提取液及固-液分离方法对固态非淀粉多糖酶类活性的影响

本试验旨在探讨固态酶制剂评估中酶的适宜提取液及固-液分离方法。采用4×3双因素完全随机设计,其中提取液分别为去离子水、乙酸-乙酸钠缓冲液(0.1 mol/L,p H 5.50)、磷酸盐缓冲液(0.05 mol/L,p H 6.00)和0.9%Na Cl溶液;溶液提取后的固-液分离方法分别为不分离、3 000 r/min离心3 min和中速滤纸过滤。每个处理5个重复,每个重复设2个平行,测定各个处理下酶的活性,并考察提取液的类型对酶制剂产品(α-半乳糖苷酶除外)溶解离心后溶液中溶质及蛋白质含量的影响。结果表明,磷酸盐缓冲液溶解木聚糖酶后活性最高(P0.05);乙酸-乙酸钠缓冲液、磷酸盐缓冲液及0.9%Na Cl溶液溶解β-葡聚糖酶后活性相当(P0.05),且均显著地高于去离子水(P0.05);去离子水溶解β-甘露聚糖酶后活性最高,其次为乙酸-乙酸钠缓冲液,两者都显著地高于磷酸盐缓冲液和0.9%Na Cl溶液(P0.05)。提取液类型对α-半乳糖苷酶活性无显著影响(P0.05)。酶制剂溶解后的固-液分离方法对木聚糖酶的测定活性无显著性影响(P0.05);提取液离心或过滤后β-葡聚糖酶活性最高(P0.05);提取液离心后β-甘露聚糖酶活性最高(P0.05);而提取液不分离时α-半乳糖苷酶的活性最高(P0.05)。提取液的种类和酶制剂溶解后的固-液分离方法对4种非淀粉多糖酶的测定活性有极显著的交互作用(P0.01)。乙酸-乙酸钠缓冲液对木聚糖酶制剂的溶解度最大(P0.05),去离子水和0.9%Na Cl溶液均对β-葡聚糖酶及β-甘露聚糖酶制剂的溶解度最大(P0.05)。然而,乙酸-乙酸钠缓冲液溶解木聚糖酶、β-葡聚糖酶、β-甘露聚糖酶后提取液中蛋白质的含量均最低(P0.05)。上述结果表明,乙酸-乙酸钠缓冲液溶解4种固态酶制剂可以最有效地将酶蛋白提取出来,α-半乳糖苷酶提取后不宜固液分离,而其他3种酶的提取液适宜进行离心分离。

Effects of Extraction Solution and Solid-Liquid Separation Method on the Activities of Non-Starch Polysaccharide Enzymes

This experiment was conducted to investigate appropriate extract solution and solid-liquid separation method for evaluating enzyme in solid feed enzyme product. Extract solution of deionized water, acetic acid-so-dium acetate buffer solution (0.1 mol/L, pH 5.50), phosphate buffer solution (0.05 mol/L, pH 6.00) or 0.9% NaCl solution and solid-liquid separation method of no separation, 3 000 r/min of centrifugation for 3 min or filtration were used in a 4×3 factorial arrangement. Each treatment contained 5 replicates with 2 determi-nation in each. The activities of enzymes in the products were determined. Then, the effects of the types of ex-tract solution on the contents of solute and protein were investigated for the enzyme products ( excludingα-gal-actosidase) dissolved and centrifuged. The results showed that the highest determined activity of xylanase was presented in the phosphate buffer solution buffer solution ( P<0.05) . The similar determined activities ofβ-glu-canase were observed in acetic acid-sodium acetate buffer solution, phosphate solution and 0.9% NaCl solution ( P>0.05) , and were significantly higher than that in deionized water ( P<0.05) . The greatest and greater de-termined activities ofβ-mannanase were observed in deionized water and acetic acid-sodium acetate buffer solu-tion, respectively, and were significantly greater than those in phosphate solution or 0.9% NaCl solution ( P<0.05) . The type of extract solution had no significant effect on the determined activity of α-galactosidase ( P>0.05) . The solid-liquid separation method had no significant effect on the determined activity of xylanase ( P>0.05). The greater determined activity of β-glucanase was observed in centrifugation or filtration (P<0.05). The greatest determined activity of β-mannanase was presented in centrifugation (P<0.05). However, the highest determined activity of α-galactosidase product was observed in no separation (P<0.05). There was a significant interaction between the type of extract solution and solid-liquid separation method in the determined activities of 4 enzyme products ( P<0.01) . The highest dry matter solubility of xylanase product was observed in acetic acid-sodium acetate buffer solution ( P<0.05) . The highest dry matter solubility ofβ-glucanase andβ-mannanase was presented in deionized water and 0.9% NaCl solution ( P<0.05) . However, the lowest protein contents of xylanase,β-glucanase andβ-mannanase were observed in acetic acid-sodium acetate buffer solution ( P<0.05) . It is concluded that acetic acid-sodium acetate buffer solution is the most efficient to extract the en-zyme protein from the product. After dissolved, the α-galactosidase product is not suitable to separate, but the other enzymes can be separated with centrifugation.