Study On Fermentation Of Glycoprotein GP-1 By Streptomyces Kanasensis ZX01 And A Novel Anti-plant-virus Agent

Plant virus disease,also known as “plant cancer”,is one of the most severe diseases causing loss of agricultural production.Microorganism is an important biological resource library full of bioactive natural products,from which development of anti-plant virus agents with high activity,low toxicity,broad spectrum and safety has been a hot spot in the field of plant virus disease control.Streptomyces kanasensis ZX01 was a novel Streptomyces with significant activity against Tobacco mosaic virus?TMV?,which was isolated from soil nearby Kanas Lake,Xinjiang province by Research and Development Center of Biorational Pesticide,Northwest A&F University.A novel glycoprotein GP-1 was isolated through bioactivity-guided method.Based on the previous study of our research group,this paper performed medium optimization,fermtation conditions control and fermentation scale-up,aiming to improve GP-1 production.In addition,a novel anti-plant virus agent with glycoprotein GP-1 as active ingredient was preliminarily developed.Main results are as follows:?1?Fermentation process in shake flask was preliminarily studied to comfirm seed medium,fermentation medium and fermentation conditions of glycoprotein GP-1 production by Streptomyces kanasensis ZX01.Seed medium: soluble starch 20 g/L,NaCl 0.5 g/L,FeSO₄·7H₂O 0.01 g/L,K₂HPO₄ 0.5 g/L,KNO₃ 1 g/L,MgSO₄·7H₂O 0.5 g/L,inoculum age of 72 h.Fermentation medium: soluble starch 13.6 g/L,yeast extract 4.2 g/L,NaCl 3.5 g/L,CaCO₃ 0.3 g/L,millet 10.00 g/L.Fermentation conditions: inoculum size of 6%,initial p H of 6.5,rotating speed of 150 rpm.After optimization,glycoprotein GP-1 production increased by 168% to reach 0.9253 mg/L.?2?Fermentation process in fermentor was further studied.The effects of temperature,agitation speed and aeration rate on GP-1 production by Streptomyces kanasensis ZX01 were conducted in 5 L bench-scale fermentor,comfirming temperature of 30?,agitation speed of 200 rpm and aeration rate 2.0 vvm as the optimal operation conditions.Four batches of verification test were carried out under this fermentation conditions in 5 L fermentor,obtaining average glycoprotein GP-1 production of 3.92 mg/L,which was about 4.2 times higher than that from shake flask.?3?Based on constant volumetric oxygen transfer coefficient?k La?,fermentation process of glycoprotein GP-1 by Streptomyces kanasensis ZX01 was scaled up from 5 L to 15 L and 70 L,resulting in average glycoprotein GP-1 production of 3.92 mg/L,4.03 mg/L and 3.82 mg/L,respectively.?4?Batch fermentation kinectics of Streptomyces kanasensis ZX01 was studied in 5 L fermentor.Kinetic models of strain ZX01 growth and glycoprotein GP-1 synthesis were established according to Logistic equation and Luedeking-Piret equation,respectively.The values predicted by the models were in good agreement with the experimental values.?5?A novel anti-plant virus agent,0.5% glycoprotein GP-1 aqueous solution?AS?,was preliminarily developed with glycoprotein GP-1 as active ingredient,through screening different surfactants,preservatives,antifreezes and defoamers.The formula of agent: 0.5% glycoprotein GP-1?active ingredient?,5% APG?surfactant?,1% Kathon?preservative?,1% glycol?antifreeze?,1% PDMS?defoamer?and 91.5% water?solvent?.?6?Indoor pot experiment and field plot experiment both indicated that 0.5% glycoprotein GP-1 AS featured great persistence on controlling TMV disease.In indoor pot experiment,control efficacy of 200 times diluent against TMV disease was 76.28%,75.73% and 67.85% on 5,10 and 15 d,respectively.In field plot experiment,control efficacy of 200 times diluent against TMV disease was 42.39%,47.40% and 43.09% on 10,20 and 30 d,respectively.In conclusion,this article studied systematically the batch fermentation of glycoprotein GP-1 by Streptomyces kanasensis ZX01,which not only improved glycoprotein GP-1 production,but also expanded fermentation scale.The results provide theoretical basis and technical support for industrial production of GP-1.In addition,0.5% glycoprotein GP-1 AS provides a reference for the comprehensive control of plant virus disease.