Expression of the lasB gene encoding an organic solvent-stable elastase in Pichia pastoris and potential applications of the recombinant enzymes in peptide synthesis

ABSTRACT The methylotrophic yeast Pichia pastoris is widely used for the expression of heterologous enzymes. While the purity of the desired expression product is of major importance for many applications, we found that recombinant enzymes produced in methanol medium were contaminated by a 37-kDa endogenous yeast protease. This enzyme was completely inhibited by phenylmethanesulfonyl fluoride (PMSF) but not by 1,10-phenanthroline, EDTA, and pepstatin A, suggesting the nature of a serine protease. Its secretion was abolished in P. pastoris strains GS115 and KM71 by specific mutagenesis of a subtilisin gene (SUB2) but not by inactivation of the gene encoding vacuolar proteinase B (PRB). Bioinformatic comparisons of Sub2 protein with subtilisins from other fungal genomes and phylogenetic analyses indicated that this enzyme is not an orthologue of the vacuolar protease cerevisin generally present in yeasts but is more closely related to another putative subtilisin found in a small number of yeast genomes. During growth of P. pastoris, Sub2 was produced as a secreted enzyme at a concentration of 10 μg/ml of culture supernatant after overexpression of the full-length SUB2 gene. During fermentative production of recombinant enzymes in methanol medium, 1 ml of P. pastoris culture supernatant was found to contain approximately 3 ng of Sub2, while the enzyme was not detected during growth in a medium containing glycerol as a carbon source. The mutant strain GS115-sub2 was subsequently used as a host for the production of recombinant proteases without endogenous subtilisin contamination.

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Peptide synthesis catalyzed by organic solvent-stable protease from Pseudomonas aeruginosa PST-01 in monophasic aqueous-organic solvent systems

Journal of Bioscience and Bioengineering ◽

10.1016/s1389-1723(00)87668-9 ◽

1999 ◽

Vol 88 (5) ◽

pp. 513-518 ◽

Cited By ~ 30

Author(s):

Hiroyasu Ogino ◽

Mitsuharu Yamada ◽

Fumitake Watanabe ◽

Humiko Ichinose ◽

Masahiro Yasuda ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Organic Solvent ◽

Peptide Synthesis ◽

Solvent Systems ◽

Solvent Stable

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An organic solvent-stable protease from organic solvent-tolerant Bacillus cereus WQ9-2: Purification, biochemical properties, and potential application in peptide synthesis

Bioresource Technology ◽

10.1016/j.biortech.2010.05.055 ◽

2010 ◽

Vol 101 (20) ◽

pp. 7991-7994 ◽

Cited By ~ 30

Author(s):

Jiaxing Xu ◽

Min Jiang ◽

Honglin Sun ◽

Bingfang He

Keyword(s):

Organic Solvent ◽

Bacillus Cereus ◽

Peptide Synthesis ◽

Potential Application ◽

Biochemical Properties ◽

Organic Solvent Tolerant ◽

Solvent Tolerant ◽

Solvent Stable

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Peptide Synthesis of Aspartame Precursor Using Organic-Solvent-Stable PST-01 Protease in Monophasic Aqueous-Organic Solvent Systems

Biotechnology Progress ◽

10.1002/bp060382y ◽

2007 ◽

Vol 23 (4) ◽

pp. 820-823 ◽

Cited By ~ 13

Author(s):

Shotaro Tsuchiyama ◽

Noriyuki Doukyu ◽

Masahiro Yasuda ◽

Kosaku Ishimi ◽

Hiroyasu Ogino

Keyword(s):

Organic Solvent ◽

Peptide Synthesis ◽

Solvent Systems ◽

Solvent Stable

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Facile fractionation of bamboo hydrolysate and characterization of isolated lignin and lignin-carbohydrate complexes

Holzforschung ◽

10.1515/hf-2020-0040 ◽

2020 ◽

Vol 0 (0) ◽

Author(s):

Xiaodi Wang ◽

Yongchao Zhang ◽

Luyao Wang ◽

Xiaoju Wang ◽

Qingxi Hou ◽

...

Keyword(s):

Solvent Extraction ◽

Organic Solvent ◽

Extraction Process ◽

Water Soluble ◽

Organic Solvent Extraction ◽

Glycoside Linkage ◽

Potential Applications ◽

Tremendous Amount ◽

Main Components ◽

Hemicellulosic Sugars

AbstractAn efficient separation technology for hydrolysates towards a full valorization of bamboo is still a tough challenge, especially regarding the lignin and lignin-carbohydrate complexes (LCCs). The present study aimed to develop a facile approach using organic solvent extraction for efficiently fractionating the main components of bamboo hydrolysates. The high-purity lignin with only a trace of carbohydrates was first obtained by precipitation of the bamboo hydrolysate. The water-soluble lignin (WSL) fraction was extracted in organic solvent through a three-stage organic solvent extraction process, and the hemicellulosic sugars with increased purity were also collected. Furthermore, a thorough characterization including various NMR techniques (31P, 13C, and 2D-HSQC), GPC, and GC-MS was conducted to the obtained lignin-rich-fractions. It was found that the WSL fraction contained abundant functional groups and tremendous amount of LCC structures. As compared to native LCC of bamboo, the WSL fraction exhibited more typical LCC linkages, i.e. phenyl glycoside linkage, which is the main type of chemical linkage between lignin and carbohydrate in both LCC samples. The results demonstrate that organic phase extraction is a highly efficient protocol for the fractionation of hydrolysate and the isolation of LCC-rich streams possessing great potential applications.

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