Cyclic dipeptides in actinomycete Brevibacterium sp. associated with sea cucumber Apostichopus japonicus Selenka: Isolation and identification / 第二军医大学学报

Objective To investigate the secondary metabolites of actinomycete Brevibacterium sp. associated with the sea cucumber Apostichopus japonicus Selenka. Methods The ethyl acetate extract of the actinomycete was purified by repeated column chromatography on silica gel, Sephadex LH-20, and high-performance liquid chromatography (HPLC) to obtain pure compounds; and the compound structures were elucidated by spectroscopic analysis (nuclear magnetic resonance, NMR; mass spectrometry, MS) and the results were compared with the previously reported data. Results Seven compounds were isolated: cyclo-(L-Pro-L-Phe) (1), cyclo-(L-Pro-L-Met) (2), cyclo-(L-Pro-L-Tyr) (3), cyclo-(L-Pro-L-Val) (4), cyclo-(L-Pro-L-Pro) (5), cyclo-(L-Val-Gly) (6), and cyclo-(L-Pro-L-Leu) (7). Conclusion This is the first report on the secondary metabolites of microorganisms associated with the sea cucumber Apostichopus japonicus Selenka, and all the seven compounds have been reported from the actinomycete Brevibacterium sp. for the first time.

A Novel Screening Method for Isolating High Cholesterol Oxidase Producing Strain / 微生物学通报

The cholesterol oxidase producing strain Brevibacterium sp.DGCDC-82 was treated with NTG (1 mg/mL)under ultrasonicztion(200 W,50 kHz).A red mutant named Brevibacterium sp.DGCCN-25 showed higher and stable production of cholesterol oxidase was obtained.The enzyme activity was increased by 140%,it is 1.24 U/mL.Then dealed with DGCCN-25 using the same method,two revertants were obtained,one was white and the other was rose pink.The enzyme activity of two revertants was obvious decrease,they are 0.17 U/mL and 0.69 U/mL.The results showed the positive correlation between COD acticity and red pigment producing by Brevibacterium sp..The relativity model can be used as a method of screening for mutation and directed evolution.

Cloning and Expression of Brevibacterium sp. DGCDC-82 Cholesterol Oxidase Gene in Escherichia coli / 微生物学通报

Based on DNA sequence encoding cholesterol oxidase reported on the NCBI,cholesterol oxidase gene was cloned from Brevibacterium sp. DGCDC-82 by PCR methods,which showed homology of 98% to the previously reported cholesterol oxidase gene from Brevibacterium sterolicum ATCC21387. Subsequently,the resulting products were digested with NcoI and EcoRI and ligated to the pET28a vector by T4 DNA ligase.The recombinant plasmid,pET28a-choB,was transformed into Escheriehia coli BL21-CodonPlus(DE3)-RP which contain extra copies of the argU and proL genes.The positive clone was induced with IPTG,and enzyme expressed in BL21-CodonPlus(DE3)-RP,the enzyme activity was about 340U/L.The expression products were analyzed by SDS-polyacrylamide gel electrophoresis indicating that about 55kD protein was obtained,which accounted for about 16% of the total cell protein.