Studying The Key Technology To Produce Chitin And Derivative With High Value By Enzymatic Methods

Chitin is the most abundant renewable natural resource like cellulose.Billions tons of chitin is produced each year in nature, whereas a large amountof chitinolytics is produced to decompose the same amount chitins. The fact thatchitin is broadly produced and stablely existed in nature shows and prove thatthe chitinolytic enzymes is existed broadly too. The diversity of environmentand species of microorganisms make it possible to find chitinolytic enzymeswith different properties.Chitin, as well as chitosan, which is deacetylse chitin, and theiroligosaccharides forms has been studied and applied in many fields, such asfood technology, phamorcology, material science, microbiology, tissueengineering, bionanotechnology and so on. So there comes the huge demand forthis production. However, the conventional production of chitin, chitosan and their oligosaccharides involves the use of strong acids and base, which creates adisposal problem due to the large amounts of toxic waste that need furthertreatment and may pollute the environment. To overcome this problem, analternative method using enzyme has been emerged, which can be in aconsiderable extent replace the non-environment friendly chemical process.Producing chitin and its related products by method of bioprocess ispromising and interesting. Because of the milder producing conditions, and lessenvironment pollution, the biological or enzymes method has become an idealmethod replacing the chemical method, which is the focus. The recent studyingfocused on the methods of biological or enzymes methods which are idealmethod with advantages such as mild producing conditions, less environmentalpollutions. On account of these backgrounds, a novel process was applied toextract chitin from shrimps materials that pepsin and citrate was used todemineralization and deprotein. And then, novel strains with the high ability toproduce chitinase or CDA was isolated from soils of Qinling Mountains that isdifferent from marine environment used to isolated these strains. After that, the cultural medium and parameter of fermentation was optimized by one-factorexperiment and response surface methodology (RSM).In this thesis, pepsin and citrate were applied for demineralization anddeprotein during produce chitin. According to the ash content, the parameter ofcitrate solution, whose concentration and time were12%and13hours, wasdecided by one factor experiment. Under this condition, the ash content of chitinwas1.5%, which is meet the requirment of industrial grade, and close to foodgrade. As for the deprotein studies, the optimal deproteinization conditions wasstudied by one factor experiment at first, and Kjeldahl method was used todetermine the protein content. The data of one factor experiments indicates thatthe proper catalytic condition might be as follows, pH1.5, temperature35℃,enzyme dose500U/g, and the process time4hours, take the cost of productioninto account. On the basis of these results, response surface methodology (RSM)was used to study the optimal process conditions, the data indicates that theoptimal condition occurs at700U/g,36.09℃, and5hours, and the maximumdeproteinization rate approaches8.47%. The strain with significant ability is the foundation and the key offermentation industrial, that is one of the most important work in the field ofdecompose chitin by microorganisms. In this study, soils in different envirementwere collected from Qinling Mountains, and the strains with high ablity toproduce chitinase or CDA screened after enrichment culture. Applying theisolation medium that colid chitin was the sole carbon source,20strains wereselected according to the scale of clarity zone around the colony in medium.These strains were filtered further by fermentation, and the chitinase activity insupernatant was determined by DNA method. The first three strains with highablitity producing chitnase were Z4, F9，and D5-23. The strain Z4was the bestone whose enzyme activity in supernantant reaches to2.3U/mL. Morphologyand sequencing of the16S rDNA information indicated that the strain wasmicrobacterium sp. The chitinase production of Z4was markedly enhanced bystatistical optimization of medium composition and culture conditions. Thesingle factor experiment shows that the glucose and yeast extract is the optimalsource of carbon and nitrogen. The effect of glucose, yeast extract and initial pH on chitinase production was studied with method of Plackett-Burman design,and was then further optimized with the method of steepest ascent and RSM.Data show that3.38g/L glucose,3.24g/L yeast extract and an initial pH of7.5were optimum for the production of chitnase. According to the results of singlefactor experiment, the optimum technological parameters were ascertained byorthogonal experiment which is culture temperature28℃, initial pH of7，thevolume of liquid60mL, inoculum size6%.Besides the optimizing the medium and fermentation parameter, themethod of molecular biology were also used to study the property of the relatedgenes because clone and express the chitinase gene in other microorganism isanother useful method to enhance the production of chitinase. For the purposeof clone the chitinase gene of Z4, several PCR primers were designed accordingthe sole chitinase gene from microbacterium genus and conserved amino acidsequence of chitinase. The genome DNA was extracted by kits and used astemplate for PCR amplification. The production was sequenced and analyzed bybioinformation methods. The results show that the sequence was part of chitnase gene, and belong to chitinase of GH18familys.In order to get new strains with powerful ability to produce CDA,28strainswere isolated and screened out from the soil samples by method of color reactionin plate medium. And then, the paranitroacetanilide was used as the substrate toanalyze the enzyme activity. The strain F2-7-3was screened out from thesestrains for the highest CDA activity, which can reach more than250U/mL. Themorphological properties and16SrDNA sequencing were studied and datasuggested that the isolated strain belonged to the evolution branch ofRhodococcus. The enzyme activity was studied further, data shows that theoptimum temperature was50℃, the optimum pH was7.The component of medium and parameters during fermentation wereoptimized respectively in order to improve the production efficient of CDA.According the results of single factor experiment and orthodox experiment,orthogonal experiment implies that the KH2PO4was a most significant influencefactor than other salts. Hence, the maximum chitinase activity of250.61U/mLwas obtained by using the optimized medium that contents of the three most important components, sucrose, yeast extract and KH2PO4were7g/L,9.15g/Land0.93mmol/L respectively. Subsequencetly, the fermentation conditions ofF2-7-3were optimized by method of the single factor analysis to improve thefermentation process, the optimum conditions obtained were: temperature30℃,pH7and liquid volume40mL in250Ml flask.New strains with excellent ability to produce enzymes and associated datewere offered in the present studies, which are essential to producechitin/chitosan and their oligosaccharide by enzymes, a kind of friendly methodto environment.