猪毛角蛋白降解菌的分离筛选及其降解特性研究

Isolation and identification of pig hair keratin-degrading strain and its degradation characteristic

为了筛选能高效降解猪毛角蛋白的微生物,该研究利用猪毛角蛋白为唯一碳氮源的培养基从土壤中分离角蛋白降解菌,并对其菌种分类及降解角蛋白的特性进行了研究。通过驯化共筛选到6株能够降解猪毛角蛋白的菌株,经过7 d发酵培养,菌株X-3对猪毛角蛋白的降解率达77.3%,降解效果最佳。结合菌株形态特征、生理生化特征及16S rDNA发育进化树分析,初步鉴定该菌株为凝结芽孢杆菌(Bacillus coagulans)。菌株X-3是一株广谱的角蛋白降解菌,对猪毛、羊毛、鸡毛、鹅毛、鸭毛的降解率均大于60%。该菌株在角蛋白底物作用下可产生角蛋白酶和二硫键还原酶,且酶活与猪毛角蛋白降解率变化存在显著的正相关性,表明菌株X-3产生的角蛋白酶和二硫键还原酶在角蛋白降解中起到非常关键的作用。在降解过程中,产生了大量的可溶性蛋白,二硫键中的硫也随之转化为硫酸盐、亚硫酸盐和巯基化合物,其中硫酸盐是主要的转化形式。该菌株的分离筛选丰富了猪毛角蛋白降解菌的微生物资源库,在氨基酸饲料生产中具有潜在的应用价值。

Pig hair keratin accumulates an abundance of potential protein and amino acids,which could be used for the source of amino acid feed.Due to the highly cross-linking by disulfide,hydrogen bonding and hydrophobic interations,Keratin is insoluble and not degraded by normal proteases such as pepsin,trypsin and papainl.Keratin degrading microbial strains can directly utilize keratin as carbon and nitorgen source,which can secret a variety of enzymes,such as keratinase,disulfide reductase.Currently,the kerain-biodegradation by microorganisms represents an alternative attractive method to improve the nutritional value of keratin wastes.In order to obtain the microorganisms with high pig-hair keratin degrading ability,the strains were isolated from environment soil by taking keratin as sole culture carbon and nitrogen source.The identification of Strain X-3 was studied and the keratin-degradation characteristics were further investigated from the aspects of thiolysis and enzymolysis.It was expected to provide theoretical guidance for the resue of feather waste resource.In this study,six strains with pig hair degrading ability were obtained,and strain X-3 could degrade pig hair keratin more effectively than other five strains.The degrading rate of pig hair keratin was up to 77.3%after 7 days fermentation,which was higher than that of the reported strains.The colonies of strain X-3 were irregular in shape,moist and rough on peptone beef medium.The cell of the strain was in the shape of rod,Gram positive and sporulating under the microscope.An aceession number MK182787 was obtained after the information of 16S rDNA sequence of strain X-3 was submitted to NCBI.The phylogenetic tree was constructed based on the sequence of 16S rDNA by the software Mega 5.0.The strain X-3 was identified as Bacillus coagulans on the basis of morphology,physiological characteristics and 16S rDNA sequence phylogenetic analysis.The wool,chicken feather,goose feather,duck feather keratin-degrading rate was 62.3%,86.7%,88.4%,72.4%,respectively after 7 days fermentation.Bacillus coagulans X-3 was a broad spectrum keratin degrading strain because it showed efficient degrading properties to different keratins.The maximum of keratinase activity(84.1 U/mL)and reductase activity(23.8 U/mL)were detected during keratin degradation.High keratinase activity and disulfide reductase activity were detected during the fermentatin process and the keratin degrading rate changed relating to enzyme activity,which indicated enzyme plays important role in the process of keratin degradation.Sulfur in the keratins was transferred into sulfate,sulfite and thiol compounds in the degradation process.The sulfur in the keratin was mainly removed into the medium in the form of sulfate.The soluable protein,sulfate,sulfite and thiol content began to increase quickly accompanied the keratin degradation.However,further rearch will be required to determine the relationship of thiolysis and pig hair keratin degradation.Up to date,Bacillus coagulans has not been reported on the pig hair keratin degrading area,thus,we provide a new efficient keratin-degarding strain to utilize pig hair.Bacillus coagulans X-3 has a great potential roles in amino acid feedstock and environmental protection for its excellent keratin-degrading property and biological safety.