| Plant protein is derived from plants,mainly including soybean protein,glutenin,etc,which has the same nutritional value as animal protein.Plant protein does not contain cholesterol and has a variety of physiological and health functions,so it is widely used in health food,health drink and other fields.In the era of pursuing great health,plant-based protein food is increasingly favored.There are many glutamine groups in plant protein,and hydrogen bonds and other structures are easily formed between these amide groups,thus causing protein aggregation and precipitation,and lowering its solubility,which affecting its application in plant protein beverages and other foods.Therefore,it is necessary to improve the properties of plant protein,enhance its solubility,improve its utilization rate,and broaden its application and market in food engineering.Protein-glutaminase(PG)is a new type of protein modifier,which can act on glutamine groups on the side chains of many proteins and polypeptides to make them deamidate,thus improving the solubility and other characteristics of proteins.PG does not produce cross-linking reaction,and does not cause changes in protein flavor.It has mild modification conditions,high specificity,wide application and good market potential.At present,there are a little studies on producing PG strains,PG mainly comes from Chryseobacterium proteolyticum,and the ability of wild strains to produce PG by fermentation is poor,which limits the industrial production and wide application of PG.The purpose of this study is to screen out wild strains with high PG production.The properties of PG produced by different strains are different,and the degree of deamidation of plant protein is different.Therefore,more new producing PG strains need to be excavated and identified.Meanwhile,the PG sequences of different strains are analyzed.Protoplast-UV mutagenesis is used to screen out high-yield and characteristic strains.By optimizing the fermentation conditions of the strains,the PG production is increased,which lays a foundation for the large-scale production and application of PG.The main research contents of this topic are as follows:1.Establishing an enrichment culture method of screening producing PG strainsAccording to the growth characteristics of the strains,the study determined the optimal enrichment plan for PG-producing strains as follows: F enrichment medium,10% inoculum size,culture for 5 days.Compared with 9 days of culture reported in the existing literature,the enrichment time of the screening method in this study is shortened,and the screening efficiency is improved.49 soil suspensions are enriched and cultured,and 72 PG-producing strains are screened out,of which 16 strains have enzyme activities greater than 2.0U/m L.The enzyme activity of producing PG wild strains reported in the literature is 0.26-0.72U/m L,and that of strain A4142 is2.15U/m L,which is 2.99 times higher than that of wild strain ZYF 120413-7 with the highest enzyme activity reported in the literature,providing excellent strains for production.2.Identification of PG-producing strains46 producing PG strains were identified.42 strains such as A4142 belong to Chryseobacterium proteolyticum,THN1 is Chryseobacterium oranimense,T9541 is Chryseobacterium sediminis,T15113 is Chryseobacterium gleum,and 4A1 is Chryseobacterium cucumeris.A total of 5 strains of different species were screened,with the enzyme activity ranging from 0.38 to 2.15 U/m L.Among them,three producing PG strains,C.oranimense THN1,C.sediminis T9541 and C.cucumeris 4A1,were reported for the first time in this study,expanding the source of producing PG strains and enriching the strain bank.3.Identification and Analysis of PGSDS-PAGE of fermentation broth of strains showed a band above 18.4KD,which was consistent with the theoretical molecular weight 19860 of PG reported in the literature.By measuring and analyzing the PG sequence of the strains,the results show that the similarity of the PG protein sequence of 42 C.proteolyticum strains such as A4142 and C.proteolyticum 9670 T reported in Japanese literature is 100%,and the similarity of the PG protein sequence of THN1,T9541,T15113,4A1 and C.proteolyticum 9670 T is more than 74%,which showed that the PG sequences produced by different strains are different.The properties of PG from different strains may be different,and the degree of deamidation of plant protein is different,thus expanding the application of PG in the field of food industry.By analyzing the enzyme production curve of the strains,the rapid enzyme production period and the highest enzyme activity point are determined,which is convenient to better guide the application of the strain in practice.4.Protoplast-UV combined mutation breedingThe conditions of protoplast preparation were optimized.The optimal conditions were determined as follows: the bacterial concentration was OD600=5,25 mg/m L lysozyme,enzymolysis was carried out at 30? for 1.5 h,and 100 ?L of 0.1% SDS was added.At this time,the product of protoplast formation rate,purity and regeneration rate was 75.94%,which was 45.76% higher than the protoplast preparation method established in the laboratory.According to the lethal rate and positive mutation rate,the UV irradiation time was determined to be 30 s.Optimizing substrate concentration for enzymatic reaction,and the enzyme activity increased to3.04U/m L,which is 32.17% higher.After three rounds of mutagenesis screening,a total of 6,351 strains were initially screened,and the strain PU0316 was obtained with an enzyme activity of 3.73 U/m L,which is increaseded by 15.48%.In this study,the white Chryseobacterium proteolyticum which can normally secrete PG were screened out,which is convenient for the fermentation and purification of PG in the later stage and enriches the strain types.5.Optimization of fermentation conditions of the strainsThrough single factor optimization,lactose was determined to be the best carbon source,and polypeptone was determined to be the best nitrogen source.The promoter could stabilize the p H of the culture medium and keep the ammonium ion at a low level.Span 80 could promote the growth of the strain.By optimizing the fermentation conditions,it was determined that the optimal culture conditions for enzyme production were 0.1% lactose,2.0% polypeptone,1.5% promoter and 240r/min rotation speed.At this time,the enzyme production of the strain was the highest,and the enzyme activity was 5.31 U/m L,increasing of 55.72%,which was the highest PG enzyme activity reported in the literature at present.According to the growth characteristics of producing PG strains reported in the literature,F enrichment medium,10% inoculation amount of soil suspension and 5days of culture were used for enrichment.Compared with 9 days of culture reported in the existing literature,the screening method in this study has shortened enrichment time,improved screening efficiency,high positive rate of strains and high enzyme activity.A high-yield strain with an enzyme activity of 2.15 U/m L was screened from the soil,which is 199% higher than the wild strain ZYF 120413-7 with the highest enzyme activity reported in the literature.Among them,C.oranimense THN1,C.sediminis T9541,C.cucumeris 4A1 are new producing PG strains,which are reported for the first time in this paper.The PG sequences of the strains were determined and analyzed,which showed that the PG sequences produced by different strains were different,the properties of PG might be different,the degree of deamidation of plant protein was different,and the optimal action conditions were different,thus expanding the application of PG in food industry.Protoplast-UV mutagenesis was used to screen white high-yield PG strains with pigment deletion,which can provide convenience for subsequent purification.By optimizing the fermentation conditions,the enzyme activity increased to 5.31U/m L,which is the highest PG enzyme activity reported in the literature at present.In this study,a high-yield PG strain was obtained through screening,which laid a foundation for large-scale industrial production of PG. |
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