was predicted to be derived from Ruminococcus bicirculans. The amino acid sequence of endoglucanase GH5 analyzed using BLASTP software exhibited the most similarity 60% with endoglucanase code CDC67342.1, which is commonly found in Ruminococcus sp. CAG: 57, a bacterial species of goat rumen. By survey the conserved region by SwissProt software, the sequence showed the highest similarity (49%) with the frame of endoglucanase 3pzt.1.A with the recovery of 53%. It was also indicated as monomer structure with a ligand of Mn ++ (Figure 3.7). Using the Phyre2 tool, the sequence displayed the highest similarity with the c3pzvB endoglucanase frame with the confidence of 100 %. Besides, it had a separated functional region and N-terminal region including separated FN3 structure (Fig 3.8). 10 3.2.2. Prediction of pI and pH values of enzyme containing FN3 module based on sequences Gene sequence encoding endoglucanase GH5 which contains FN3 module was estimated to have pH optimum at acidic pH, be stable at below 55°C and have high homogeneous pI values in both unknown functionally regions (X domain, FN3 module) and the active site (Egc). By using of pI values of general enzyme molecule as well as each the homogeneous module, the expression and optimization of some conditions for enzyme hydrolysis become more convenient. 3.3. Cloning of XFn3Egc gene 3.3.1. Analysis of optimal triplet code of XFn3Egc sequence The sequence encoding endoglucanase GH5 (XFn3Egc) was optimized to have the best utilization rate of 97% compared to 46% before optimization. After optimization, 86% of the sequence showed relevance in the range of 91-100%, compared to the sequence before optimization with only 49%. The gene sequences before and after optimization for expression on E. coli are described in Figure 3.10. The optimized XFn3Egc gene was artificially synthesized and inserted into pET22b (+) at the NcoI+XhoI restriction site to generate a vector named pET22-XFn3Egc. Figure 3.7. Prediction of conserved regions by SwissProt Figure3.8. Prediction of conserved regions by Phyre2 Mn 11 Figure 3.10. Gene sequence of XFn3Egc before (A) and after performing the codon optimization for expression in E. coli (B) (yellow region is FN3 sequence; blue region is actived region; red letters are optimal sequences) 12 3.3.2. Design of pETSUMO expression vectors containingFn3, Egc, Fn3Egc, XFn3, XFn3Egc genes The genes Fn3, Egc, Fn3Egc, XFn3, were amplified from the pET22-XFn3Egc template BY PCR, then the target genes and XFn3Egc were cut by two restriction enzymes NcoI-XhoI and inserted into pET22b (+) at the same restriction sites to create recombinant vectors respectively.After that, the genes in expression vectors were sequenced and expressed in E. coli. However, they were expressed at a low level and almost existed in inclusion body. Therefore, the genes were transferred from pET22b(+) to pETSUMO using NcoI and XhoI restriction enzymes to generate pET22SUMO- Fn3, pET22SUMO-Egc, pET22SUMO-Fn3Egc, pET22SUMO- XFn3Egc and pET22SUMO-XFn3. All the plasmids then were transformed into E. coli DH10B for cloning. The transformant colonies were inoculated in LBA medium to screen E.coli strains containing recombinant plasmids. These plasmids are linearized by using single restriction enzymes,whereas, when they were cut by two restriction enzymes, the generated genes showed the correct sizeas calculated. Thus, the expression vectors carrying the target genes have been successfully designed. 3.4. Expression of recombinant E. coli strains carrying the target genes After determiningthe optimal conditions for expression, the recombinant E. coli BL21 (DE3) strains were inoculated in LB medium containingampicillin at 25°C, induced with 0.5 mM IPTG, and cultured for 5 hours. The result of protein analyzing by polyacrylamide gelelectrophoresis showed that all 5 types of recombinant proteins were expressed at high level and had the correct size as calculated. Almost proteins were found in the soluble 13 form except the Egc without FN3 existing in insoluble fraction(Figure 3.18). Figure 3.18. Analysis of SFn3, SEgc, SFn3Egc, SXFn3Egc, SXFn3 expressed in E. coli BL21 (DE3) strains in total, soluble and insoluble fractionson 12.6% polyacrylamide gel containing SDS. Negative control: pETSUMO; Marker: unstained protein standard (A) (B) Figure 3.19. Analysis of proteinsin soluble fractions by non-denaturing polyarylamide gel electrophoresis (A) andzymogram (B); Marker: unstained protein standard (Thermo scientific); cellulase: possitive control (Sigma) Total Soluble Insoluble 14 