Screening,Identification,Cloning And Expression Of A Novel Heparinase-producing Strain

Heparin is a class of highly sulfated linear glycosaminoglycans,which has anti-coagulation,anti-thrombosis,anti-virus,anti-inflammation and anti-tumor effects,but studies have shown that in the process of heparin application,there are often many different side effects,such as heparin-induced thrombocytopenia,allergic reactions and so on.Low molecular weight heparin and ultra-low molecular weight heparin obtained by pyrolysis of unfractionated heparin not only have higher anticoagulant activity,but also have fewer side effects.Therefore,more and more attention has been paid to their research and preparation.At present,low molecular weight heparin products are mainly prepared by the chemical method.The treatment effect of this method is too intense,which can easily lead to changes in the structure of heparin and loss of biological activity.In contrast,the reaction conditions of heparin enzymatic pyrolysis are mild,which does not affect the structural characteristics of heparin itself,and the yield of products is higher;the reaction is completed in one step,and the produces almost no impurities,which is more conducive to the separation and purification of downstream.Therefore,the enzymatic production of heparin drugs has important industrial application prospects.Heparinase is a class of proteins that can specifically cleave heparin and heparin sulfate glycoside bonds.There are two key problems to be solved in the production of heparin drugs by heparinase lysis:the narrow source of heparinase and the high cost of heparinase preparation.Therefore,it is particularly important to screen new microorganisms with a high yield of heparinase and construct a high expression system of soluble heparinase.To solve the above problems,Raoultella sp.NX-TZ-3-15,a new heparinase-producing microorganism,was isolated from soil and its fermentation conditions were optimized.The whole genome of Raoultella sp.NX-TZ-3-15 was sequenced and analyzed,two novelheparinase gene sequences H1（684 bp）and H2（699 bp）were obtained.These genes were cloned by seamless cloning and assembly technology.The recombinant plasmid was constructed and introduced into E.coli to express soluble heparinase efficiently.The rusults of this paper are shown as the followings:（1）In this paper,a novel heparinase-producing strain was screened and isolated from soil samples attached to the slaughterhouse.The strain was identified as Raoultella sp.by morphological observation,16S ribosomal DNA identification and BLAST homology comparison and stored as Raoultella NX-TZ-3-15.（2）The fermentation medium composition and culture conditions affecting the growth and metabolism of Raoultella NX-TZ-3-15 were studied,and the optimum fermentation medium（1%maltose,1%soybean peptone,0.25%KH₂PO₄,0.025%MgSO₄·7H₂O,0.2%heparin sodium）,and the optimum culture conditions（initial pH 9.5,10%inoculation,37°C,200 rpm,24 h）were obtained.（3）Raoultella sp.NX-TZ-3-15 was sent for genome-wide sequencing.Bioinformatics technology was used to analyze the sequencing results,and the novel heparinase gene sequences H1 and H2,were obtained.The target genes H1 and H2 were obtained by using genome DNA of Raoultella sp.NX-TZ-3-15 as the template of PCR.The recombinant plasmids pBENT-H1and pBENT-H2 were successfully constructed with the plasmid pBENT through seamless cloning and assembly technology,and transferred into E.coli BLR（DE3）competent cells.The recombinant bacteria were verified by colony PCR,single digestion of recombinant plasmid and sequencing.The results showed that the cloning was successful.（4）The results of induced expression of recombinant bacteria showed that the target proteins expressed by E.coli BLR（DE3）pBENT-H1 and E.coli BLR（DE3）pBENT-H2 were soluble,mainly expressed in the supernatant,and the molecular weight of the expressed proteins was 25 kDa.The enzyme activity of E.coli BLR（DE3）pBENT-H1 was 1650 U/L,which was4.34 times higher than that of Raoultella sp.NX-TZ-3-15,but there was almost no heparinase activity in E.coli BLR（DE3）pBENT-H2.In the follow-up study,E.coli BLR（DE3）pBENT-H1 was selected as the research object.（5）The induction conditions of E.coli BLR（DE3）pBENT-H1 were studied.The activity of recombinant heparinase reached to the highest level（2140 U/L）under the optimal conditions as follows:cultivation at 30°C in LB medium,induction with 0.25 mM IPTG for 8 h after inoculating at 37°C for 3.5 h(OD₆₀₀ of bacterial solution was about 0.7)with rotary shaking at200 rpm.