Hindawi Publishing Corporation Enzyme Research Volume 2014, Article ID 197938, 20 pages http://dx.doi.org/10.1155/2014/197938 Research Article Molecular Cloning and Optimization for High Level Expression of Cold-Adapted Serine Protease from Antarctic Yeast Glaciozyma antarctica PI12 Norsyuhada Alias,1 Mu’adz Ahmad Mazian,1 Abu Bakar Salleh,1 Mahiran Basri,2 and Raja Noor Zaliha Raja Abd. Rahman1 1 Enzyme and Microbial Technology Research Centre, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia (UPM), 43400 Serdang, Selangor, Malaysia 2 EnzymeandMicrobialTechnologyResearchCentre,FacultyofScience,UniversitiPutraMalaysia(UPM),43400Serdang, Selangor, Malaysia Correspondence should be addressed to Raja Noor Zaliha Raja Abd. Rahman; [email protected] Received 4 March 2014; Revised 6 May 2014; Accepted 14 May 2014; Published 30 June 2014 Academic Editor: Raffaele Porta Copyright © 2014 Norsyuhada Alias et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Psychrophilic basidiomycete yeast, Glaciozyma antarctica strain PI12, was shown to be a protease-producer. Isolation of the PI12 protease gene from genomic and mRNA sequences allowed determination of 19 exons and 18 introns. Full-length cDNA of PI12 protease gene was amplified by rapid amplification of cDNA ends (RACE) strategy with an open reading frame (ORF) of 2892 bp, coded for 963 amino acids. PI12 protease showed low homology with the subtilisin-like protease from fungus Rhodosporidium toruloides (42% identity) and no homology to other psychrophilic proteases. The gene encoding mature PI12 protease was cloned into Pichia pastoris expression vector, pPIC9, and positioned under the induction of methanol-alcohol oxidase (AOX)promoter. The recombinant PI12 protease was efficiently secreted into the culture medium driven by the Saccharomyces cerevisiae -factor ∘ signal sequence. The highest protease production (28.3 U/ml) was obtained from P. p a stor i s GS115 host (GpPro2) at 20 Cafter72 hours of postinduction time with 0.5% (v/v) of methanol inducer. The expressed protein was detected by SDS-PAGE and activity staining with a molecular weight of 99 kDa. 1. Introduction green algae that lived in cold environment are also listed as impending sources of cold enzymes. The dominant taxa Eukaryotic as well as prokaryotic organisms can produce are ascomycetous and basidiomycetous yeasts and melanized enzymes adapted to cold. The majority of the cold-adapted fungi [2, 7]. enzymes that have been characterized were originated from The properties that characterized and distinguished cold bacteria living in the Polar Regions in Antarctic and Antarctic adapted enzymes from enzymes of higher temperature origin seawaters [1, 2]. Other possible sources of cold enzymes are their increased turnover number ( cat)andinherent ∼ ∘ / are offered by the psychrophiles that inhabit at 5 Cin catalytic efficiency ( cat ) at low temperatures, which is other permanently cold environments such as the deep considered to be an adaptive strategy of the psychrophiles to sea, glaciers and mountain regions, in soils, and fresh or compensate for low metabolic fluxes and slow reaction rates saline waters related with cold-blooded animals such as fish at their physiological temperatures [8]. Cold-active enzymes or crustaceans and artificial sources such as refrigeration are more thermolabile and sensitive to other denaturants appliances and equipments [2–6]. Although the studies of relative to their thermophilic and mesophilic counterparts cold adapted enzymes are focusing more on psychrophilic that confer the conformational flexibility to the active site9 [ ]. and psychrotrophic bacteria; yeast, fungi, and unicellular As temperature decreases, enzymes demonstrate a declining 2 Enzyme Research catalytic rate owing to reduction of structural flexibility and been deposited in the GenBank database under accession undergo cold denaturation [7]. number JX896956 [14]. Psychrophilic enzymes possess remarkable prospective in a wide variety of industrial applications. Our interest 2.3. Nucleic Acid Isolation. A single colony of the yeast was is focused on cold-adapted protease as a useful biocata- inoculated into 50 mL yeast peptone dextrose broth (YPD) lyst in industry such as low temperature clothes washing ∘ andincubatedfor10daysat4Cwithoutshaking.Thecell and enzyme-added detergents to hydrolyze macromolecular pellets were frozen in liquid nitrogen and ground to a powder stains [10]. An example of commercially available cold- in a ceramic mortar. Genomic DNA was extracted using active protease is from Novozyme (trade name Savinase) a phenol-chloroform method as described elsewhere [19]. which is sold