| ?-L-rhamnosidase(EC 3.2.1.40)is a glycosidase which can specifically cleave a non-reducing terminal rhamnosyl group.To date,?-L-rhamnosidase is widely used in food processing,pharmaceutical industry and chemical engineering,and has a very broad market prospect.Microbial ?-L-rhamnosidase generally requires the presence of an inducer to secrete it,but ?-glucosidase could also be produced at the same time.This may cause great trouble in the subsequent purification and application.Therefore,it is very urgent to obtain a highly efficient ?-L-rhamnosidase.A strain named Aspergillus niger NCU-317 capable of secreting ?-L-rhamnosidase was isolated and screened in our earlier study.This strain also could produce extra ?-glucosidase after adding the inducers.So,this study intends to solve the problems shown above through molecular biology technology.The aim of this study was to construct a genetic engineering strain which can secrete active recombinant a-L-rhamnosidase on the basis of Aspergillus niger NCU-317 encoding gene.The main results of this study are as follows:1.Aspergillus niger NCU-317 was inoculated on solid media instead of traditional liquid meida,and the total RNA was completely extracted.Then,designing specific primers and using TdT terminal transferase to obtain the target fragment on the basis of RACE technology,what's more,nested and touch-down PCR were used to improve the stability and reliability of RACE technology.Results showed that improved RACE technology could be very efficient and stable to extract the encoding gene from Aspergillus niger.2.The full length of ?-L-rhamnosidase gene(?-L-Rha)was successfully obtained using the cloned cDNA sequence of Aspergillus niger NCU-317 as a template.The fulllength cDNA of ?-L-rhamnosidase is 3018 bp contained a 2748 bp complete open reading frame,the 5' non-coding region was 216 bp and the 3' non-coding region was 72 bp.Bioinformatic analysis of the sequence showed that the encoded protein consisted of 915 amino acids with a predicted theoretical molecular weight of 103.94 kD and an isoelectric point of 5.59.The secondary protein structure was dominated by random coils;the amino acid sequence of Aspergillus niger NCU-317 had the highest homology with the bacterial family ?-L-rhamnosidase family protein which can reach to 99.6% through UniProt alignment.3.The prokaryotic recombiant expression vector pET25b-?-L-Rha was constructed,and Rossetta,which could recognize the rare codon,was used as the host strain.After IPTG induction experiment,the ?-L-rhamnosidase secreted by the positive Rossetta-engineering strain was determined to be in the form of inclusion body.The inclusion body was renatured through dilution dialysis method,but the enzyme assay using rutin and naringin as substrate can hardly detect the enzyme activity.4.The pPIC9K-?-L-Rha recombinant vector was constructed and Pichia pastoris GS115 was used as the host strain.The recombinant vector was linearized with Bgl II and electroporated into Pichia pastoris GS115.Eight transformants that conformed to the Bgl II linearized phenotype were screened,but it is almost impossible to determine the enzyme activity.So,eight positive genetic engineering strains were inducted for 120 h,subsequently,seventy percent ammonium sulfate precipitation and dialysis operation was used to process the recombinant enzyme.In the end,almost no enzyme activity can be detected.5.The original gene from Aspergillus niger NCU-317 and the encoding sequence derived from JMU-TS528 were separately optimized,and the enzyme activity was detected in the optimized JMU-TS528 genetically engineered GS115.The enzyme activity was 626.21 U/mL at 45?. |
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