The Analysis Of Sugar Beet Pulp's Hydrolysate And Properties Of Xylanase Through Site-directed Mutagenesis

The sugar beet pulp is the rest product of manufacting cane sugar. Corase fiber, as one component part of sugar beet pulp, is preserved absolutely. At present, there are third primary processing methods as following:first, be used as the coarse fodder of animals;second, like primary processing method, the sugar beet pulp is considered as an agricultural wastes were burned or discarded directly; third, the traditional method of sugar beet pulp hydrolysis is mainly use strong acid, strong alkali, high temperature and so on. It is high cost and little application prospect. As a result, many remain compositions, like corase fiber, pectin, nitrogen free extract and crude protein were wasted on a large scale without any positive role of adding economic efficiency and increasing the added value of industrial products.Thermophilic fungus Thermomyces lanuginosus DSM10635 used in this article is a heat-resistant strain. The xylanase from this strain was determined as 13305 IU / mL. Diluted the crude enzyme solution for 500IU. Then the sugar beet pulp was hydrolyzed by dilute xylanase solution at 60℃for 24 hours. Centrifuged the hydrolysate, and supernatant was concentrated by vacuum evaporation at 50℃to remained 20%(w/w) dry matter, then decolorized by active carbon filter. the sugars were analyzed by HPLC. Finnally, the products were mainly polysaccharide, oligosaccharide, glucose, xylose and arabinose. Xylanase was used to avoid side reaction of hydrolysis. Due to the enrichment of protein and other impurities, a large number of free amino acids were generated through acid hydrolysis. And it also took place color reaction because of the monosaccharide from hydrolysis. Finally, a large number of production was consumed. In comparison, there are many advantages of xylanase hydrolysis law, such as mild conditions with the hydrolysis,high efficiency and hydrolysis time is easy to control, etc. With the growing demand of society for their extensive, will promote the industry's overall development.Although the xylanase was used in many large fields, and has many enzymes features that other reagents cannot have, many using problems still exist. For example, low enzyme activity; low level of yield and high cost of production, especially there are a number of factors can affect the enzyme hot stability directly. Because of the hot environment could protect the bacterial strain from harmful microorganisms in the process of growth and quicken the chemical reaction . By the reference texts, the main way to enhance thermostability as following: increasing the amount of disulfide bond or forming the electrovalent bond between protins with different kinds of electric charge.In this study, the thermostability of xylanase was enhanced by site-specific mutagenesis, through synthetic oligonucleotides acting as mutagens. The fouth base of the alanine was changed into threonine, as a result, the residue of xylanase's protin will be changed. Finally found that the xylanase from this site-specific mutagenesis strain also with optimal temperature 70℃and optimal pH 6.5, there is any change in the inactivation curve. As the result, the fouth base mutagenesis without any help of thermostability.