The soluble fractions of recombinant proteinswere determined cellulase activity on the CMC plates. The results illustrated that only SXFn3Egc clearly exhibitied the hydrolysis activity on CMC substrate. On the other hand, by using zymogram assays, all 4 proteins (SFn3, SFn3Egc, SXFn3Egc, SXFn3) were migrated to the correct position on the polyacrylamide gel stained by Coomassie brilliant blue despite they were separated in the gel under non-reducing conditions. On the gel stained by Congo red, a bright band can be visualized in SXFn3Egc lane which was similar to the one in thelane of positive control (Figure 3.19). Thus, XFn3Egc enzyme was successfully expressed by XFn3Egc gene with the correct size as calculated and demonstrated the CMC hydrolysis. The domain FN3 was determined to increase the solubility of catalytic region. The X domainalso contributes to increasethe cellulase activity of catalytic region. 3.5. Recombinant proteinspurification and determination of cellulase activity 3.5.1. Purification of recombinant proteins The recombinant proteins were completely elutedby elution buffercontaining 400 mM imidazole. The results of analyzing the eluted fractions by SDS-PAGE showed that only one band was detected which was similar in size to the target proteins.The purity of recombinant proteins evaluated by the Quantity One 1-D Analysis softwarewas over 99%. Figure 3.20. SDS-PAGE analysis of the fractions collecting from SFn3 purification (F1-F9) Figure 3.22. SDS-PAGE analysis of the fractions collecting from SFn3Egc purification (F1-F7) 15 Figure 3.24. SDS-PAGE analysis of the fractions collecting fromSXFn3Egc purification(F1- F10) Figure 3.26. SDS-PAGE analysis of the fractions collecting fromSXFn3 purification(F1-F8) 3.5.2. Evaluation of the enzyme activity after purification 3.5.2.1. Evaluation of hydrolysis activity of recombinant proteins on CMC After purification, SFn3, SFn3Egc, SXFn3Egc, SXFn3 were evaluated their hydrolysis in CMC. In which, SXFn3Egc and SFn3Egcclearly exhibited catalytic activity. The hydrolysis zones of SXFn3Egc was larger than the hydrolysis zoneof SFn3Egc (having higher enzyme activity). 3.5.2.2. The analysis of the ability of SFn3 and SXFn3 to promote CMC hydrolysis activity Mixing of functionally unknown proteins (SFn3, SXFn3) and enzymes containing catalytic region increased CMC hydrolysis activity. The combination of SFn3, SXFn3 with SFn3Egc led to the increase in enzyme activity which accounted for 74.5% and 49.9%, respectively while the CMCase activity illustrated an increase of 27 % by mixing SXFn3, SFn3 and SXFn3Egc (Figure 3.29). The results showed that the FN3 domain significantly affected the hydrolysis rate of enzymecompared to the single enzyme. This can be assumed 16 that the FN3 domain mayinteracttoCMC and help to increase the affinity of enzymeforits substrate. Figure 3.29. Hydrolysis activities of single proteins and mixture of proteins after purification in CMC; Possitive control (ĐC+): Cellulase (Sigma) 3.5.2.3. The SFn3 and SXFn3 increased activity of enzymes to hydrolysis of filter paper On the filter paper, the hydrolysis activity of SXFn3Egc mixing with SFn3, SXFn3 demonstrated an increase of 86.8% and 13.6% respectively, compared to single SXFn3Egc. The combination of SFn3Egc with SFn3 or SXFn3 also help to increase the enzyme activity in comparison with single SFn3Egc, however, the difference was not statistically significant. This result showed that both SFn3 and SXFn3 helped to increase cellulose hydrolysis activity of enzyme in filter paper. SFn3 and SXFn3 do not exhibit cellulase activity, therefore, these proteins only help SXFn3Egc and SFn3Egc to increase enzyme activity. The domain FN3 increased catalytic activity of protein containing FN3 module in filter paper by two hypothesized reasons: (1) FN3 module loosedthe cellulose crystalline structures of filter CMC substrate 17 paper for enzymeseasilyaccessing into cellulose fibers then hydrolysis; (2) FN3 module increased affinity between enzymes and cellulose and helped the enzyme to anchor to substrate. Figure 3.30. Hydrolysis activities of single protein and mixture of proteins after purificationinfilter paper; Possitive control (ĐC+): Cellulase (Sigma) 3.5.3. FN3 module increased affinity of enzyme for the substrate 3.5.3.1. CMC There was an increase in the cellulase activity of SXFn3Egc, SFn3Egc in CMC treated by SFn3 and SXFn3(Figure 3.31). By using CMC treated by SFn3 or SXFn3, SXFn3Egc exhibited the catalytic activity stronger than SFn3Egc. The activity of SXFn3Egc and SFn3Egc in CMC