Enhancing _Paenibacillus_ sp. Cold-Active Acetyl Xylan Esterase Activity through Semi-Rational Protein Engineering  
[PERSON]  
1 Institute of Basic Sciences, Pukyong National University, Busan 48513, Republic of Korea;  
[EMAIL_ADDRESS] (K.J. ), [EMAIL_ADDRESS] (S.N. )1  
[PERSON]  
1 Institute of Basic Sciences, Pukyong National University, Busan 48513, Republic of Korea;  
[EMAIL_ADDRESS] (K.J. ), [EMAIL_ADDRESS] (S.N. )1  
[PERSON]  
2 Department of Microbiology, Pukyong National University, Busan 48513, Republic of Korea;  
[EMAIL_ADDRESS] (S.Y.J. ); [EMAIL_ADDRESS] (G.R.Y. )2  
[PERSON]  
2 Department of Microbiology, Pukyong National University, Busan 48513, Republic of Korea;  
[EMAIL_ADDRESS] (S.Y.J. ); [EMAIL_ADDRESS] (G.R.Y. )3  
[PERSON]  
3 Department of Chemistry, Pukyong National University, Busan 48513, Republic of Korea;  
[EMAIL_ADDRESS] (J.K.), [EMAIL_ADDRESS] (H.J.K. );  
[EMAIL_ADDRESS] (H.J.K. );  
be seamlessly integrated into existing pharmaceutical manufacturing processes, lowering costs and improving efficiency [1]. Ongoing research and development efforts in enzymatic synthesis hold substantial potential for the synthesis of novel and intricate pharmaceutical compounds, contributing to filling the biocatalytic toolbox [1]. In this regard, psychrophilic organisms present strong candidates for novel enzyme identification [10; 11]. Carbohydrate esterases (CE) catalyze the deacetylation of acetylated saccharide residues in hemicellulose and pectin [12; 13]. As such, CE facilitates the breakdown of plant biomass into simpler sugars for biofuels or value-added product production. Hemicellulose, a heterogeneous polysaccharide, constitutes a substantial portion of plant cell walls, typically including short chains of xylan, mannan, galactan, and arabian. Among these polymeric xylans, the major component (20-35%) in hemicellulose, are acetylated at the C2 or C3 positions of xylose residues and require deacetylation for xylose production. Among CE enzymes, CE1-7, 12, and 16 are acetyl xylan esterases (AXEs). These AXEs have broad industrial applications, such as biobleaching, semi-synthetic \(\beta\)-lactam antibiotic production, and animal feed production [12]. Recently, the cold-active _Paenibacillus_ sp. AXE (PbAcE) was isolated and characterized, along with its X-ray crystal structure [14]. PbAcE, displaying higher activity at 4 \({}^{\circ}\)C than that at 25 \({}^{\circ}\)C, belongs to the CE7 family, which catalyzes the hydrolysis of ester linkages between xylose and acetic acid, and is able to deacetylate cephalosporiorn antibiotics [15]. Similarly to other AXEs [16; 17; 12; 18], PbAcE exhibits considerable potential for industrial applications. Moreover, its cold-active nature offers distinct advantages, enabling bioconversion processes at low temperatures. This characteristic reduces energy costs and minimizes contamination risk [19]. PbAcE shares common structural and catalytic characteristics with CE7 family members. The PbAcE X-ray crystal structure revealed a donut-shaped hexameric structure comprising a trimer of dimers. Because of this shape, the substrates can access active sites via the tunnel formed in the central ring of the hexameric structure, and the active site of each subunit faces toward the center of the ring [14]. Each PbAcE subunit has typical \(\alpha/\beta\) hydrolase folds with a catalytic triad of Ser185-His303-Asp274. PbAcE, similarly to other members of the CE7 family, exhibits broad substrate specificity. The substrates are mostly acetate esters, including glucose penta-acetate and xylan acetate, tertiary alcohol esters, and antibiotics (such as cephalosporiorn C [CPC], cefotaxime and, 7-amino cephalosporioranic acid [7-ACA]) [14]. PbAcE demonstrates diverse activities across different substrates, likely attributable to its substrate-binding pockets (Figures 1a and S1).