Polyester synthesis activity of immobilized Humicola insolens (HiC) was systematically Studied with three-series of substrates varying in (i) omega-hydroxyalkanoic acid (omega HA), (ii) alpha,omega-n-alkane diol, and (iii) alpha,omega-n-alkane diacid chain length. Covalent immobilization of HiC on Amberzyme oxirane (AO) resin (i.e., AO-HiC) was prepared. HiC-AO's activity for omega HA substrates with 6, 10, 12, and 16 carbons was C16 > C12, where C10-omega HA and C6-omega HA were not polymerized. In contrast, N435's activity for omega HA substrates was C 16 = C 12 > C10, where C6-omega HA was not polymerized. HiC-AO activity for copolymerization of sebacic acid (C10-diacid) with alpha,omega,n-alkane diols with 3-, 4-, 5-, 6-, and 8-carbon chain lengths was C8 > C6, where C3, C4, and C5 diols were not polymerized. N435's relative activity for diol substrates was C8 = C6 = C5 > C4 > C3. HiC-AO activity for copolymerizations of 1,8-octanediol with alpha,omega-n-alkane diacids with 6-, 8-, 9-, 10-, and 13-carbon chain lengths was C13 = C10, where HiC showed little activity for C6, C8, and C9 diacid copolymerization. N435 displayed similar activity for all these diacid chain lengths. Thus, N435 has a broader Substrate promiscuity than HiC-AO, This is most apparent for shorter chain length omega HA, diol, and diacid monomers. These trends were similarly observed for a series of small molecule esterification reactions. Comparison of HiC-AO- and N435-catalyzed C16-HA homopolymerization at 8 h gave polymers with M-n 40.4 and 25.5 kg/mol, respectively. Furthermore, HiC-AO- and N435-catalyzed copolymerization of 1,8-octanediol/C13-diacid polymerizations at 8 h gave polymers with M-n of 11.0 and 9.6 kg/mol, respectively.