Characterizaton of recombinant Bacillus halodurans CM1 xylanase produced by Pichia pastoris KM71 and its potental applicaton in bleaching process of bagasse pulp Haniyya 1 , Lina Mulyawat 1 , Is Heliant 1,* , Phitsanu Pinmanee 2 , Kanokarn Kocharin 3 , Duriya Cantasingh 2 , Thidarat Nimchua 2 1 Center for Bioindustrial Technology, Agency of Assessment and Applicaton of Technology (BPPT), Building 611/614, LAPTIAB‐BPPT, PUSPIPTEK Area, Setu, South Tangerang 15314, Indonesia 2 Enzyme Technology Research Team, Biorefnery and Bioproduct Technology Research Group, Natonal Center for Genetc Engineering and Biotechnology,113 Phahonyothin Road, Khlong Nueng, Khlong Luang, Pathum Thani 12120, Thailand 3 Microbial Cell Factory Research Team, Biorefnery and Bioproduct Technology Research Group, Natonal Center for Genetc Engineering and Biotechnology, 113 Phahonyothin Road, Khlong Nueng, Khlong Luang, Pathum Thani 12120, Thailand *Corresponding author: is.heliant@bppt.go.id SUBMITTED 9 July 2020 REVISED 19 October 2020 ACCEPTED 10 December 2020 ABSTRACT Thermoalkalophilic xylanases promise potental applicaton in pulp biobleaching to reduce the use of toxic chlorinated chemical agents, which are harmful to the environment. In this study, a thermoalkalophilic endoxylanase gene (bhxyn3) originatng from Indonesian indigenous Bacillus halodurans CM1 was cloned into yeast expression vector pPICZα A and expressed in Pichia pastoris KM71 under the control of AOX1 promoter. Recombinant P. pastoris expressed the highest fnal level of xylanase (146 U/mL) on BMGY medium afer fve days of cultvaton. Optmizaton of xylanase producton on a small scale was carried out by varying the methanol concentratons and the optmal xylanase producton by the recombinant P. pastoris was observed in the culture with 2% (v/v) methanol afer four days of the inducton phase. The recombinant xylanase (BHxyn3E) was thermotolerant and alkalophilic, with an optmal temperature at around 55‐65 °C and under pH 8.0. The enzyme actvity was slightly induced by K + , Fe 2+ , and MoO 4 2‐ . Enzymatc bleaching of bagasse pulp with no prior pH adjustment (pH 9) using BHxyn3E at 200 U/g oven dried pulp increased the lightness index (L*) and changed substantally the color a index (a*); however, the treatments did not change the whiteness index in a signifcant way. Therefore, further optmizaton and assessment such as adjustment of incubaton temperature and pH in biobleaching were needed to reduce the use of harmful chemical agents in industrial applicatons. KEYWORDS Bacillus halodurans; biobleaching; cellulase‐free xylanase; Pichia pastoris; thermoalkalophilic xylanase Indonesian Journal of Biotechnology VOLUME 26(1), 2021, 15‐24 | RESEARCH ARTICLE 1. Introducton Remarkable interest has been focused on integrating hy drolytic enzymes, such as xylanases, that degrade xylan components in plant cell walls into noncomplex sugars in industries, particularly in the pulp and paper industry. Xylanases (EC 3.2.1.8) are produced by microorganisms and considered as the key enzymes that hydrolyze β1,4 xylans to lead to the degradation of complex polysaccha rides (Jeya et al. 2009). On the other hand, the enzymes have widespread potential applications to be used in such a wide range of industrial fields (textile, feed, beverage, and biofuels) since its ability to facilitate the removal of xy lan through partial hydrolysis of lignin carbohydrate com plexes, thereby enabling lignin removal from raw materi als allowing it to be processed in the next stages of man ufacturing. In the pulp and paper process, this fragmenta tion of xylan polymer allows residual lignin attached to the remaining xylan complex, to be exposed by the efficient used bleaching agents. Thus, pretreatment of pulp using xylanases can reduce the load of chlorine and chlorine based compounds in the subsequent chemical bleaching stages. As a side effect, the utilization of cellulasefree xy lanase enable to enhance several physical pulp properties, such as brightness, tensile strength, and tear factor with out affecting the cellulose fiber strength of paper products (Zhao et al. 2015). Currently, xylanases that are intended to be used in the industrial bleaching process must be robust to operate in extreme conditions since most operations were conducted under high temperature and strong alkaline pH for at least anhour long. Hence it must be adaptable in such an ex treme environment. Xylanases are produced by members of bacteria and fungi, commonly in group of bacterial xy lanases display an active at mesophilic temperatures (be low 50 °C) in acidic or neutral pH (Walia et al. 2017). Indones J Biotechnol 26(1), 2021, 15‐24 | DOI 10.22146/ijbiotech.57701 www.jurnal.ugm.ac.id/ijbiotech Copyright © 2021 THE AUTHOR(S). This artcle is distributed under a Creatve Commons Atributon‐ShareAlike 4.0 Internatonal license.