Li Yinyin, Lin Yue, Jiang Yingzi, Mehwish Hafiza Mahreen, Rajoka Muhammad Shahid Riaz, Zhao Liqing
College of Chemistry and Environmental Engineering, Shenzhen University, Shenzhen, 518060, Guangdong, China.
Health Science Center, Shenzhen University, Shenzhen, 518060, China.
Appl Biochem Biotechnol. 2022 Oct;194(10):4971-4984. doi: 10.1007/s12010-022-03917-5. Epub 2022 Jun 9.
Heparin is a class of highly sulfated, acidic, linear, and complex polysaccharide that belongs to the heparin/heparan sulfate (HS) glycosaminoglycans family. Enzymatic depolymerization of heparin by heparinases is a promising strategy for the production of ultra-low molecular weight heparins (ULMWHs) as anticoagulants. In the present study, a novel heparinase-producing strain Raoultella NX-TZ-3-15 was isolated and identified from soil samples. Herein, the heparinase gene MBP-H1 was cloned to the pBENT vector to enable expression in Escherichia coli. The optimized conditions made the activity of recombinant heparinase reach the highest level (2140 U/L). The overexpressed MBP-H1 was purified by affinity chromatography and a purity of more than 90% was obtained. The condition for biocatalysis was also optimized and three metal ions Ca, Co, and Mg were utilized to activate the reaction. In addition, the kinetics regarding the new fusion heparinase was also determined with a V value of 11.29 μmol/min and a K value of 31.2 μmol/L. In short, due to excellent K and V, the recombinant enzyme has great potential to be used in the clinic in medicine and industrial production of low or ultra-low molecule weight heparin.
肝素是一类高度硫酸化、酸性、线性且复杂的多糖，属于肝素/硫酸乙酰肝素（HS）糖胺聚糖家族。通过肝素酶对肝素进行酶促解聚是生产作为抗凝剂的超低分子量肝素（ULMWHs）的一种有前景的策略。在本研究中，从土壤样品中分离并鉴定出一种新型的产肝素酶菌株拉乌尔菌属NX-TZ-3-15。在此，将肝素酶基因MBP-H1克隆到pBENT载体中，以便在大肠杆菌中表达。优化后的条件使重组肝素酶的活性达到最高水平（2140 U/L）。通过亲和层析对过表达的MBP-H1进行纯化，获得了纯度超过90%的产物。还对生物催化条件进行了优化，并利用三种金属离子Ca、Co和Mg来激活反应。此外，还测定了新型融合肝素酶的动力学，V值为11.29 μmol/min，K值为31.2 μmol/L。简而言之，由于具有优异的K值和V值，该重组酶在医学临床以及低分子量或超低分子量肝素的工业生产中具有巨大的应用潜力。
