黑麦草玉米谷蛋白水解产物中分离的一种五肽的生物活性

1、黑麦草玉米谷蛋白水解产物中分离的一种五肽的生物活性Bioactivity of a Pentapeptide Isolated from Corn Gluten Hydrolysate on Lolium perenne L. 1a natural herbicideCorn gluten hydrolysate (CGH)Five dipeptidesgreaterisolate and identify2 bioactivityPetri dish bioassaySephadex G-15 gel filtration and two-step C18 reversed phase hi

2、gh performance liquid chromatography a pentapeptide+0.5 mg/mL of the pentapeptide50% of root length in the perennial ryegrass bioassayinhibit3 Corn gluten meal (CGM) is a byproduct of corn (Zea mays L.) from the wet-milling process and has been patented as a natural preemergence herbicide. It inhibi

3、ts root growth of germinating seeds but does not damage plants that have formed a mature root system. Because CGM contains approximately 10% nitrogen by weight, it is useful as a natural preemergence herbicide and fertilizer for various plant production systems.4 Corn gluten hydrolysate (CGH), a wat

4、er-soluble material derived from the action of bacterial proteinase, was found more herbicidally active against three grass species than CGM both in the soil and Petri dish bioassays. It also has a greater nitrogen content and could potentially be used to fortify the CGM to improve it as a natural w

5、eed and feed material . Its high water solubility makes it sprayable and easy to apply as a herbicide in some crop production systems. It is also useful as a source for the isolation and identification of the bioactive compounds. 5Materials and Methods Perennial Ryegrass Bioassays1ml test solution P

6、etri dish with fiter paper10 seedsCoveredSealed with Parafilmincubated in a growth chamber90mol/s/m2 ，25/15day/night, an 8h photoperiod6 after 7 days: The longest root and shoot of each seedling were measured. 7 measurements found to have root lengths within 1S.D The seven measurements average root

7、length of the control （received D.D. H2Oonly） The value was expressed as the percentage of the control length. 1007Extraction and Purification Gluten hydrolysate powderD.D. H2Oa 10% solution0.2m membrane filter filtrate designated as GH-f8Sephadex G-15 gel filtration column:calibrated with blue dext

8、ran and NaCl aqueous solutioneluted with D.D. H20 at a flow rate of 1.6 mL/min. a Gilson model 201 fraction collectorcollectEighty fractions with 270 drops a YSI conductivity bridgeConductivity9 30 ml GH-f was loaded onto the column eluted with D.D. H20 at rate of 16 mL/minSelected tubes bioassayed

9、for their bioactivityTubes 50-56 were found to contain the root- and shoot-inhibiting compound(s) freeze dried and subjected to the following purification steps10 12 mg of 53GH-g15-eHPLC0-25% MeOH in 50min, 25-100% MeOH in 25 min, maintained at 100% MeOH 15 min, and 100-0% MeOH in the next 10 min at

10、 flow rate of 4 mL/min.UV absorbance at 280 nmcollected at 10-min intervals for the first 50 min and at 25 min intervals for the next 50 min.Seven fractionssubjected to the perennial ryegrass bioassayFour fractions were found to have greater than 90% root inhibition. The third fractionwas chosen for

11、 further purification 11 12 mg of the bioactive 53GH-g15-e was injected onto a semipreparative Delta Pak with a MeOH in water linear gradient of from 0 to 15% in 30 min at a flow rate of 4 mL/min. The sample was monitored by UV absorbance at 214 nm. Five fractions were collected based on the five ma

12、jor peaks with retention time between 19 and 29 min on the chromatogram.12 One peak was identified as bioactive and subjected to further purification by using a C18 RP narrow bore. with a linear AB gradient from 5 to 25% B in 40 min. B : 0.08% TFA in acetonitrile rate:200 L/minEluent A :0.1% trifiuo

13、roacetic acid in D.D. H2013Peptide sequencingAA composition The molecular weight A major peak14Validation of the Inhibitory Activity of the Pure Compound To confirm the bioactivity of the isolated compound, peptide with the same sequence as the bioactive peak isolated from CGH was synthesized by the

14、 Protein Facility at Iowa State University and was tested for its inhibitory activity. An aqueous solution in a concentration series of 0,0.5,1.0,1.5, 2.0, and 4.0 mg/mL was prepared and subjected to the perennial ryegrass bioassay. 15Statistical Analysis least significant difference (LSD) 16 Result

15、s and Discussion 17 The bioassay results of the fractions suggested that there are probably at least two other groups of compounds in CGH hydrolysate which have root inhibiting activity. All six bioactive peptides were proved to inhibit root formation at a concentration lower than that of either CGM or CGH, and they were demonstrated as naturally occurring, growthregulating compounds. 18They have potential for use in combination with nontoxic, compatibl