| The area of oceans account for more than70percent of the earth’s surface and sea water accounts for97percent of total water on earth. Marine environment is an important part of the global ecological environment and marine carbon cycle plays a critical role in the total carbon cycle of biosphere Marine microorganisms decompose the organic carbon stored through photosynthesis of marine phytoplankton and photosynthetic bacteria into carbon dioxide, and thus play an important role in the marine carbon cycle. Xylan is a main component of hemicellulose and also the second abundant carbon source on the earth, thus represents an important kind of marine organic carbon. Compared to cellulose, the structure of xylan is more complex and needs the synergy of many xylanolytic enzymes to be complete degraded. β-1,4-endo-xylanase (EC3.2.1.8) can cleave the β-1,4-xylosidic bonds of the main chain of xylan and plays a key role in the xylan breakdown. Therefore, it has significance in the marine carbon cycle. Marine environment is generally characterized by high salinity and high fluidity, and the deep sea (more than2000m deep) is specifically characterized by high pressure, low temperature, low transmission of light and oligotrophy. Marine organisms have evolved to adapt the extreme environment and preserve many gene resources which may have great values. Therefore, oceans are reservoir not only for species but also for genes. Sequence analysis and function identification of these gene resources are significant for the study of biochemical processes in the marine environment.Glaciecola mesophila KMM241is a cold-adapted marine bacterium isolated from an invertebrate. Our previous study showed that G. mesophila KMM241could produce more than one kind of xylanases and possesses. We have cloned and characterized a xylanase XynA from this strain and studied the potential of XynA in improving the quality of wheat bread. In this study, the xylanase XynB from G. mesophila KMM241was studied, including gene cloning and sequence analysis, heterogenous expression and biochemical propery analyses In addition, the function of the N-terminal domain of XynB was studied and elucidated The main research results are listed as below:1. Gene cloning and sequence analysis of gene xynBThe16S rRNA gene sequence of G. mesophila KMM241had more than99%identity to that of Pseudoalteromonas atlantica T6c The whole genome of strain T6c was sequenced and released in GenBank, which revealed that there is a gene encoding a hypothetical endo-1,4-[3-xylanase in the genome Based on the5’ end and3’ end sequence of this gene, a pair of specific primers for PCR were designed, and the total sequence of xylanase gene xynH was amplified through PCR using the genomic DNA of strain G. mesophila KMM241as template. Sequence analysis showed that gene xynB is2739bp in length, encoding a protein of912amino acids, which is the precursor of xylanase XynB. XynB precursor is composed of a signal peptide (Metl-Ala43), a N-terminal domain (NTD, Glu44-Arg584) with unknown function and a GH family8catalytic domain(CD, Ser585-Glu912) Sequence alignment showed that only three enzymes have hotnology with the whole amino acid sequence of XynB. They are all hypothetical glycoside hydrolases from strains P. atlantica T6c (98%), Gaciecola chathamensis S18K6(80%) and Glaciecola polaris LMG21857(73%), respectively, none of which has been studied. The amino acid sequence of the catalytic domain of XynB showed highest similarity of41%to a hypothetical xylanase. Among characterized xylanases, XynB had highest identity (39%) to the xylanase Xyn8from an uncultured bacterium (ABB71891) The multiple sequence alignment revealed that XynB contained glutamate and aspartate residues Glu586, Asp646and Asp786, which are considered to be crucial for catalytic activity of GH family8. The amino acid sequence of the NTD of XynB showed no homology to any functional sequence in the data bases, suggesting that the NTD may be a domain with new structure. These analyses of XynB sequence indicated that XynB may be a novel multidomain xylanase of GH8.2. Heterologous expression and characterization of Xyn BObtaining the active enzyme through heterologous expression is very important for the study of the enzymatic properties of XynB. An expression plasmid pET-22b-xynB was constructed and successfully expressed in E. coli BL21(DE3). The best condition for XynB expression was determined through optimization experiments. Strain E. coli BL21(DE3) was induced by0.5mM IPTG and cultured at15℃for96hours. At this condition, a highest intracellular enzyme activity of4.2U/ml was obtained. For the purification of recombinant XynB, the E. coli strain was cultured for48hours when the proportion of target protein was relatively high. The recombinant XynB was purified from intracellular proteins through sonication, DEAE-Sepharose ion exchange chromatography and Ni-affinity chromatography. SDS-PAGE analysis showed that the Mr of XynB is about100kDa, which is in accordance with the calculated Mr of XynB (97.1kDa) without the predicted signal peptide. Therefore, the recombinant XynB is a multidomain xylanase containing the NTD and the CD.Study on the specificity of substrate of recombinant XynB showed that it is a specific endo-xylanase which could efficiently hydrolyze beechwood xylan and oat spelt xylan, but had no enzymatic activity on CMC, starch, laminarin, chitosan and mannan. The optimum pH for XynB is6.0-7.0. XynB is relatively more active in alkaline condition than in acid condition, which lost its enzymatic activity quickly in acid condition and retained most of its activity in alkalescent conditions. XynB was relatively stable in a wide pH range (pH6.0-10.0), retaining more than80%activity after incubation at pH6.0-10.0,15℃for1h. The optimal temperature for XynB was35℃. It retained7.5%and14.6%activity at0℃and5℃, respectively. XynB exhibited very low thermostability, retaining less than40%activity after60-min incubation at35℃and losing all