Studies On Regulation Of Microbial Alkali-tolerant Xylanase Synthesis And Its Mode Of Substrate Hydrolysis

Microbial xylanases have received a great deal of attention in the last twenty yearsmainly due to their potential application in the pulp and paper industry and food industry. Aalkali-tolerant xylanase producing strain Bacillus pumilusWL-11 was screened from the soilsamples collected from Taizhou pulp and paper mill. Bio-synthesis and its regulation ofWL-11 xylanase were intensively studied in this thesis, as well as its substrate hydrolysis andmode of action. The main results are as following:(1) Fifteen soil samples were collected from Taizhou pulp and paper mill. The ecologicaldistribution of microbial xylanase producers isolated from these samples was analyzed, theresults showed that the strains isolated from the soil samples collected in the wheat strawstackyard are mainly fungi which produce acidic xylanase, while from the soil samples in theeffluent of pulp and paper mill the bacteria which produce alkaline xylanase are the majority.A strain library containing 226 bacterial and fungal xylanase producers was successfullyconstructed, two bacterial producers Pseudomonas sp WXULI-11and WL-11 and three fungalproducers Aspergillus niger XY04B, Aspergillus niger XY07C and Aspergillus glaucusXY12D were screened out from this library. All these five strains can over-produce xylanasewithout or with only small amounts of cellulolytic activity.(2) Production conditions for xylanases of three fungal strains were preliminarilyinvestigated and compared with production characteristic of xylanase of Pseudomonas spWLUN024, a mutant derived from parent strain WXULI-11. The results showed that the fourmicrobial xylanases are all inducible by xylan, wheat bran and some other xylosic materiel.The best inducer was xylan (Sigma) while xylose is or isn't an inducer for different strains.The production of xylanase by different strains varies with various nutritional andenvironmental factors, which can prove that microbial xylanases are biodiversity from thisview of point.(3) Strain WL-11 is identified as Bacillus pumilus according to its morphological andtaxonomic characteristics and its 16S rDNA sequence analysis. An endo-β-1,4-xylanase A(Xyn A) present in culture filtrates of WL-11 was purified to homogeneity by (NH4)2SO4salting-out, CM-Sephadex, Sephadex G-100 and Sephadex G-75 chromatographies. Theenzyme has a molecular weight of 26.0 kDa, an isoelectric point of 9.5, apparent Km of16.6mg/mL, Vmax of 1263 μmol/(min·mg) and is optimally active between pH 7.2 and 8.0and stable at the range of pH 6.0 to 10.4. The enzyme is optimally active at 45-55 °C andstable at less than 45 °C, while its half-life of activity are 35 min and 15 min at 55 °C and 60°C respectively. The enzyme activity can be severely inhibited by the metal ions such as Cu2+,Hg2+ and Zn2+, and be activated by Ca2+, Mg2+, Ba2+, Fe2+, Sn2+, Fe3+ and Li+, while it is notsignificantly affected by Al3+, Mn2+ and Co2+.(4) Zymogram analysis showed that WL-11 mainly secretes extracellular xylanase Afree-of cellulase. Thin-layer chromatography (TLC) analysis showed that the crude xylanasepresent in culture filtrates of WL-11 liberates mainly xylotriose (Xyl3) and otheroligosaccharides including xylobiose (Xyl2), and xylotetraose(Xyl4) and xylopentose(Xyl5)from Oatspelt xylan(Sigma), while xylose is undetectable. This result showed that WL-11produce no or very lower activity xylosidase, so it is suitable for enzymic preparation ofxylo-oligosaccharide.(5) Effects of various carbon sources on the production of xylanase by WL-11 wereinvestigated. The results showed that WL-11 xylanase is inducible by xylan,xylo-oligosaccharide and wheat bran. The induction of xylanase activity is dependent oninducer concentration and is not fully repressible by glucose and starch. The optimumconcentration for xylanase production of wheat bran is range from 50 to 60 g/L. Thebiosynthesis of xylanase by WL-11 can be enhanced by addition of 10 g/L of slow-releasinginducer, such as xylan to the medium containing 40 g/L of wheat bran, while it can beinhibited by addition of 10 g/L of quick-releasing inducers, such as xylose andxylo-oligosaccharide. Biosynthesis of xylanase by WL-11 can be induced by lowconcentration (3 g/L and 10 g/L) of xylose, while it is inhibited by high concentration ofxylose (30 g/L).(6) Effects of various nitrogen sources on production of xylanase by WL-11 are different.WL-11 can overproduce xylanase by using inorganic nitrogen sources except KNO3, while itis relatively low by organic ones except peptone. Ammonia sulfate and peptone are optimalfor the production of xylanase at the concentration of 2 g/L (calculated as N content).(7) The results showed that production of xylanase and cell growth of WL-11 is poor byusing 0.02 mol/L of various amino acids as sole nitrogen source. Addition of 0.005 mol/L ofamino acid, such as Pro, His and Gly to the medium containing 0.02 mol/L of (NH4)2SO4 candramatically increase xylanase production by WL-11, while it shows no significantstimulatory effect on cell growth.(8) The aeration rate 0.55 vvm and agitate speed 500r/min is the best choice for xylanaseproduction by WL-11. The enzyme activity could reach the highest level 1270 U/mL in 24 hunder the optimal conditions in 5 L jar experiment, whereas that in 36 h in shaking-flaskexperiment.(9) Strain WL-11 can not only produce xylanase, but also secrete some amount ofprotease. Effects of various carbon sources and nitrogen sources on production of xylanaseand protease were studied. The results showed that different carbon source can induce orinhibit xylanase production by different inducible effect, but it has no significant effect onproduction of protease;Nitrogen sources can not regulate xylanase production directly,whereas it can regulate xylanase production via adjusting protease expression. There is acertain direct correlation between the production level of protease and xylanase. When theamount nitrogen source was kept constant, the relative production amount of proteasecompared to xylanase production can be decreased by raising the ratio of carbon to nitrogen;Whereas the amount of carbon sources was kept constant, it has no significant effect on therelative production amount of protease by raising such ratio.(10) Hydrolysis of xylans from Oat spelt, Birch wood and Beech wood by purifiedxylanase A was studied. The result showed there is difference of the process of hydrolysis ofdifferent xylan. The products formed during the hydrolysis process of different xylan byxylanase A were analyzed by HPLC method. It was found that the xylotriose is the mainoligomer in the different xylan hydrolysate. Xylanase A has strongly ability to hydrolyze thepentose in hard wood xylan hydrolysate, while it has poor ability to hydrolyze the pentose inOat spelt xylan hydrolysate.(11) Products of hydrolysate of self-prepared xylan from corncobs was analyzed by TLCand HPLC methods. The results showed that 80% of xylo-oligosaccharides are xyl2, xyl3 andxyl4 in the hydrolysate and no xylose is found.