Cloning, expression and characterization of the serine protease gene fromChaetomium thermophilum

Abstract

Aims: Microbial proteases play an essential role in scientific research and commercial applications. This study is to clone, sequence, and express a thermostable protease gene from the thermophilic fungi Chaetomium thermophilum and to generate yeast strains expressing C. thermophilum protease suitable for industrial applications. Methods and Results: Degenerate primers were designed based on the conserved domain of other identified serine proteases and cDNA fragment of C. thermophilum gene pro was obtained through reverse transcriptase‐polymerase chain reaction (RT‐PCR). The full‐length cDNA of 2007 bp was generated using RACE amplification. The cDNA contains an open reading frame of 1596 bp encoding 532 amino acids. Sequence analysis of the deduced amino acid sequence revealed high homology with the catalytic domains of the subtilisin serine proteases. The C. thermophilum gene pro was expressed in Escherichia coli BL21 (DE3) and Pichia pastoris, respectively and soluble protein was obtained in P. pastoris. The expressed protease was secreted into the culture medium by the yeast in a functional active form and the purified recombinant protease exhibits optimum catalytic activity at pH 8·0 and 60°C. The enzyme is stable at 60°C. The integration of gene pro into P. pastoris genome is stable after 10 generations and the yeast transformants showed a consistent protease expression. Conclusions: Gene pro encoding a serine protease from C. thermophilum was cloned, sequenced, and overexpressed successfully in P. pastoris. The expressed protease was purified and the properties of the recombinant protease are characterized. Significance and Impact of the Study: Chaetomium thermophilum is a soil‐borne thermophilic fungus and the protease cloned from it is stable in a high temperature and a wide rage of pH. The overexpression of the enzyme in a mesophilic micro‐organism offers a potential value for scientific research and commercial applications.

Cloning, expression, and characterization of serine protease from thermophilic fungus Thermoascus aurantiacus var. levisporus

“…It was found that the sequence of Tapro gene had high similarity to that from Aspergillus niger (XM 001391433) and Penicillium marneffei (XM 002147391). Similar to other subtilisins reported previously (Catara et al, 2003;Shi et al, 2008;Li and Li, 2009), T. aurantiacus var. levisporus protease gene Tapro encodes a putative enzyme that consists of a putative catalytic domain with an active sites formed by three residues of Asp183, His215, and Ser384.…”

“…In the activity assays, the expressed protease exhibited remarkable thermal stability and a high activity and it is one of the most thermostable proteases isolated from fungi (Shenolikar and Stevenson, 1982;Gaur et al, 1989;Morton et al, 2003;Wang et al, 2006;Merheb et al, 2007;Liu and Yang, 2007;Yang et al, 2007;Peña-Montes et al, 2008;Li and Li, 2009). Most of proteases reported before are not stable when treated at a high temperature or a wide range of pH, and their optimum reaction temperature is relatively lower, for example, the protease from Stenotrophomonas maltophilia was found to be stable up to 40°C for only 5 min (Miyaji et al, 2005), the purified protease of Aspergillus nidulans was stable below 60°C, while the inactivation rate increase dramatically above this temperature (Peña-Montes et al, 2008), the protease of T. aurantiacus remained stable only up to 50°C (Merheb et al, 2007), the Malbranchea pulchella protease, named thermomycolase, was stable at 45°C when concentrated from the culture medium (Maheshwari et al, 2000).…”

“…levisporus is still lower than that of proteases isolated from thermophile bacteria (Manavalan et al, 2007) and hyperthermophilic archaea, most applications of enzymatic action occur at 40-60°C. The thermostable enzymes from thermophilic fungi may be more suitable for commercially applications than those from hyperthermophiles (Maheshwari et al, 2000;Li and Li, 2009).…”

“…However studies on the proteases from thermophilic fungi are very limited, only few proteases gene have been cloned, such as, protease from thermophilic fungi Thermomonospora fusca YX (TfpA), Chaetomium thermophilum and have been expressed in P. pastoris, respectively (Kim and Lei, 2005;Li and Li, 2009).…”

Halofi̇li̇k Lakti̇k Asi̇t Bakteri̇leri̇ni̇n Üretti̇ği̇ Hi̇droli̇ti̇k Enzi̇mler

“…Ekmek üretiminde ise proteazlar, hamurun yo¤unlu¤unu azaltmak için, hamurda bütünlü¤ü sa¤lamak için, ekmekteki gluten gücünü düzenlemek için, doku kontrolünü ve tad› gelifltirmek için ilave edilmektedir (39). Proteazlar ayn› zamanda t›bbi teflhis, ilaç, deterjan, bira sanayi ve biyomoleküler uygulamalarda kullan›lmaktad›r (40,41).…”