Screening Of Xyloglucanase Production Strains And Studies Of Its Fermentation Process And Enzymatic Properties

Xyloglucanases （XEG）（EC3.2.1.151） are an important glycoside hydrolase, also beingnamed as xyloglucan endo-β^-1,4-glucanases, which are the biocatalysts for hydrolysis ofbackbones of xyloglucan. XEG have huge potential application in industries of bio-ethanol,food, pharmaceutical, textile, paper, detergents, and also play an important role in plantgrowth. At present, the research of XEG on abroad has contained the transformation of plants,the structure and function of XEG, the high-level expression of gene which encoding XEGand so on. In contrast, currently domestic research on the XEG is still at the initial stage.There is few relevant reports, which makes the study of XEG imperative.In this study, we established a rapid method about how to screen strains which couldgenerate XEG. We studied Paenibacillus xylanilyticus with the highest XEG activity,including the XEG activity optimization, the separation and purification of XEG, theenzymatic properties and mass spectrometry analysis. The main contents are as follows:Firstly, the xyloglucan which extracted from pumpkin as the sole carbon source wasprepared for first screening. The xyloglucan which had been dyed with Remazol brilliant Blueas the sole carbon source and indicator was used for second screening. The strains werefermented and second screened after proving its function of producing XEG and having theXEG activity by observing its transparent circle. The experimental results obtained4wildbacteria from collected samples. Meanwhile,10strains from3000strains which preserved inlaboratory were screened. The colony characteristics and morphology were observed, and iwas identified16S rDNA sequence in the NCBI, which determined the species of the strains.A wild strain Paenibacillus xylanilyticus which had the highest XEG activity was selected forfurther study.Subsequently, the highest XEG activity2.06±0.09U/mL was obtained from P.xylanilyticus fermented in the basis enzyme production medium, and the basis medium wasoptimized by single factor experiments. Response surface methodology （RSM） was used tostudy and optimize the fermentation process for XEG production from P. Xylanilyticus. Theresults showed that the optimal parameters for the fermentation process conditions were asfollows: the initial pH7.46, Yeast extract2.26%, Peptone1.00%, xylose0.27%, NaCl0.60%.The XEG activity produced from P. Xylanilyticus was10.88±0.96U/mL, and close totheoretical prediction value （11.04U/mL）, and forecast accuracy was98.71%, indicating thehigh credibility. The activity was improved5-6times than before.After that, we separated and purified XEG from the fermentation broth. After precipitatedbetween10%-80%ammonium sulfate saturation, Desalting colume, DEAE FF ion exchange,Sephacryl S^-100gel filtration, cation exchange chromatography, the final protein was verifiedon SDS-PAGE with one band, which proved that the XEG was purified to homogeneite. Inthe end, a21.41-fold purification with a yield of11.34%was obtained, and the specificactivity reached236.31U/mL. The target protein molecular weight was39kDa.Meanwhile, the study of enzyme properties showed that:â‘ the optimum temperatureof XEG was50℃, and there was still90%activity reserved after heated for2h at60℃, while there was still25%activity reserved at65℃in the same condition, and it was not stablehigher than65℃.â‘¡the optimum pH was7.0, and retained at least80%activity for2h inthe pH range from5.5-8.5.â‘¢the XEG was activated by1mmol/L Zn²⁺、Ca²⁺、Fe²⁺、Fe3+and5mmol/L Fe²⁺, strongly inhibited by5mmol/L Cu²⁺and1mmol/L Mn²⁺.â‘£at pH5.0and50℃the Kmand Vmaxfor xyloglucan were0.80mg/mL and6.71U/mL, the kcatwas11.21s^-1.⑤it got the highest specificity to xyloglucan.At last, the mass spectrum was obtained through the mass spectrometry, and it was provedthat this target enzyme was xyloglucanase.