Expression, purification and characterization of K5 lyase in Escherichia coli / 生物工程学报

K5 polysaccharide of high molecular weight (HLW) can be splitted into low molecular weight (LMW) K5 polysaccharide by K5 lyase which can be used as the substrate of partial synthesis low molecular heparin sulfate (HS). To prepare recombinant K5 lyase (Elma) and analyze its biological activity. The gene of Elma was cloned by PCR amplification and was ligated with pET-28a. Then the recombinant expression vector pET-28a-Elma was transformed into Escherichia coli BL21 (DE3). After induction with 0.2 mmol/L IPTG at 16 degrees C for 5 h, Elma was successfully expressed, SDS-PAGE analysis demonstrated that the enzyme constituted more than 30% of the total cell proteins. After Ni(2+)-NTA affinity and G-75 chromatography, the purity of enzyme was more than 95%. Enzymatic activity was determined according to the change of absorbance at 232 nm per ml of the sample. The reduction of the polysaccharide molecular weight could be detected by PAGE electrophoresis. Elma can partially split HA and HS. Its optimal reatcion temperature is 37 degrees C and the optimal reaction pH is 7.0.

High-level expression of fusion protein GGH in Pichia pastoris GS115 by constructing a double plasmid co-expression system / 生物工程学报

In order to make a large-scale preparation of(GLP-1A2G)2-HAS (GGH), the double-plamid pPICZalphaB and pPIC9K co-expression system was introduced into Pichia pastoris GS115. Firstly, the GGH fusion gene was amplified by PCR to create the recombinant expression plasmid pPICZalphaB-ggh, which was transformed into P. pastoris GS 115/F2 that was integrated by another recombinant expression plasmid pPIC9K-ggh. The immunology method combined with high concentration antibiotic was used to screen recombinant strain P. pastoris GS115/F3 capable of high-level expression of GGH protein. The GGH fusion protein expressed by GS115/F3 increased 49.7% compared with the GS 115/F2 in the expression conditions of 3% methanol inducing 80 h at 30 degrees C. At the same time, the quantitative PCR results showed that GGH gene dose in GS115/F3 increased 26.7% with respect to that of GS 115/F2. Furthermore, the Western blotting experiment indicated that the recombinant GGH possess the two antigenicities of GLP-1 and HSA.

Cloning, expression and characterization of N-acetylornithine aminotransferase from Corynebacterium crenatum and its effects on L-arginine fermentation / 生物工程学报

N-Acetylornithine aminotransferase (EC 2.6.1.11, ACOAT) catalyzes the conversion of N-acetylglutamic semialdehyde to N-acetylornithine, the forth step involved in the L-arginine biosynthetic pathways. We studied the enzyme properties to set up reliable theoretical basis for the arginine fermentation optimization. ACOAT encoding gene argD was cloned from an industrial L-arginine producer Corynebacterium crenatum SYPA 5-5. Analysis of argD sequences revealed that only one ORF existed, which coded a peptide of 390 amino acids with a calculated molecular weight of 41.0 kDa. The argD gene from C. crenatum SYPA 5-5 was expressed both in Escherichia coli BL21 and C. crenatum SYPA. Then ACOAT was purified by Ni-NTA affinity chromatography and its specific enzyme activity was 108.2 U/g. Subsequently, the expression plasmid pJCtac-CcargD was transformed into C. crenatum SYPA and the specific activity of ACOAT was improved evidently in the recombinant C. crenatum CCD. Further fermentative character of CCD1 was also analyzed. The results showed that the L-arginine producing ability of the recombinant strain was 39.7 g/L improved by 14.7%.

Metabolic flux analysis of L-serine synthesis by Corynebacterium glutamicum SYPS-062 / 生物工程学报

Corynebacterium glutamicum SYPS-062 was an L-serine producing strain stored at our lab and could produce L-serine directly from sugar. We studied the effects of cofactors in one carbon unit metabolism-folate and VB12 on the cell growth, sucrose consumption and L-serine production by SYPS-062. In the same time, the metabolic flux distribution was determined in different conditions. The supplementation of folate or VB12 enhanced the cell growth, energy synthesis, and finally increased the flux of pentose phosphate pathway (HMP), whereas the carbon flux to L-serine was decreased. The addition of VB12 not only increased the ratio of L-serine synthesis pathway on G3P joint, but also caused the insufficiency of tricarboxylic acid cycle (TCA) flux, which needed more anaplerotic reaction flux to replenish TCA cycle, that was an important limiting factor for the further increasing of the L-serine productivity.

The Activity Study of Aminodeoxychorismate Synthase of Differernt Corynebacterium glutamicum / 中国生物工程杂志

The two pabAB genes encoding aminodeoxychorismate synthase(ADC synthase)from a L-serine producing strain Corynebacterium glutamicum SYPS-062 and model strain Corynebacterium glutamicum ATCC 13032 were ampilified by PCR.The result of nucleotide sequence analysis showed that both pabAB fragments were 1863bp,encoding 620 amino acids.16 bases differences that resulted in the changes of 7 amino acids were found in the pabAB of SYPS-062.The two pabAB were inserted into pET-28a to yield the recombinant expression vector pET-28a-pabAB and then transfromed into BL21(DE3).Upon IPTG induction,soluble ADC synthase was over-produced by E.coli BL21(DE3)harboring the expression construct.Recombinant ADC synthase purified by Ni-NTA affinity chromatography showed a single band about 67kDa on SDS-PAGE gel,and activity of aminodeoxychorismate synthase analysis show that the enzyme specific activity of SYPS-062 is 46.6% lower than ATCC 13032.