Research For Producing Mucopolysaccharide A By Bacterial Fermentation Method From Streptococcus Zooepidemicus

Mucopolysaeccharides usually refer to a class of microbialpolysaccharides which consist of basic unit of aminohexose and uronicacid. They are widespread in animals, especially with high quantity inconnective tissues. Mucopolysaeccharides are also calledglycosaminoglycan because of their solution with high viscosity.Mucopolysaeccharides can be categorized into dermatan sulfate, heparinsulfate, keratan sulfate, chondroitin sulfate and hyaluronic acid based onmonosaccharide constituents, molecular weight and functional groups.Mucopolysaeccharides are of special physiological functions andpharmacological features and closely related to human life.Streptococcus zooepidemicus BU100is proved to be a strain whichcan steady produce a novel mucopolysaeccharide A. It is isolated fromcattle nasal mucosa after a series of mutation screening. An early studypreliminarily determined the fermentation conditions, isolation andpurification methods of mucopolysaeccharide A, and its structure. Thus,this thesis mainly focused on the optimization of fermentation conditions,the improvement of isolation and purification methods and gene synthesis of mucopolysaeccharide A.In the first part of this thesis, by using of orthogonal test, wedetermined the optimal ratio of the culture medium, that is, sucrose15,nitrogen sources (fish peptone-yeast extract1:1)10,powdered beef5,KH₂PO₄2, NaH₂PO₄2,NaHCO₃2,MgSO₄·7H₂O2. Meanwhile, singlefactor method was applied to optimize shaking culture, includingtemperature-30℃, initial PH-5.0and rotational speed of shakingtables-160r·min^-1. Based on the optimal medium, we obtainedmucopolysaeccharide A with the yield of10.09g·L^-1. A subsequent5L-scale fermentation improved the yield to20.48g·L^-1.In the second part of this thesis, we improved the device of diatomiteplate filtration for mucopolysaeccharide A's purification. A buffer devicewas added to the inlet of the liquid. The improved device can obtainclarified crude solution after twice filtration. The absorbance of final cellconcentration is0.002at600nm which means the removal rate of cellreaches95.10%. The loss of mucopolysaeccharide A is21.40%and thequantity of residual protein measures up to the standard (＜0.1%).In the third part of this thesis, we extracted the complete genomefrom BU100. Cloning of related genes responsible for biosynthesis ofmucopolysaeccharide A is based on the design of primers which refer tothe sequences of sz-hasA, a conserved sequence of sz-hasA andnon-coding region located in the upstream and downstream of sz-hasA. Unfortunately, we did not get the expected products, only obtained a genefragment with a length of673bp. Sequencing study found that thisfragment is not an ideal PCR amplified products, however the results canbe regarded as a basis and reference for subsequent study.