Expressed Cellobiohydrolase Enzyme of Thermobifidia fusca in Pichia pastoris as Host Can Act on Cotton Substrate

Document Type : Research Articles

Authors

• Karim Imangholiloo ¹
• Nasrin Moshtaghi ²
• Seyed Hasan Marashi ²
• Abdolreza Bagheri ²
• Ahmad Sharifi ³

¹ Agricultural Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran

² Biotechnology and Plant Breeding Department, Ferdowsi University of Mashhad, Mashhad, Iran

³ Ornamental Biotechnology Department, ACECR, Mashhad Branch, Mashhad, Iran

https://doi.org/10.22067/jcmr.2022.74925.1029

Abstract

Cellulose which is extremely produced by plants, can be used for biofuel production but this function needs chemical or enzymatic digestion. Cellulose hydrolysis of plant wastes for ethanol production requires a mixture of three enzyme groups, including endoglucanases, exoglucanases, and beta-glucosidases. The cellobiohydrolase enzyme (Cel6B) from Thermobifidia fusca has been used for cellulase activity extensively. This research aimed to express recombinant Cel6B enzyme in Pichia pastoris. For this purpose, cel6B gene in control of AOX1 promoter (methanol inducible) was introduced into Pichia pastoris. Amplification of cel6B gene was performed by PCR technique and then introduced into the Phil-S1 yeast vector. The recombinant construct contained the cel6B gene sequence and PHO1 signal sequence as secretion signal was transferred into Pichia pastoris GS115 strain. The transformed yeast cells expressed the recombinant Cel6B to yield 2.104 U (µmol/min)/ml of culture medium. Purified recombinant enzyme showed the best activity at 60 °C and pH 4.5 and this was agreed with optimum conditions for recombinant Cel6B enzymes which were produced in other systems. The purity of the enzyme was examined by SDS–PAGE technique, and a single band with a molecular weight about 59.6 kDa was observed. As cel6B gene sequence was not optimized for expression in the Pichia pastoris yeast, this could be one of the reasons for low level activity of recombinant Cel6B enzyme. This thermostable enzyme can be used for cellulolytic digestion of biomaterials in biofuel production research and other uses.

Keywords

• Cellulose
• Cel6B
• yeast
• AOX1 promoter
• Expression

20.1001.1.20089147.2022.14.1.1.1

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