Fiber Pile Of Capsule Of Bacterium Cellulose Enzyme Complex So9733-1 Proteomics Analysis And Enzyme Gene Cloning, Characterization

Myxobacteria are Gram-negative gliding bacteria, which exhibit complicated multicellular social behavior and produce large numbers of bioactive compounds that have unique structures and diverse functions. The principal habitats of myxobacteria are soil, dung, decaying plant material, and the bark of living and dead trees. Based on their degradation ability for biomacromolecular, myxobacteria can be divided into two groups:cellulolytic myxobacteria and bacteriolytic myxobacteria.Within cellulolytic group, there are two genuses named Sorangium and Byssophage, in which Sorangium cellulosum is the type species of Sorangium.Studies on S. cellulosum have mainly been focused on its excellent production ability of diverse and novel bioactive secondary metabolites, whereas the degradation ability on macromolecules, the unique and basic property of S. cellulosum, have received less attentions and none of the involving enzymes has been characterized so far. S. cellulosum can grow on simple inorganic salt medium （e.g. CNST medium） with sterilized filter paper as the sole carbon source and can degrade complex polysaccharides such as cellulose, solulable starch and xylan efficiently.Our previous studies showed that a high-molecular weight component about 1500-2000 kDa with multi-enzymatic activities （e.g. endo-glucanase, exo-glucanase, xylanase, Glucosidase et al.） was linked to the cellular surfaces and might organize into a large complex.In this paper, we isolated and identified the components of the multi-enzyme complex using proteome technique, and we proceded to clone and characterization the enzymes in the complex. The work will help us to understand the organization strategy and polysaccharides degrading mechanism of the complex.First, we analyze the components of the multi-enzyme complex utilizing classical proteome technique:Two-dimensional polyacrylamide gel electrophoresis （2D-PAGE） and mass spectrometry （MS）. We successfully separate the proteins in the complex by 2D-PAGE. It was found that the isoelectric point （pI） of proteins are mainly on the acid side, and on the alkaline side, there are rare proteins.30 protein spots were excised from coomassie brilliant blue-stained gel, conducted in-gel digestion, and then analyzed using MALDI-TOF. Among the available mass spectrums of the 21 proteins, only one matched the endo-glucanase from Ralstonia solanacearum.We proceed to excise the 30 protein spots mentioned above, and then analyzed using tandem mass spectrometry （linear trap quadrupole, LTQ）. Finally, base peak chromatograms of 5 proteins were obtained, and one protein was identified to beα-amylase with the peptide sequence of R.VVVARPAPGAAIR.R.Considering the limitations of the classical proteome technique （extremely acid, alkaline, or hydrophobic protein etc. can’t be separated by 2D-PAGE）, we used shot gun proteomics to globally isolate and identify the components of the multi-enzyme complex from So9733-1. Finally, a number of cellulase, amylase, mannanase, and xylosidase were identified, and it is correspondent with the phenomenon that Sorangium cellulosum can efficiently degrade complex polysaccharides such as cellulose, solulable starch and xylan.By shot gun proteomics analysis of the multi-enzyme complex, two peptides （K.EPFEVCIDDIR.L and K.YDALAYFYHNR.S） were identified to match the cellulase （YP₀01612873, coded by gene sce2234） from So ce56. Among all the predicted cellulases of So ce56, only this protein has CBM domain. Using the sequnces of the two peptides and gene sce2234, we designed PCR primers and obtained a 670-bp cellulase-related fragment by Touch-down PCR. The nested insertion-specific primers were designed based on the 670-bp fragment, and the flanking regions of the fragment were obtained using thermal asymmetric interlaced （TAIL）-PCR. By assembling the sequences, the full-length of the ORF （named celA） was obtained.The gene celA consist of 2,373 bp encoding a 790-amino-acid protein with a calculated molecular mass of 84.9 kDa and a theoretical isoelectric point of 6.63. A 41-residue signal peptide was present at the N terminus of the protein predicted by SignalP. The mature CelA protein contained CBM₄₉ at N-terminus, a conserved Cellulase N-domain and a catalytic domain of the glycoside hydrolase family 9. The ORF encoding the mature cellulase was subcloned into pET-28a（+） and expressed in Escherichia coli BL21 （DE3）. After induction with 0.5 mM IPTG at 18℃for 20 hr, the recombinant CelA was expressed in the supernatant of the cell lysates with cellulase activity.Characterization analysis suggested that the optimum pH and temperature for the crude enzyme were 7.0 and 35℃, respectively. Crude CelA exibit activities ofβ-1,3-1,4-glucanase,β-1,4-glucanase,β-1,3-glucanase, and xylanase. Ca2+ enhanced CelA activity by about 70% at 1 mM concentration and Cu2+, Zn2+ strongly inhibited the CelA activity.We also amplified the ORF coding the mature cellulase without the domain CBM₄₉, and expressed in Escherichia coli BL21 （DE3）. After induction with IPTG, the supernatant of the cell lysates had no cellulase activity. We presumed that CBM₄₉ was essential for the enzyme to function properly.In the proteomics analysis of the multi-enzyme complex, a peptide （R.GIVPVLWDTGTDIK.R） was obtained to match the protein （YP₀01611539, coded by gene sce0902） from So ce56. Based on the sequnce of the gene sce0902, a 384-bp DNA fragment was amplified from So9733-1 by Touch-down PCR. To obtain the whole sequence of the gene （named amyA）, the TAIL-PCR technique was employed to amplify the 5’and 3’flanking sequences of the fragment.Finally, the full-length of the gene amyA containing 1,329 bp was obtained. The gene encoded a 442-amino acid protein and a putative 38-amino acid signal peptide. The estimated molecular weight of Amy was 46.2 kDa and the theoretical isoelectric point was 4.59. The mature protein contained a single catalytic domain similar to proteins from the glycoside hydrolase family 5 and the endoglucanases of COG2730. The gene coding the mature AmyA was ligated into the expression vector pET-22b（+）, and expressed in Escherichia coli BL21 （DE3）. After induction with 0.5 mM IPTG at 18℃for 20 hr, the recombinant protein was expressed in cell lysate supernatant with amylase activity.The recombinant protein was purified to electrophoretic homogeneity by Ni-affinity chromatography and subsequently characterized. The recombinant AmyA had optimum activity at pH 6.5 and 40℃. AmyA exibit high activity of amylase, and after incubation for 12 hr, activities ofβ-1,3-1,4-glucanase,β-1,4-glucanase, (3-1,3-glucanase, and xylanase were detected. AmyA was sensitive to mental irons and chemicals:K+, Ca2+ and Mg2+ at 1 mM concentration can slightly inhibit the activity while Cu2+, Zn2+ and EDTA at 1 mM concentration strongly inhibited the enzyme activity.A 422-bp xylanase fragment was obtained in our previous research. Using the antibodies of the fragment, the xylanase was confirmed to be organized in the purified cell-bound cellulolytic enzyme complex by Western blotting and located on the cell surface protuberances by immunofluorescent staining. We cloned the xylanase gene （xynA） by Tail-PCR based on the 422-bp xylanase fragment. Characterization analysis suggested that the purified recombinant XynA was a cold-active xylanase with low thermostability.The gene was composed of 1,209 bp and had only 52.27% G+C content, which is much lower than that of myxobacterial DNA reported （67-72%）. It is supposed that xynA may originate from other bacterial species with lower G+C content than that of S. cellulosum. The ORF encoded a predicted protein of 402 amino acids with a calculated molecular mass of 45.8 kDa and a theoretical isoelectric point of 4.83. SignalP analysis suggested that a 41-residue signal peptide was present at the N terminus of the protein. The mature XynA protein contained a single catalytic domain of the glycoside hydrolase family 10. Furthermore, homology modeling using SwissMode programs showed a typical （β/α）8 structure in the XynA catalytic domain.The xylanase gene fragment coding the mature xylanase, was subcloned into pET-22b（+） and expressed in Escherichia coli BL21 （DE3）. After induction with 1 mM IPTG at 18℃for 20 hr, the recombinant XynA was expressed in the supernatant of the cell lysates. The recombinant protein was purified by Ni-affinity chromatography.The optimum temperature for the XynA was 30℃, and XynA exhibited 33.3% activity at 5℃and 13.7% activity at 0℃. Approximate 80% activity was lost after 20-min preincubation at 50℃. These results indicated that XynA was a cold-active xylanase with low thermostability. At 30℃, the Km values of XynA on beechwood xylan, birchwood xylan and oat spelt xylanwere 30.74,32.77 and 41.87 mg/ml, respectively. The purified XynA displayed optimum activity at pH 7.0. The activity of XynA was enhanced by the presence of Ca2+. The recombinant XynA hydrolyzed beechwood xylan, birchwood xylan and xylooligosaccharides （xylotriose, xylotetraose, and xylopentose） to produce primarily xylose and xylobiose. As a typical cold-active xylanase, XynA is most active at low and intermediate temperatures and could offer advantages over the currently used xylanases in many of the low to moderate temperature processes, in particular in the food industry.Multi-clone antibody was produced with the purified XynA as the antigen, which could be used to analyse the location of the protein in So9733-1.As the cellulase CelA, amylase Amy A, and xylanase XynA are components of the multi-enzyme complex, molecular cloning and characterization of the enzymes will help us to reaveal the organization and function of the complex.