as an encapsulated detergent [11]. Proteases Removal of RNA from genomic DNA was performed by the are degradative enzymes which catalyze the hydrolysis of ∘ addition of 15 L RNase (10 mg/mL) and incubated at 37 C proteins and are commonly found among animal tissues, for 45 min. Total RNA was extracted by the guanidium isoth- plant, and microorganisms [12]. Proteases execute a large iocyanate method using Trizol reagent (Invitrogen, USA) variety of functions and take part in numerous biochemical according to the manufacturer’s instructions. Approximately reactions in living organisms, including formation of spore 4 g of total RNA was digested with 0.5 gofRNAse-free and germination, coagulation, cascade reactions, posttransla- DNaseina10L reaction mixture containing 1x reaction tion reactions, modulation of gene expression, enzyme modi- buffer (20 mM Tris-HCl, pH 8.4; 50 mM KCl and 2mM fication, and secretion of various protein enzymes biocatalyst. MgCl2) prior to removal of DNA. The mixture was incubated Over one-third of all known proteolytic enzymes are serine ∘ ∘ at 37 C for 15 min. DNaseI was later heat-activated at 65 C peptidases which constitute the largest group of peptidases for15min.DNAandRNAwerequantifiedusingUltraspec [13]. 2100 prospectrophotometer (Amersham Biosciences, USA). Glaciozyma antarctica (formerly known as Leucosporid- The integrity of total RNA was assessed by running an aliquot ium antarcticum) is an obligate pyschrophilic yeast that of the RNA sample on a denaturing agarose gel stained inhabit cold, marin, and terrestrial Antarctic ecosystems. with ethidium bromide (EtBr) and by spectrophotometric Glaciozyma antarctica strain PI12 has an optimum growth ∘ ∘ analysis (Ultraspec 2100 pro Amersham Biosciences, USA). temperature of 12 C[14]andcangrowupto18C. The reclas- Total RNA was reverse-transcribed with an oligo(dT)20 and sification of this yeast from L. antarcticum to G. antarctica SuperScript III Moloney Murine Leukemia Virus Reverse was proposed by Turchetti et al., 2011 [15]. Previous study Transcriptase(Invitrogen,USA)followingthemanufacturer’s on this unique yeast has isolated several cold-active proteins, instructions. namely, antifreeze protein, -amylase, and chitinase [16–18]. In this work, we described the morphological characteristics of G. antarctica through observation under scanning electron 2.4. Cloning of G. antarctica PI12 Protease Gene. Puta- microscope (SEM) and transmission electron microscope tive partial protease gene of 1321 bp was derived through (TEM), cloning of genomic DNA and cDNA sequences a genome survey sequence library of G. antarctica.The encoding the PI12 protease gene, phylogenetic study, and, gene has 27% similarity to subtilisin-like protease from expression and optimization of the recombinant PI12 pro- Rhodosporidium toruloides (EMS20811). DNA walking of tease expression in Pichia pastoris expression system. partial putative protease gene was conducted using DNA Walking SpeedUp Premix Kit (Seegene, Korea) accord- ing to the manufacturer’s instructions. Three target spe- 2. Materials and Methods cific primers (TSP) were designed from the upstream region of known sequences with the following condi- 2.1. Culture and Isolation of Microorganism. Glaciozyma tions: 18–23 nucleotides long with 40% < GC content < ∘ antarctica strain PI12 was isolated from the Antarctic marine 60% for TSP1 (5 -AGGGTCAAGACGTTGCAGT-3 ), 55 C ∘ ∘ ∘ water near Casey Station (66 21 25 S; 110 37 09 E). The ≤ Tm ≤ 60 CforTSP2(5-ATGCGAAGTCAGAAG- ∘ ∘ stock culture was kept in 20% (v/v) glycerol and stored at CAGGATC-3 )while60C ≤ Tm ≤ 65 CforTSP3(5- ∘ −80 C prior to experimentation. GCAGCCAAATACCTGGAAGCAC-3 ). RACE was per- formed using the SMART RACE cDNA Amplification Kit (Clontech, USA). Two gene-specific primers (GSPs) were 2.2. Microorganism Identification. The isolated strain was synthesized for the 5 -and3-RACE reactions based on grown on different types of solid media (nutrient agar, the sequence of the RT-PCR products as follows: GSP sabouroud dextrose agar, and potato dextrose agar) for 10 I: 5 -ACCAGTGTCCAGCACCCCAATCTTAATCC-3 and ∘ days at 4 C and the culture characteristics (colony colour, GSP II: 5 -TCATCAGTGGGACGAGCATGTCGT-3 .Both shape, and texture) were determined. Simple and negative primers were paired with universal primers provided in staining were performed to identify the cell morphology, the kit to amplify the upstream and downstream region arrangement, and size of the psychrophilic yeast. SEM and of the gene of interest. The PCR products were subcloned TEM were conducted to study the surface features and the into pGEM-T vector (Promega, USA) and sequenced.