treated by SFn3 illustrated an increase of 31.5% and 23.8%, respectively. However, the activity of two enzymes in CMC treated by SXFn3 slightly raised to7.3% and 5.9%, respectively. Thus, SFn3 and SXFn3 demonstrated the ability to promote catalytic activity by increasing the affinity of the enzyme for CMC. Based on protein bands visualized in the native gel with the presence of CMC as substrate, both SFn3 and SXFn3 showed the Filter paper substrate 18 ability to bind and hydrolyze CMC. SFn3 and SXFn3 may increase the affinity of enzyme for its substrate, so the hydrolysis activity of enzyme was stronger than this of the single enzyme. The mixture of SXFn3Egc and SFn3 completely reacted with CMC, soit was not visualized on the gel compared to the mixture of SXFn3Egc with SXFn3 (Figure 3.32). This is a reason why SFn3 increased the catalytic activity of enzyme better than SXFn3. Figure 3.31. The role of SFn3 and SXFn3-treated CMCin catalyticactivities of SFn3Egc and SXFn3Egc Figure 3.32.Analysis of the ability of SFn3, SXFn3 to increase the affinity between enzyme and CMC; M: Standard protein scale (Thermo Scientific) C M C a d so rb ed W it h S F n 3 C M C a d so rb ed W it h S F n 3 C M C a d so rb ed W it h S X F n 3 C M C a d so rb ed W it h S X F n 3 Substrate In PBS CMC 1% in PBS 19 3.5.3.2. Filter paper The catalytic activity of SXFn3Egc revealed an increase by using the filter paper pretreated by SFn3 and SXFn3. In particular, the activity of SXFn3Egc in filter paper treated by SFn3 washigher than in the filter paper treated by SXFn3 (Figure 3.33). However, the activity of the SFn3Egc in filter paper treated by SFn3 and SXFn3 insignificantly changed compared to native filter paper. This indicated that the absorption of SFn3 and SXFn3 on the filter paper surface (especially SFn3) may help to increase catalytic activity of SXFn3Egc. Figure 3.33. The role of SFn3 and SXFn3-treated filter paper incatalytic activitiyof SFn3Egc and SXFn3Egc; FP: Filter paper (Whatman No. 1) 3.5.3.3. Analysis of the ability of SFn3 and SXFn3 to loose crystalline structure in filter surface The filter paper was treated by SFn3 and SXFn3 then scanned by electron microscopy at 500x and 1000x magnifications. Many loosen, separated and exfoliated cellulose fibers were seen in the SNF3 and SXFn3-treated papers compared to the native filter papers (Figure 3.34). At 5000x magnification, the surface of filter papers treated by FN3 were not as smooth as the untreated samples. F P a d so rb ed ư it h S F n 3 F P a d so rb ed w it h S X F n 3 F P a d so rb ed ư it h S F n 3 F P a d so rb ed w it h S X F n 3 Substrate 20 Therefore, SFn3 and SXFn3 affected the catalytic activity of enzymes by loosing the surface of the filter papers. Figure 3.34. Scanning electron micrographs of filter papers (Whatman No. 1) untreated with SFn3 or SXFn3 (column 1) treated with SFn3 (column 2), treated with SXFn3 (column 3) at the magnifications: 500 times (row A), 1000 times (row B) and 5000 times (row C) 3.5.3.4. The effect of temperature, pH on the adsorption of SFn3 and SXFn3 on cellulose substrate In order to increase the adsorption of FN3 on the filter paper as well as cellulolytic activity, we assessed the effect of pH, temperature on the filter paper absorption of the proteins. The result showed that the adsorption of SFn3 and SXFn3 on filter paper reached the highest rate at pH 4 and decreased at pH 6 and pH 8. Besides, the absorption of both SFn3 and SXFn3 on filter paper at 40°C or 60°C was stronger than the absorptionat 20°C. 3.6. Evaluation of some properties of enzyme SXFn3Egc 3.6.1. Effect of pH 21 The optimum pH value for SXFn3Egc activity was 4. The enzymes showed strong activity in the pH range 3.0-5.0. At pH 3 and pH 5, the activity of SXFn3Egc was approximately 80% compared to its activity at pH 4. The catalytic activity showed a gradual decrease in the pH range 7.0-9.0. 3.6.2. Effect of temperature In the temperature range 30 - 60 o C, SXFn3Egc had different levels of activity: From 30 o C to 40 o C, enzyme activity increased and reached the highest value at 40 o C; in the range 45-50 o C, the enzyme still exhibited strong activity (accounted for 84-88% of activity at the optimal temperature). However, SXFn3Egc activity decreased gradually at 55-60 o C. 3.6.3. Evaluation of the thermal stability The SXFn3Egc demonstrated thermostability by retaining the highest residual activity after 30 minutes and decreased gradually from 60 to 90 minutes in the temperature range 30 - 60°C. 3.6.4. Effect of some metal ions and chemicals The results o