玉米胚乳中产生的重组纤维生物水解酶 II (Cel6A) 的特异性活性和实用性。

IF 4.6 Q2 MATERIALS SCIENCE, BIOMATERIALS ACS Applied Bio Materials Pub Date : 2024-04-01 Epub Date: 2024-03-07 DOI:10.1007/s11248-024-00376-y
Enio Duque Y Duque, Milena Aguirre, Nathan C Hood, Elizabeth E Hood
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引用次数: 0

摘要

Cellobiohydrolase II(CBH II)是一种外切葡聚糖酶,是木腐真菌毛霉(Trichoderma reesei)的真菌混合酶的一部分。因此,很难对其进行纯化,也很难建立特异性的活性测定方法。这种酶的基因由水稻 Os 谷蛋白启动子驱动,被转化到具有组织培养能力的 High II 玉米中,酶在种子的胚乳中积累。将从组织培养中获得的转基因品系与 Stine 近交系玉米的雄性和雌性精英品系僵茎 16083-025 (雌性)和 Lancaster MSO411 (雄性)进行杂交,以便将来在其杂交种中生产。该酶在 6 代回交中的积累量增加了 27-266 倍,T1 和 T2 代之间增加了 27-266 倍,T2 和 T3 代之间增加了 2-10 倍,T4-T6 代增加较少。近交系的种质每一代都会取代组织培养的玉米品种种质,最终目的是培育出带有转基因的高产杂交种。CBH II 酶从 T5 近交系雄性谷粒中纯化了 10 倍,达到均一,回收率为 47.5%。比活性被测定为每微克蛋白质 1.544 个单位。玉米衍生的 CBH II 可用于棉花的生物抛光,去除表面纤维以提高可染性,并增加玉米粉中的葡萄糖以提高基于淀粉的第一代工艺的乙醇产量。
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Specific activity and utility of recombinant cellobiohydrolase II (Cel6A) produced in maize endosperm.

Cellobiohydrolase II (CBH II) is an exo-glucanase that is part of a fungal mixture of enzymes from a wood-rot fungus, Trichoderma reesei. It is therefore difficult to purify and to establish a specific activity assay. The gene for this enzyme, driven by the rice Os glutelin promoter, was transformed into High II tissue culture competent corn, and the enzyme accumulated in the endosperm of the seed. The transgenic line recovered from tissue culture was bred into male and female elite Stine inbred corn lines, stiff stalk 16083-025 (female) and Lancaster MSO411 (male), for future production in their hybrid. The enzyme increases its accumulation throughout its 6 generations of back crosses, 27-266-fold between T1 and T2, and 2-10-fold between T2 and T3 generations with lesser increases in T4-T6. The germplasm of the inbred lines replaces the tissue culture corn variety germplasm with each generation, with the ultimate goal of producing a high-yielding hybrid with the transgene. The CBH II enzyme was purified from T5 inbred male grain 10-fold to homogeneity with 47.5% recovery. The specific activity was determined to be 1.544 units per µg protein. The corn-derived CBH II works in biopolishing of cotton by removing surface fibers to improve dyeability and increasing glucose from corn flour for increasing ethanol yield from starch-based first-generation processes.

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来源期刊
ACS Applied Bio Materials
ACS Applied Bio Materials Chemistry-Chemistry (all)
CiteScore
9.40
自引率
2.10%
发文量
464
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