The Purification,Physicochemical Properties And Anticoagulant Mechanism Of Marine Bacilllus Subtilis Anticoagulant Protein

Thrombotic diseases are also called thromboembolic diseases."The Lancet"reported on the causes of death in China over the past 27 years（1997-2017）.Stroke and ischemic heart disease were the main causes of death in the Chinese population.In addition,thromboembolic diseases are a complication in many cancers and have become the second leading cause of death among cancer patients.Anticoagulant drugs have been widely used in clinic,but there are still many defects.For example,short half-life,bleeding,hypersensitivity,and drug interactions limit its scope of application.At present,most of the various anticoagulant substances are isolated from animals and plants,but there are relatively few derived from microorganisms.Microbes have the characteristics of easy cultivation,short growth cycle,and environmental friendliness,therefore,they are favored by researchers.Compared to land,the ocean has the potential to produce unique,unmatched,biologically active compounds.There are few reports of anticoagulant active substances derived from natural microorganisms,and they are mainly concentrated on fungi（Lachnum singerianum）,bacteria（Oceanimonas）,and streptomyces.There are few reports on anticoagulant active substances derived from marine Bacillus subtilis.In the early stage,we obtained a strain of Bacillus subtilis ZHX capable of producing anticoagulant protein from the mangrove silt in Beihai city,Guangxi,China.First,the method for detecting anticoagulant activity was optimized.Then,ultrasound-assisted and fermentation conditions were optimized to increase the yield of anticoagulant protein.The anticoagulant protein was separated and purified,and the physicochemical properties of the anticoagulant were researched.The anticoagulant properties and anticoagulant mechanism of anticoagulant protein were preliminarily explored.The main conclusions are as follows:（1）Optimization and perfection of conditions of anticoagulation activity detection method.By optimizing the buffer type and concentration,fibrinogen（FIB）concentration,thrombin（TB）concentration,and reaction time,the optimal reaction conditions were obtained:fibrinogen（0.2%,w/v）were prepared by dissolving them in PBS（0.05 M PBS,p H 7.4）containing Na Cl（0.09%,w/v）.A 40μL sample or the control（PBS buffer）was mixed with 20μL thrombin（10 NIH/m L）in a plate well,followed by adding 140μL fibrinogen quickly.Each solution was then mixed vigorously for 10 s and incubated for 10 min at 37℃.Finally,the absorbance of the solution at 405nm(OD₄₀₅)was measured.（2）The effect and mechanism of ultrasound on anticoagulant protein produced by B.subtilis ZHX.The growth curve of B.subtilis ZHX showed that the“diauxic growth（DG）”phenomenon,which was mainly caused by the preferential consumption of glucose（main carbon source）.At different stages of cell growth,ultrasonic waves were applied to obtain the best ultrasonic treatment time for the logarithmic phase of cell growth,especially the exponential prophase（5 h）.Optimize the ultrasonic parameters（ultrasonic power,duty cycle and ultrasonic duration）,and perform ultrasonic treatment under the best conditions,comparing the changes before and after ultrasonic treatment.It was found that under optimal conditions（25k Hz,ultrasonic power of 140 W,duty cycle of 40%,duration of 5 min）,ultrasonic treatment did not significantly change the cell morphology（P>0.05）,but a small number of cells broken.Ultrasound increases cell permeability and promotes substrate utilization.Compared with the control group,the anticoagulant activity increased by 1.72 times（from 32 U/m L to55.36 U/m L）,the fermentation time was shortened by 3 h,and the glucose consumption completion time（from 10 h to 9 h）was shortened by 1 h.Ultrasound can significantly increase the production of B.subtilis ZHX anticoagulant protein.During ultrasonic treatment,cell growth phase,cell morphology,and cell integrity were significant to cells.Low-intensity ultrasound can produce a stable cavitation,which can effectively stimulate the exchange of substances and energy,promote metabolism,promote cell growth,and increase productivity.（3）Optimization of fermentation conditions for anticoagulant protein produced by B.subtilis ZHX.The single-factor method and orthogonal design experiment were used to optimize the fermentation medium by Bacillus subtilis.The best medium composition:sucrose 45 g/L,peptone 40g/L,Mg SO₄ 7H₂O 3.5 g/L,K₂HPO₄·3H₂O 3 g/L,KH₂PO₄ 1 g/L.The optimized culture medium was simple and convenient to operate,and conducive to kinetic analysis in the fermentation process.On this basis,the single-factor method was used to optimize the fermentation process,and the optimal fermentation conditions were obtained:filling volume 50/250 m L,speed 220 r/m,initial p H 6,inoculation volume 4%（v/v）,Temperature 30℃,fermentation time 60 h.The results show that the optimized culture conditions can significantly improve the ability of Bacillus subtilis to produce anticoagulant protein（the highest anticoagulant activity was 236U/m L）and shorten the fermentation time（initial fermentation time was 66 h,and optimization time was 60 h）.（4）Isolation,purification and physical and chemical properties of anticoagulant protein derived from marine microorganism B.subtilis ZHX.After analysis by ammonium sulfate precipitation,anion exchange chromatography,gel filtration chromatography and gel permeation chromatography,pure anticoagulant protein was isolated from marine microorganism B.subtilis ZHX.The molecular weight was 27.2 k Da.Analysis of its physical and chemical properties showed that the optimal reaction temperature of the anticoagulant protein was 50℃,and the temperature stability was 25～37℃;the optimal reaction p H was 9,and had good stability under alkaline conditions（especially p H 9～10）.Cu²⁺and Fe³⁺had significant inhibitory effects on anticoagulant proteins.When the metal ion was less than 10 m M,Zn²⁺,Ca²⁺and Mn²⁺have a positive effect on the anticoagulant activity.Especially,when the concentration of metal ion Zn²⁺were 5 m M and 10 m M,the anticoagulant activity increased by 40.49%and33.17%,respectively.（5）Study of anticoagulant properties and mechanism of anticoagulant proteins in vitro.Electrophoretic pure anticoagulant protein does not cause platelet aggregation and inhibited on platelet aggregation.The inhibition rate of 35 U/m L anticoagulant protein on platelets is not significantly different from heparin sodium（50 U/m L）.The red blood cell aggregation experiment showed that the anticoagulant protein did not cause the aggregation.The hemolysis rate of the red blood cell（RBC）with anticoagulant activity of 80U/ml was 1.8%±0.0167,there was no transparent circle on the agar plate of electrophoresis pure anticoagulant protein,which indicated that anticoagulant protein was certain safety on RBC.Anticoagulant protein can significantly prolong the experiment of plasma calcium recovery time and thrombin time,and was a good anticoagulant.The anticoagulant protein can degrade fibrinogen,the bandγwas preferentially degraded,the second was the Bβchain,and the last one was Aαchain,and the starting time of degradation was 30 min,60 min and 180 min,respectively,which was time-dependent.The anticoagulant protein did not react with thrombin.Its anticoagulation mechanism mainly included:（1）degrading fibrinogen（coagulation factorⅠ）,reducing the content of fibrinogen,and exerting an anticoagulant effect;（2）blocking the coagulation reaction by inhibiting the interaction between coagulation factor and platelet receptors;（3）The anticoagulation pathway may be related to the endogenous coagulation pathway.In this study,the anticoagulant protein from Bacillus subtilis has certain safety.It is expected to provide a reference for anticoagulant active substances from marine microorganisms,as well as a scientific basis and reference for the development of anticoagulant drugs with safety.