Optimization Of Enzyme Production Conditions And Characteristics Of Agarase From Microbulbifer Sp.

Agar which has good gel property and stability is a kind of linear polysaccharide extracted from red algae.Agaro-oligosaccharides,which are the enzymatic degradation product of agar,have a variety of biological activities and are widely used in food,medicine,cosmetics and other fields.In this study,a marine bacterium Microbulbifer sp.with agar degrading ability was isolated from the marine sediments of Pearl Bay Mangrove in Fangchenggang City,Guangxi.Its physiological and biochemical properties such as gram staining,catalase and oxidase were determined and its 16S r DNA sequence was analyzed.The effects of various factors on agarase production by the strain were studied,such as carbon source,nitrogen source and temperature,etc.The response surface methodology was used to optimize the enzyme production conditions.Enzymatic properties such as the optimum reaction temperature and temperature stability of agarase were studied.The main results obtained in this paper are as follows:（1）An agar-degrading bacterium Microbulbifer sp.was isolated and identified,and its basic physiological and biochemical properties were measured.An agar-degrading bacterium was obtained through enrichment,preliminary screening and rescreening.It was identified by 16S r DNA identification as a bacterium of the genus Microbulbifer.The strain was a Gram-positive bacterium,aerobic,salt-tolerant,had no effect on denitrification,and could not hydrolyze starch and cellulose.The catalase test,oxidase test and lipase test were all positive.The strain could grow with agar as the sole carbon source,and the initial agarase activity was 1.60 U/m L.According to the results of thin layer chromatography,the agaro-oligosaccharides produced by this agarase degrading agar were neoagarotetraose and neoagarohexaose.It was speculated that the agarase wasβ-agarase,which might belong to the GH16 family of enzymes.（2）The conditions for the strain to produce agarase were optimized.The results of single factor experiment showed that the suitable conditions for the strain to produce agarase were as follows:the best carbon source was agar,the best nitrogen source was ammonium chloride,the culture temperature was 35℃,the initial p H value of the medium was 7.0,the agar concentration was 0.3%,the inoculation volume was 3%,and the incubation time was 6 h.The enzyme production conditions of the strain were optimized by response surface methodology.The optimal enzyme production conditions were determined as follows:the culture temperature was 33.8℃,the agar concentration was 0.3%,and the initial p H of the medium was 6.8.Under the optimized enzyme production conditions,the agarase activity reached the maximum value of 7.15 U/m L,which was 4.5 times higher than that before optimization.（3）Some properties of the agarase were determined.The optimum reaction temperature of agarase in crude enzyme solution was 40℃.The loss of agarase activity was small in the range of 25-60℃.The agarase had good thermal stability at 30,40 and 50℃,and still retained more than 80%relative enzyme activity after holding for 2 hours.The optimum p H of the enzyme reaction was9.0.The agarase activity was stable in the range of p H 6.0-9.0.The optimum substrate concentration of the agarase was 0.4%.The agarase has good substrate specificity.Mn²⁺,Co²⁺,Mg²⁺could promote the agarase activity,while Ca²⁺and Ag⁺inhibited the activity of agarase.K⁺and Na⁺of 1 mmol/L promoted the agarase activity,but the K⁺and Na⁺of 10 mmol/L inhibited the activity.Zn²⁺had no significant effect on the agarase.To sum up,in this paper,a Gram-positive agar degrading bacterium（Microbulbifer sp.）was isolated.β-agarase from the strain can degrade agar to neoagarotetraose and neoagarohexaose.The activity of agarase increased by347.87%（7.15 U/m L）after the optimization of enzyme production conditions.The agarase secreted by the strain has good thermal stability（30-50℃）and acid-base stability（p H 6.0-9.0）.Therefore,the agarase has great potential application value.