Screening Of The Strains Producing Chitin Deacetylase

Chitin deacetylase(CDA) is an enzyme that catalyzes the hydrolysis of acetamine groups of N-acetyl-D-glucosamine in chitin,convering it to chitosan.As the substrate of the enzyme mentioned above,chitin is slightly soluble,which makes it difficult to determine the enzyme activity of CDA, thus it hampered the progress in CDA research.A simple and effective method of determining the enzyme activity was put forward with p-nitroacetanilide as substrate and paranitroanilinum as production.The FAP(Features Absorption Peak) of p-nitroacetanilide and paranitroanilinum were taken into account.By analyzing absorption spectrum of various buffer solution,interference of buffer on the enzyme activity were excluded.The stability and repeatability of absorption values in the enzyme reaction system was assured by simulating system.In comparing to the traditional method,the determination was greatly simplified with identical outcome.Using culture media with chitin as sole carbon source and addition of p-nitroacetanilide as color indicator,we isolated 459 strains with yellow circle surrounding from 15 soil samples.Then,268 strains was isolated after second screening by discolorating filter paper chips at faster speed with darker color. Screened after being incubated by shake flasks together with the method mentioned above,a strain with CDA chitin deacetylase productive ability together with optimal stabilization,named strain 11-3,was finally obtained. Gas chromatography showed acetic acid existed in the production of reaction between crude enzyme and colloidal chitin,which displayed the deacetylation characteristic of the enzyme.The result of physiological and biochemical characteristics conbined,studies indicate that strain 11-3 may be members of genus Rhodococcus sp.There's no report on this genus culture with the ability of producing CDA so far.Influence factors on the enzyme production of Strain 11-3 were acquired by researching on various factors.The result was as follows:It could utilize many kinds of materials as carbon resource;Saccharose was the best of all. When came to Nitrogen resource,peptone was the best with the addition of ammonium sulfate and corn slurry.Sodium acetate promoted enzyme production while ammonium acetate was to the contrary.FeSO₄ strains had a stronger effect on inhibiting enzyme production;KH₂PO₄ promoted enzyme production,the impact of MgSO₄,CaCl₂,and MnSO₄ on enzyme production was insignificant.The optimal medium composition(g/L) was:sucrose 5, corn pulp 5,ammonium sulfate 2,peptone 2,KH₂PO₄ 1.5,sodium acetate 1, pH 7.5.The optimal culture conditions were:broth volume 30-35 mL in 250 mL Erlenmeyer flasks,inoculation time 24 h,inoculation volume 5%, agitation speed 180 r/min at 30℃,fermentation time 60 hours.After optimization of enzyme production conditions,enzyme production increased from the 57 U/mL initially to 5035 U/mL,nearly 100 times concerning the initial yield,which accounted for the great potential of the strain 11-3.Part of the enzyme characteristic of strain 11-3 was investigated and excellent thermal stability displayed:when treated in 45℃for 1h,more than 90%activity remained.The best temperature was 50℃and the optimal pH was 7.0.Higher enzyme activity remained with pH variation was 7.0-10.0. Ag⁺ was of effective inhibition to it.A simple and effective method of determining the enzyme activity was put forward in the thesis.Rhodococcus 11-3 with stable performance,higher production CDA vitality and excellent thermal stability was acquired. Foundation was laid for further research.