Identification Of Acid Xylanase Procuding Strain Of Streptomyces, And Optimization Of Enzyme Production, Cloning And Expression

Hemicellulose as the second renewable natural resource polysaccharide complexes on earth, the content of it is next to that of cellulose in straw. However, the great majority straw is wasted as agricultural deposalor is burned making a great threat to the environment. China is rich in straw resources, but how to effectively utilize hemicellulose in the straw and transform it into available energy resource is the key problem to be solved. Xylanase is the major hemicellulase which could degrade hemicellulose, and is quite commonly used in paper industry, food processing industry, feed industry. This research screened xylan-decomposing bacteria from soil samples and rumen contents of Tan sheep and identified several strains with high yield of xylanase. Afterwards, fermentation condition was optimized for the highest-yield strain and the produced xylanase from this strain was studied for enzymatic characteristics. Meanwhile, degradations of three kinds of coarse feeds(wheat straw/ maize straw/ alfalfa meal) were tested using xylanase from the highest-yield strain. In addition, taking advantage of molecular biological cloning technology, xylanase gene was cloned and expressed in E.coli BL21(DE3), and then enzymatic characteristics of the recombinase were studied for its potential application in degradation of coarse feedstuffs. The results showed that:1. Six xylanase-producing strains were screened from soil samples and rumen contents of Tan sheep using Congo red staining and liquid fermentation method. Among of them, WL003 from soil was selected for its highest enzymatic activity, which is 20.68 U/mL. Strain WL003 was identified as Streptomyces griseorubens, a kind of gram-positive bacterium, according to its physiological and biochemical characteristics and the construction of phylogenetic tree as well.2. Stain WL003 was suitable for growing at pH 6.0, 32℃, 180 rpm with wheat straw as carbon source and meatext as nitrogen sourse. WL003 reached its peak of enzyme production at the 7th day. Addition of Tween-80 improved the enzymatic activity of xylanase to 50.82 U/mL, which was 2.46 times of the original activity. The xylanase had the highest degradation effects on wheat straw and lowest on alfalfa meal.3. The xylanase secreted by Streptomyces griseorubens WL003 showed optimal temperature of 65℃ and optimal pH of 4.5. After treating at 30~50℃ for 10 minutes, the residual xylanase still hold more than 80% activity, and 88% activity after treating at pH 3.5~11.0 for 1.5 h. Tween-80 could activate the activity of xylanase, while Cu2+, SDS and β-mercaptoethanol significantly inhibited the activity.4. Xylanase gene xyn130 from Streptomyces griseorubens WL003 was cloned. The integral open reading frame of this gene was 1320 bp, encoding 439 amino acids, with theoretic molecular weight of 47.25 kDa and isoelectric point at 5.86. Xylanase is a hydrophilic and stable protein, containing 3 glycosylation sites and 20 phosphorylation sites. Besides, XYN130 belongs to GH10 family and possesses the typical domain with(β/α)8 for this family. Glu-170, Glu-278 and Asn-343 are the possible catalytic residues of XYN130.5. The expression vector pET-xyn130 was built, and successfully expressed in E.coliBL21(DE3). The positive clone reached its highest xylanase activity after cultivating at 0.2 mM IPTG for 6 hours induction. The recombinase showed optimal temperature of 60℃ and optimal pH of 5.5. The xylanase was well tolerated the temperature range of 30℃ to 75℃ for 10 min and pH range of 3.5 to 11.0 for 1.5 h, while xylanase activity dropped sharply when temperature was above 75℃ and pH was lower than 3.0 and higher than 12.0. Cu2+, SDS, β-mercaptoethanol, EDTA, Mn2+ and Zn2+ significantly inhibited the xylanase activity, with Cu2+ being the worst and SDS the second.Based on above results, it could be concluded that an acid xylanase producing Streptomyces griseorubens WL003 was screened from soil samples by using Congo red staining. Xylanase activity of fermentation liquid increased 2.46 times after the optimization. Crude enzyme had the highest ability of degrading wheat straw and the lowest of degrading alfalfa meal. Xylanase gene xyn130 from Streptomyces griseorubens WL003 was successfully cloned(NO. KU860088). The gene is 1320 bp long, and encodes 439 amino acids, and has the theoretic molecular weight of 47.25 kDa and isoelectric point at 5.86. The expression vector pET-xyn130 was successfully expressed in E.coliBL21(DE3). The recombinase showed optimal activity at 60℃ and pH 5.5, respectively. SDS could significantly inhibit the xylanase activity, and Cu2+ could completely inactivatethe xylanase.