As a result of cloning of the inuA, inu1, aglC, and fopA genes encoding endoinulinase (endoINU), exoinulinase (exoINU), α-galactosidase C (AGLС) and sucrase (SUC), respectively, into the recipient strain Penicillium verruculosum B1-537 (ΔniaD), recombinant producer strains were obtained that are capable of producing target recombinant enzymes with a high yield (32‒50% of the total extracellular protein). Enzyme preparations of endoINU, exoINU, AGLC, and SUC were obtained and characterized. Using chromatographic methods, endoINU, exoINU, SUC, and AGLC with a molecular weights of 62, 56, 67, and 76 kDa, respectively, were isolated in a homogeneous form (according to polyacrylamide gel electrophoresis). The homogeneous endoINU had a high specific activity against Jerusalem artichoke inulin (56 U/mg). ExoINU was active towards inulin (17 U/mg), sucrose (850 U/mg), raffinose (41 U/mg), and stachyose (15 U/mg). SUC decomposed sucrose (10.5 U/mg), raffinose, and stachyose (3.8 and 1.4 U/mg, respectively). AGLC had raffinase and stachyase activities (31 U/mg and 30 U/mg, respectively), exhibited no activity towards sucrose, but had a high level of activity towards the synthetic substrate, p-nitrophenyl-α-D-galactoside (311 U/mg). The kinetic parameters (k_cat and K_m) of the hydrolysis of the corresponding substrates by homogeneous enzymes were determined. The temperature optimum was 50‒55°C for endoINU, 55‒65°C for exoINU, 65°C for AGLC, and 35°C for SUC. EndoINU, exoINU, AGLC and SUC exhibited its maximum activity at pH 6.5, 4.5, 4.5‒5.0, and 5.5‒6.0, respectively. The thermal stability of the enzymes was studied at different temperatures. EndoINU exhaustively hydrolyzed inulin with the formation of fructooligosaccharides with a degree of polymerization of 3‒8. ExoINU quantitatively converted inulin into glucose-fructose syrup (GFS) with a Glu : Fru ratio of 1 : 3, and sucrose into GFS with a Glu : Fru ratio of about 1 : 0.63 (SUC provided the same results in the sucrose hydrolysis). Soy galactooligosaccharides (raffinose and stachyose) were converted to sucrose and monosaccharides (glucose, galactose, and fructose) under the action of AGLC. The combined action of SUC, and AGLC resulted in a complete conversion of raffinose, stachyose and sucrose to monosaccharides. The same results were achieved using ExoINU. This enzyme can be considered promising for biotechnological applications due to its broad substrate specificity, which allows it be used both for the production of GFS from inulin and sucrose, and for the destruction of soybean galactooligosaccharides.

将分别编码内切菊粉酶 (endoINU)、外切菊粉酶 (exoINU)、α-半乳糖苷酶 C (AGLС) 和蔗糖酶 (SUC) 的 inuA 、 inu1 、 aglC 和fopA基因克隆到受体菌株Penicillium v ​​erruculosum B1- 537 (Δ niaD )，获得了能够高产量生产目标重组酶（占总胞外蛋白的 32-50%）的重组生产菌株。获得并表征了 endINU、exoINU、AGLC 和 SUC 的酶制剂。使用色谱方法，以均质形式分离出分子量分别为62、56、67和76 kDa的endoINU、exoINU、SUC和AGLC（根据聚丙烯酰胺凝胶电泳）。均质的 endINU 对菊芋菊糖具有高比活性 (56 U/mg)。ExoINU 对菊粉 (17 U/mg)、蔗糖 (850 U/mg)、棉子糖 (41 U/mg) 和水苏糖 (15 U/mg) 具有活性。SUC 分解蔗糖（10.5 U/mg）、棉子糖和水苏糖（分别为 3.8 和 1.4 U/mg）。AGLC 具有棉子酶和水苏酶活性（分别为 31 U/mg 和 30 U/mg），对蔗糖没有活性，但对合成底物对硝基苯基-α- D-半乳糖苷（311 U /毫克）。测定均相酶水解相应底物的动力学参数（k _cat和K _{m ）。}endINU 的最佳温度为 50-55°C，exoINU 的最佳温度为 55-65°C，AGLC 的最佳温度为 65°C，SUC 的最佳温度为 35°C。EndoINU、exoINU、AGLC 和 SUC 分别在 pH 6.5、4.5、4.5-5.0 和 5.5-6.0 时表现出最大活性。研究了酶在不同温度下的热稳定性。EndoINU 彻底水解菊粉，形成聚合度为 3-8 的低聚果糖。ExoINU 将菊粉定量转化为 Glu: Fru 比例为 1: 3 的葡萄糖果糖浆 (GFS)，将蔗糖定量转化为 GFS，Glu: Fru 比例为约 1: 0.63（SUC 在蔗糖水解中提供相同的结果）。大豆低聚半乳糖（棉子糖和水苏糖）在AGLC的作用下转化为蔗糖和单糖（葡萄糖、半乳糖和果糖）。SUC 和 AGLC 的联合作用导致棉子糖、水苏糖和蔗糖完全转化为单糖。使用 ExoINU 也取得了相同的结果。由于其广泛的底物特异性，这种酶被认为在生物技术应用方面很有前景，这使得它既可以用于从菊粉和蔗糖生产 GFS，也可以用于破坏大豆低聚半乳糖。