the activity after20-min incubation at45℃. These results indicated that XynB had cold-adaptaion properties. Co2+(1mM and5mM) had the strongest inhibitory effect on the activity, while Zn2+, Ni2+, Mn2+and Li2+at a concentration of5mM had significantly inhibitory effects. XynB may have advantages in some industrial applications as two heavy metal ions Fe3+and Cu2+at a concentration of1mM had no evident inhibitory effect on its activity NaCl had no stimulative effect on the activity of recombinant XynB, on the contrary, NaCl in a low concentration (below0.5M) had slight inhibitory effect on its enzymatic activity (more than85%residual activity) However, XynB showed salt-tolerant ability, retaining approximately40%activity in4.0M NaCl, reflecting the adaptation of XynB to marine saline environment. XynB had lower Km and higher kcat/Km to beech wood xylan (5.82mg/ml,104.64ml/mg.s) than to oat spelt xylan (11.86mg/ml,6003ml/mg.s), indicating that XynB had stronger alffinity and higher catalytic efficiency to beech wood xylan than to oat spelt xylan XynB could not hydrolyze xylotetraose and xylotriose, and could hydrolyze xylohexaose and xylopentaose. Xylohexaose was completely hydrolyzed, producing xylobiose, xylotriose and xylotetraose The hydrolysis efficiency to xylopentaose was relatively low, producing a small amount of xylotriose and xylobiose. No xylose was detected in all hydrolysis process. These results showed that XynB is a strict endo-β-1,4-xylanase exhibiting an action pattern with a demand of at least five sugar moieties for effective cleavage.Xylanases have potential applications in a wide range of industrial processes, such as food processing, animal feeds, paper and pulp, textiles and bioremediation, etc. As a strict endo-xylanase, XynB may have potential in some of these industrial processes. Especially, XynB is thermolabile, which may have advantage in the food industries where a high temperature for enzyme inactivation is not allowed. As a salt-tolerant enzyme, xynB may have potential in the biotechnological processes where the catalysis environment is highly salty, such as in the processing of sea food and saline food3. Heterologous expression of the NTD and its binding ability to insoluble polysaccharidesIn order to study the function of the NTD in xylan degradation by XynB, the NTD was expressed in E. coli. The expression vector for NTD was constructed through pET prokaryotic expression system. The NTD was successfully expressed in E. coli BL21(DE3). The expression conditions were optimized to obtain soluble NTD. E. coli BL21(DE3) was induced by0.5mM IPTG and cultured at15℃for20hours. The recombinant NTD was purified from intracellular proteins through sonication and Ni-affinity chromatography. To test whether the NTD has a function of binding the substrate during xylan degradation by XynB, the binding ability of the recombinant NTD to insoluble oat spelt xylan was studied. The result showed that the recombinant NTD exhibited strong binding ability to insoluble oat spelt xylan. In addition, the recombinant NTD also exhibited strong binding ability to avicel, but little binding ability to chitosan and chitin. These results suggested that the NTD may contain a carbohydrate binding module (CBM) As the amino acid sequence of the NTD showed no homology to other CBMs, the NTD may contain a new type of CBM.4. Crystallization of the reconibinant NTDBecause the NTD has no homology to any protein sequence with known function, it is hard to predict the structure of the NTD. In order to further study the structure and function of the NTD, we decided to analyze the structure of the NTD by crystallization. Since a large quantity of protein with high purity is needed for crystallization, we first prepared the recombinant NTD of high purity through Ni-affinity chromatography, Q Sepharose Fast Flow chromatography and gel filtration chromatography. The purified protein of NTD was concentrated to8mg/ml and applied to crystal growing apparatus. Proper conditions for NTD crystallization were determined through preliminary screening and snowflake crystals were obtained, which is small and with low resolution. Therefore, proper conditions for NTD crystallization were further optimized. After optimization, rodlike crystals are now growing on the condition of0.1M Tris-HCl, pH9.0,30%(w/v) PEG6000, which has been testified to be protein crystals by SDS-PAGE These crystals are still growing. When their size is big enough, their resolution can be determined by X ray diffection. These results provide a good foundation for the study of the stucture and the polysaccharide-binding mechanism of the NTD.5Improvement of the baking quality by the psychrophilic xylanase XynAAlthough a lot of xylanases have improvement on breadmaking and are even widely used in baking industry, only one GH10xylanase has been reported to be effective in baking. In this study, two GH10xylanases, cold-adapted Xyn A and mesophilic EX1, were compared for their effectiveness in breadmaking. the optimum dosage of the two enzymes to improve the quality of wheat flour dough and bread was270U/kg flour for EX1and0.9U/kg flour for Xyn A, showing that the dosage of cold-adapted xylanase was much smaller. With the optimum dosage, Xyn A and HX1had the same dough-softening ability,50%reduce in BU However, Xyn A was more effective in reducing DDT than EX1. Xyn A and EX1showed similar effect on improvement of bread volume (around30%increase). Although they exhibited similar anti-staling effect on bread based on decrease in bread hardness, Xyn A showed more affect on reducing firming rate, and EX1more on reducing initial crumb hardness. These results showed that the two GH10xylanases have respective advantages in improving the quality of dough and bread, and suggested their potential as additives in breadmaking. It is found for the first time that cold-adapted GH10xylanase has potential in improving the baking quality of bread. |
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