Combination of site-directed mutagenesis and yeast surface display enhances Rhizomucor miehei lipase esterification activity in organic solvent.

To increase the activity of Rhizomucor miehei lipase (RML) in organic solvent, multiple sequence alignments and rational site-directed mutagenesis were used to create RML variants. The obtained proteins were surface-displayed on Pichia pastoris by fusion to Flo1p as an anchor protein. The synthetic activity of four variants showed from 1.1- to 5-fold the activity of native lipase in an esterification reaction in heptane with alcohol and caproic acid as substrates. The increase in esterification activity may be attributed to the four mutations changing the flexibility of RML or facilitating the reaction. In conclusion, this method demonstrated that multiple sequence alignments and rational site-directed mutagenesis combined with yeast display technology is a faster and more effective means of obtaining high-efficiency esterification lipase variants compared with previous similar methods.

Enhancing thermostability of a Rhizomucor miehei lipase by engineering a disulfide bond and displaying on the yeast cell surface.

To increase the thermostability of Rhizomucor miehei lipase, the software Disulfide by Design was used to engineer a novel disulfide bond between residues 96 and 106, and the corresponding double cysteine mutants were constructed. The R. miehei lipase mutant could be expressed by Pichia pastoris in a free secreted form or could be displayed on the cell surface. The new disulfide bond spontaneously formed in the mutant R. miehei lipase. Thermostability was examined by measuring of hydrolysis activity using 4-nitrophenyl caprylate as a substrate. The engineered disulfide bond contributed to thermostability in the free form of the R. miehei lipase variant. The variant displayed on the yeast cell surface had significantly increased residual hydrolytic activity in aqueous solution after incubation at 60 degrees C for 5 h and increased synthetic activity in organic solvent at 60 degrees C. These results indicated that yeast surface display might improve the stability of R. miehei lipase, as well as amplifying the thermostability through the engineered disulfide bond.

Regulation of avilamycin biosynthesis in Streptomyces viridochromogenes: effects of glucose, ammonium ion, and inorganic phosphate.

Effects of glucose, ammonium ions and phosphate on avilamycin biosynthesis in Streptomyces viridochromogenes AS4.126 were investigated. Twenty grams per liter of glucose, 10 mmol/L ammonium ions, and 10 mmol/L phosphate in the basal medium stimulated avilamycin biosynthesis. When the concentrations of glucose, ammonium ions, and phosphate in the basal medium exceeded 20 g/L, 10 mmol/L, and 10 mmol/L, respectively, avilamycin biosynthesis greatly decreased. When 20 g/L glucose was added at 32 h, avilamycin yield decreased by 70.2%. Avilamycin biosynthesis hardly continued when 2-deoxy-glucose was added into the basal medium at 32 h. There was little influence on avilamycin biosynthesis with the addition of the 3-methyl-glucose (20 g/L) at 32 h. In the presence of excess (NH4)2SO4 (20 mmol/L), the activities of valine dehydrogenase and glucose-6-phosphate dehydrogenase were depressed 47.7 and 58.3%, respectively, of that of the control at 48 h. The activity of succinate dehydrogenase increased 49.5% compared to the control at 48 h. The intracellular adenosine triphosphate level and 6-phosphate glucose content of S. viridochromogenes were 128 and 129%, respectively, of that of the control at 48 h, with the addition of the 40 mmol/L of KH2PO4. As a result, high concentrations of glucose, ammonium ions, and inorganic phosphate all led to the absence of the precursors for avilamycin biosynthesis and affected antibiotic synthesis.

[Cloning and expression of lumbrokinase gene in Pichia pastoris].

Lumbrokinase gene F238 was amplified by RT-PCR from the total RNA of earthworm (Eisenia fetida). The gene including signal peptide sequence was inserted into pUCm-T vector to construct pUCm-T-F238. The product was sequenced. The GenBank accession number was DQ202401. Lumbrokinase F238 comprised 738bp and included an open reading frame that encoded a polypeptide of 245 amino acid residues, containing a signal peptide of 7 amino acid residues and a mature peptide of 238 amino acid residues. Both nucleotide and amino acid sequences homologies were 99% after the sequence was compared with Lumbricus rubellus F-III-2. There were two base pair mutations, which subsequently caused two amino acid mutations. The characteristics and structure of F238 was analysed and predicted with biology softwares and databases. The pl of F238 was 4.61. It had eleven Cysteines, which formed three disulfide bonds. Its secondary structure mainly consisted of beta-sheet. Lumbrokinase F238 had serine active center. It was a protease in trypsin family of serine protease superfamily. Lumbrokinase gene F238-m without signal peptide sequence was obtained by PCR using pUCm-T-F238 as template. The expression vector pPIC9-F238-m was constructed by inserting gene F238-m into yeast expression and secretion plasmid pPIC9. Plasmid pPIC9-F238-m was linearized with BgIII and then transformed into Pichia pastoris strain GS115 cell by electroporation method. Phenotypes of transformants were screened in MM and MD plates to ensure the integration of lumbrokinase gene F238-m into yeast chromosome DNA. Methanol was added to a final concentration of 0.5% for the expression of recombination protein every 24h to maintain induction. The result of SDS-PAGE showed that the molecular weight of the expression product was about 28 kDa, in correspondence with the theoretical molecular weight. After the induction of expression, the fibrinolytic activity of the supernatant was measured using artificial fibrin plates. Then the engineering strain of high activity was obtained, and the fibrinolytic activity was up to 100 U/mL.