Screening Of Inulinase - Producing Strains And Optimization Of Their Enzyme - Producing Conditions

Inulin is a polyfructan, consists of linear β-2, 1-linked D-fructofuranose molecules terminated by a glucose residue attached through a sucrose type linkage.Inlin accounts for a very high share of the Jerusalem artichoke tube, about 80% in dry weight. Jerusalem artichoke produces large amounts of biomass, and can be grown on barren land. It is considered as a relatively abundant and inexpensive material for the production of fructose and fructooligosaccharides. Enzymatic processing fructose or fructooligosaccharides has advantages, such as mild conditions, easy preparation, low cost, high security, non-population. Therefore, the use of enzymatic production of fructose and oligofructose are more suitable for industrial production.Inulinases are potentially useful enzymes for hydrolyzing inulin. Microorganisms are the main sources for the production of inulinases, the hydrolysate of exo-inulinase and endo-inulinase are fructose and fructooligosaccharides respectively. It can hydrolyze inulin into fructose or fructooligosaccharides. Fructose possess high sweetness and low caloric, in addition, fructose metabolism in humans is not insulin dependent. Therefore, it becomes an ideal food for diabetic and obese patients. Fructooligosaccharides are excellent prebiotics. They can selectively stimulate the growth of bifidobacteria and lactobacilli, improve the absorption of minerals and help maintain the health of the cardiovascular system. Therefore the exploitation and utilization of inulinases have important significance, and great application prospects in food industry and medical project.More than 60 strains include bacterial, fungal and yeast were isolated from Jerusalem artichoke rhizosphere soil. Through further screening, a bacterial strain C-56 and a fungal strain C-40 with high inulinase activity were screened. The strain C-56 was identified as Burkholderia genus, named as Burkholderia sp. C-56; the strain C-40 was identified as Cladosporium genus, named as Cladosporium sp. C-40. So far, most of the inulinase produced from Strains in the nature is exo-inulinase. However in this study, the enzyme obtained from Cladosporium sp. C-40 is endo-inulinase.The optimal carbon sources, nitrogen sources, and inorganic salt were identified as inulin, yeast extract, MgSO4·7H2O for Burkholderia sp. C-56, and inulin, yeast extract, ZnSO4·7H2O for Cladosporium sp. C-40, through single factor test, respectively.The further optimization of fermentation conditions were studied by Taguchi method. Statistical analysis of the experimental data indicated that inulin was the most significant factor for inulinase production. The optimal medium compositions of Burkholderia sp. C-56 for maximizing the inulinase production were established as 50 g/L inulin, 20 g/L yeast extract, 6 g/L MgSO4·7H2O, initial pH 6.0. Under optimum conditions, 29.34 ± 1.95 U/m L of inulinase activity were obtained, which increased about 3.69 times compared with the initial enzyme activity(6.25 ± 0.84 U/m L). The optimal medium compositions of Cladosporium sp. C-40 were established as 50 g/L inulin, 20 g/L yeast extract, 6 g/L ZnSO4·7H2O, initial pH 5.0. Under optimum conditions, 30.68 ± 1.42 U/m L of inulinase activity were obtained, which increased about 2.16 times compared with the initial enzyme activity(9.71 ± 0.97 U/mL).Fermentation dynamics of inulinase by the two strains were studied. The growth of Burkholderia sp. C-56 entered into stationary phase after 48 h, also the highest inulinase activity was obtained at 48 h. Because of the value of I/S was range from 0.2 to 0.5 during the whole fermentation process, the inulinase of Burkholderia sp. C-56 was exo-inulinase. The growth of Cladosporium sp. C-40 entered into stationary phase after 96 h, the inulinase activity reached a plateau after 72 h, and the highest inulinase activity was obtained at 96 h. Because of the value of I/S was above 10 during the fermentation process, the inulinase of Cladosporium sp. C-40 was endo-inulinase.To make a primary research on the enzyme properties of Burkholderia sp. C-56 and Cladosporium sp. C-40, the effect of temperature, pH and concentration of substrate on activity of inulinase was studied. The optimal reaction pH of inulinase from Burkholderia sp. C-56 was 5.0. The enzyme was stable and kept more than 90% activity within pH4.5~pH5.5. The optimal temperature was 55℃. The optimal reaction pH of inulinase from Cladosporium sp. C-40 was 4.5. The enzyme was stable and kept more than 90% activity within pH4.0~pH5.0. The optimal temperature was 55℃.