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|標題:||Investigation on the production of chitinase from Streptomyces spp.
In this study, three potential Streptomyces spp. were screened from the soil. By comparison on the chitinase activity, one of the strains S-1 was selected for its highest chitinase production in pH 8.0. The initial fermentation condition for chitinase production was set at pH 8.0 and 28℃.Chitinase activity was assayed in a reaction mixture containing 0.5% collidal chitin in 0.05M phosphate buffer of pH=6.0 and enzyme solution.One unit of chitinase activity was defined as the amount of enzyme which produced one μmole of reducing sugar per min.
Mass transfer effect in the shaken flask is a critical factor affecting aerobic cell cultivation, whereas only few literatures concerned about it. In this study, the sodium sulfite method was empolyed to measure the gas-liquid mass transfer coefficient KLa in both the baffled and unbaffled shaken flasks. The experimental data were used respectively to implement two mathematical models for each kind of flasks. Furthermore, a neural network estimator (NNE) for KLa prediction was established from the calculated data of the two mathematical models. By comparison on the data of these models, the NNE model was found capable to predict KLa in both kinds of shaken flasks. This implies that the NNE can be a useful tool for shaken flask operation.
By comparisons of colloid chitin and chitin powder as substrates on chitinase production, the chitin powder was found capable to attain the same chitinase activity as colloid chitin. Consequently, for the ease of medium preparation, chitin powder was used in the cultivation. Study on the seeding effect respective from the spore and the mycelium of the Streptomyces spp. S-1, it was found that with mycelium seeding at 1.5% chitin powder substrate, a maximum chitinase activity of 0.25 U/ml can be obtained. Four operation conditions in the shaken flasks were used to investigate the KLa effects on chitinase production. The result showed that at 200 rpms rotational speed and 200 ml working volumes in the 500 ml Erlenmeyer shaken flask (i.e. at a medium KLa: 47.54 hr-1), a highest chitinase activity of 0.27 Us/ ml was obtained.
Analysis on the oligosaccharide contents of the fermentation broth and the enzyme hydrolysates, only (GlcNAc), (GlcNAc)2 and (GlcNAc)5 were found on the fermentation broth, whereas (GlcNAc), (GlcNAc)2, (GlcNAc)3 along with (GlcNAc)5 were found on the enzyme hydrolysate. It implies that the streptomyces spp. may produce chitobiose , endo-chitinase or exo-chitinase.
本研究從泥土中挑選出具幾丁酵素活性的鏈黴菌來進行醱酵條件探討，而我們從泥土中挑選具較高幾丁酵素活性的菌株共有3株鏈黴菌；進一步比較幾丁酵素活性及其最適的培養pH，我們挑選3株菌中較高活性的S-1來進行探討，此菌株經鑑定確定為Streptomyces spp.，其在28℃下的最適初始培養pH為8.0。一單位(unit)的幾丁酵素活性定義為：在37℃、200rpm下，在以0.5%膠態幾丁值為基質的phosphate buffer(0.05M、pH=6.0)中，每分鐘釋放1μmole還原醣的幾丁酵素量。
以不同狀態的膠態幾丁質及幾丁質粉末來進行醱酵時，在相同的基質濃度時，幾丁質粉末的醱酵可以達到與膠態幾丁質相當的幾丁酵素活性，且定量上較為方便，故採用幾丁質粉末來當作我們醱酵的基質；另外分別進行以孢子接菌及菌絲接菌的不同基質濃度醱酵比較，由結果是以1.5%幾丁質濃度的菌絲接菌培養的酵素活性最高，達到0.25 U/ml，但是1.5% 和1.0%的幾丁質濃度醱酵，所得到的幾丁酵素活性大致上差不多，故我們仍是以1.0%的幾丁質濃度配合菌絲接菌來當作我們進行醱酵探討的條件。我們以此條件進行六組不同的氣液質傳係數探討，得到的結果是以200rpm，200ml培養基於500ml錐形瓶的KLa(=47.54 hr-1)醱酵結果最好，幾丁酵素活性可以達到0.27U/ml。我們並且進行醱酵液及經酵素水解後溶液的幾丁寡醣的方析，由結果可以知道，在醱酵液中殘留的都是幾丁單醣、幾丁貳醣及少量的幾丁五醣；而在幾丁酵素的反應液中，除了發現經幾丁酵素水解後，幾丁單醣、幾丁貳醣、幾丁三醣及幾丁五醣的濃度改變，因此可以推測此鏈黴菌具有幾丁貳醣酵素、內切幾丁酵素或外切幾丁酵素。
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