Celestino Klecius R Silveira, Cunha Ricardo B, Felix Carlos R
Laboratório de Enzimologia, Departamento de Biologia Celular, Universidade de Brasília, Brasília, DF, CEP 70910-900, Brazil.
BMC Biochem. 2006 Dec 5;7:23. doi: 10.1186/1471-2091-7-23.
In the barley malting process, partial hydrolysis of beta-glucans begins with seed germination. However, the endogenous 1,3-1,4-beta-glucanases are heat inactivated, and the remaining high molecular weight beta-glucans may cause severe problems such as increased brewer mash viscosity and turbidity. Increased viscosity impairs pumping and filtration, resulting in lower efficiency, reduced yields of extracts, and lower filtration rates, as well as the appearance of gelatinous precipitates in the finished beer. Therefore, the use of exogenous beta-glucanases to reduce the beta-glucans already present in the malt barley is highly desirable.
The zygomycete microfungus Rhizopus microsporus var. microsporus secreted substantial amounts of beta-glucanase in liquid culture medium containing 0.5% chitin. An active protein was isolated by gel filtration and ion exchange chromatographies of the beta-glucanase activity-containing culture supernatant. This isolated protein hydrolyzed 1,3-1,4-beta-glucan (barley beta-glucan), but showed only residual activity against 1,3-beta-glucan (laminarin), or no activity at all against 1,4-beta-glucan (cellulose), indicating that the R. microsporus var. microsporus enzyme is a member of the EC 3.2.1.73 category. The purified protein had a molecular mass of 33.7 kDa, as determined by mass spectrometry. The optimal pH and temperature for hydrolysis of 1,3-1,4-beta-glucan were in the ranges of 4-5, and 50-60 degrees C, respectively. The Km and Vmax values for hydrolysis of beta-glucan at pH 5.0 and 50 degrees C were 22.39 mg.mL-1 and 16.46 mg.min-1, respectively. The purified enzyme was highly sensitive to Cu+2, but showed less or no sensitivity to other divalent ions, and was able to reduce both the viscosity and the filtration time of a sample of brewer mash. In comparison to the values determined for the mash treated with two commercial glucanases, the relative viscosity value for the mash treated with the 1,3-1,4-beta-glucanase produced by R. microsporus var. microsporus. was determined to be consistently lower.
The zygomycete microfungus R. microsporus var. microsporus produced a 1,3-1,4-beta-D-glucan 4-glucanhydrolase (EC 3.2.1.73) which is able to hydrolyze beta-D-glucan that contains both the 1,3- and 1,4-bonds (barley beta-glucans). Its molecular mass was 33.7 kDa. Maximum activity was detected at pH values in the range of 4-5, and temperatures in the range of 50-60 degrees C. The enzyme was able to reduce both the viscosity of the brewer mash and the filtration time, indicating its potential value for the brewing industry.
在大麦麦芽制造过程中，β-葡聚糖的部分水解始于种子萌发。然而，内源性1,3-1,4-β-葡聚糖酶会被热灭活，剩余的高分子量β-葡聚糖可能会导致严重问题，如啤酒醪液粘度和浊度增加。粘度增加会损害泵送和过滤，导致效率降低、提取物产量减少、过滤速率降低，以及成品啤酒中出现凝胶状沉淀。因此，非常希望使用外源β-葡聚糖酶来降低麦芽大麦中已存在的β-葡聚糖含量。
接合菌小真菌微小毛霉变种在含有0.5%几丁质的液体培养基中分泌大量β-葡聚糖酶。通过对含有β-葡聚糖酶活性的培养上清液进行凝胶过滤和离子交换色谱法分离出一种活性蛋白。这种分离出的蛋白能水解1,3-1,4-β-葡聚糖（大麦β-葡聚糖），但对1,3-β-葡聚糖（海带多糖）仅表现出残余活性，对1,4-β-葡聚糖（纤维素）则完全没有活性，这表明微小毛霉变种的酶属于EC 3.2.1.73类别。通过质谱测定，纯化后的蛋白分子量为33.7 kDa。水解1,3-1,4-β-葡聚糖的最佳pH值和温度分别在4 - 5和50 - 60℃范围内。在pH 5.0和50℃条件下水解β-葡聚糖的Km和Vmax值分别为22.39 mg·mL-1和16.46 mg·min-1。纯化后的酶对Cu2+高度敏感，但对其他二价离子的敏感性较低或不敏感，并且能够降低啤酒醪液样品的粘度和过滤时间。与用两种商业葡聚糖酶处理的醪液所测定的值相比，用微小毛霉变种产生的1,3-1,4-β-葡聚糖酶处理的醪液的相对粘度值始终较低。
接合菌小真菌微小毛霉变种产生了一种1,3-1,4-β-D-葡聚糖4-葡聚糖水解酶（EC 3.2.1.73），它能够水解同时含有1,3-和1,4-键的β-D-葡聚糖（大麦β-葡聚糖）。其分子量为33.7 kDa。在pH值4 - 5和温度50 - 60℃范围内检测到最大活性。该酶能够降低啤酒醪液的粘度和过滤时间，表明其在酿造工业中的潜在价值。
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