Galliher P M, Cooney C L, Langer R, Linhardt R J
Appl Environ Microbiol. 1981 Feb;41(2):360-5. doi: 10.1128/aem.41.2.360-365.1981.
Heparinase production by Flavobacterium heparinum in complex protein digest medium, with heparin employed as the inducer, has been studied and improved. The maximum productivity of heparinase has been increased 156-fold over that achieved by previously published methods to 375 U/liter per h in the complex medium. Rapid deactivation of heparinase activity, both specific and total, was observed at the onset of the stationary phase. Nutritional studies on growth and heparinase production showed an obligate requirement for L-histidine and no vitamin requirement. L-Methionine partially relieved the L-histidine requirement. A defined medium containing glucose, ammonium sulfate, basal salts, L-methionine, and L-histidine was developed for growth and heparinase production. The growth rate in this medium was 0.21 h-1, which is 40%, higher than that in complex medium. The maximum volumetric productivity of heparinase in the defined medium was increased to 1,475 U/liter per h, providing a 640-fold increase over that achieved by previously published methods. No rapid deactivation was observed. An examination of alternate inducers for heparinase showed that heparin degradation products, hyaluronic acid, heparin monosulfate, N-acetyl-D-glucosamine, and maltose, induce heparinase in complex medium. An Azure A assay was modified and fully developed to measure the heparin concentration during fermentation and the heparinase specific activity of crude extracts of F. heparinum obtained from sonication, thus negating the need for further purification to measure activity."
研究并改进了肝素黄杆菌在复合蛋白消化培养基中以肝素作为诱导剂生产肝素酶的过程。肝素酶的最大生产率比先前发表的方法提高了156倍，在复合培养基中达到每升每小时375单位。在稳定期开始时，观察到肝素酶活性（包括比活性和总活性）迅速失活。关于生长和肝素酶生产的营养研究表明，对L-组氨酸有绝对需求，对维生素无需求。L-甲硫氨酸部分缓解了对L-组氨酸的需求。开发了一种含有葡萄糖、硫酸铵、基础盐、L-甲硫氨酸和L-组氨酸的确定培养基用于生长和肝素酶生产。该培养基中的生长速率为0.21 h-1，比复合培养基中的生长速率高40%。确定培养基中肝素酶的最大体积生产率提高到每升每小时1475单位，比先前发表的方法提高了640倍。未观察到快速失活现象。对肝素酶的替代诱导剂进行的研究表明，肝素降解产物、透明质酸、硫酸肝素、N-乙酰-D-葡萄糖胺和麦芽糖在复合培养基中可诱导肝素酶。对Azure A测定法进行了改进并充分完善，以测量发酵过程中的肝素浓度以及通过超声处理获得的肝素黄杆菌粗提物的肝素酶比活性，从而无需进一步纯化即可测量活性。
