α‐(1,4)‐Amylase,but not α‐ and β‐(1,3)‐glucanases,may be responsible for the impaired growth and morphogenesis of Paracoccidioides brasiliensis induced by N‐glycosylation inhibition

The cell wall of Paracoccidioides brasiliensis, which consists of a network of polysaccharides and glycoproteins, is essential for fungal pathogenesis. We have previously reported that N‐glycosylation of proteins such as N‐acetyl‐β‐d ‐glucosaminidase is required for the growth and morphogenesis of P. brasiliensis. In the present study, we investigated the influence of tunycamicin (TM)‐mediated inhibition of N‐linked glycosylation on α‐ and β‐(1,3)‐glucanases and on α‐(1,4)‐amylase in P. brasiliensis yeast and mycelium cells. The addition of 15 µg/ml TM to the fungal cultures did not interfere with either α‐ or β‐(1,3)‐glucanase production and secretion. Moreover, incubation with TM did not alter α‐ and β‐(1,3)‐glucanase activity in yeast and mycelium cell extracts. In contrast, α‐(1,4)‐amylase activity was significantly reduced in underglycosylated yeast and mycelium extracts after exposure to TM. In spite of its importance for fungal growth and morphogenesis, N‐glycosylation was not required for glucanase activities. This is surprising because these activities are directed to wall components that are crucial for fungal morphogenesis. On the other hand, N‐glycans were essential for α‐(1,4)‐amylase activity involved in the production of malto‐oligosaccharides that act as primer molecules for the biosynthesis of α‐(1,3)‐glucan. Our results suggest that reduced fungal α‐(1,4)‐amylase activity affects cell wall composition and may account for the impaired growth of underglycosylated yeast and mycelium cells. © 2013 The Authors. Yeast published by John Wiley & Sons Ltd.     

Malting Performance of Normal Huskless and Acid‐Dehusked Barley Samples

Sulphuric acid dehusked barley had a higher germinative energy and lower microbial infection than normal huskless (naked) barley, suggesting that the pericarp layer harboured microbial infection which may have limited the germination rate. Dehusking the normal huskless barley using sulphuric acid resulted in lower microbial infection, and increased germinative energy. The normal huskless barley sample had a higher β‐glucan content than the acid‐dehusked barley and had slower β‐glucan breakdown during malting. This resulted in the release of seven times more β‐glucan during mashing, and the production of wort of higher viscosity. The normal huskless barley sample had a higher total nitrogen content than the acid‐dehusked barley but both samples produced similar levels of amylolytic (α‐ and β‐amylase) activity over the same malting period. No direct correlation was found between barley total nitrogen level and the amylolytic activity of the malt. When barley loses its husk at harvest, the embryo is exposed and may be damaged. This may result in uneven germination which can reduce malting performance and hence malt quality.     

浸泡条件对燕麦发芽过程中酶活性的影响

为探究浸泡条件对燕麦发芽过程中淀粉酶、蛋白酶以及纤维素酶活力的影响,通过在不同的浸泡温度、时间和含有不同金属离子的浸泡水溶液中浸泡燕麦籽粒,测定燕麦中各酶活力随发芽时间的变化情况。研究结果表明,燕麦浸泡最优条件为浸泡温度15℃,时间8 h,浸泡水溶液添加2 mmol/L MgSO₄。在此浸泡条件下,16℃发芽10 d,发芽燕麦中的总淀粉酶、α-淀粉酶、蛋白酶和纤维素酶活力较高,分别达到54.39、25.3、0.55和1.35 U/g。      

大孔树脂纯化叶多酚及其对胰淀粉酶的抑制作用

研究栘叶多酚的大孔吸附树脂纯化工艺,并考察其纯化前后对胰淀粉酶的抑制作用。通过静态吸附解析实验筛选出吸附性能和解吸性能良好的D-101型大孔吸附树脂,进一步研究其纯化多酚的工艺条件和技术参数。结果显示:上样溶液质量浓度为25 mg/m L,上样流速为1 m L/min,以10 BV进行上样,以50%的乙醇溶液为解析剂,洗脱流速为3 m L/min,洗脱体积为8 BV进行洗脱,栘叶浸膏中多酚的含量由原来的39.7%提高到91.89%。纯化后,栘叶多酚对胰淀粉酶的抑制率高于纯化前,且纯化前后对胰淀粉酶的抑制率均高于100 mg/m L大黄提取物对胰淀粉酶的抑制效果,说明栘叶多酚对胰淀粉酶有明显的抑制效果,且纯化有利于提高栘叶多酚对胰淀粉酶的抑制作用。      

紫薯麦芽复合饮料加工工艺的研究

以紫薯和麦芽为原料制备复合饮料,利用麦芽里含有的淀粉酶水解原料中的淀粉,采用单因素实验和正交实验研究酶解温度、酶解时间、紫薯和麦芽质量比对饮料中还原糖含量和感官评分的影响,探讨还原糖含量和感官评分的相关性。结果表明,在不同加工条件下,还原糖含量的变化趋势并不总是与感官评分一致。优化的工艺条件是:酶解温度为75℃,酶解时间为1h,薯麦质量比为5:7,制得的复合饮料还原糖含量为10.45%,感官评分为21分。      

一种中温淀粉酶在解淀粉芽孢杆菌LT的重组表达

解淀粉芽孢杆菌可用作多种食品工业酶的生产菌株,但其极低的转化效率影响了这类工业菌株的重组改造及优化。通过对1398解淀粉芽孢杆菌和一种地衣芽孢杆菌Jbac基因组的数据分析,发现这些微生物存在IV型DNA限制修饰系统。对这些微生物转化经甲基化和未经甲基化的质粒,证实外源DNA甲基化修饰会降低DNA的转化效率;利用将外源质粒进行相同类型甲基化的这种新转化策略,表达质粒pMK4E-dx被转入解淀粉芽孢杆菌LT中,发酵结果显示,其中重组菌株LT-J1/pMK4-dx和LT-J2/pMK4-dx分泌的中温淀粉酶活达到145.9和153.6 U/mL,分别为原菌株发酵淀粉酶活的3.65和3.84倍。     

酶法生产可可粉饮料的研究

可可粉在饮料中不稳定,可以通过酶的作用降低可可粉的粒径,改善可可粉的溶解性。分别用淀粉酶、纤维素酶、蛋白酶、木聚糖酶对可可粉进行处理,发现淀粉酶、纤维素酶对可可粉有较强的作用,蛋白酶对可可粉几乎没有作用,木聚糖酶和纤维素酶、淀粉酶协同作用才有效果。通过正交实验得到酶解的最佳工艺是:在可可粉的天然pH下,加入0.5%(w/w)的淀粉酶、2%(w/w)的纤维素酶,0.2%(w/w)的木聚糖酶,在50℃下作用4h。      

耐盐米曲霉制曲及复合酶分泌条件的研究

该文研究了耐盐米曲霉制曲的产酶特性,并且通过对耐盐米曲霉在不同作用条件下制得的曲料的蛋白酶活力和淀粉酶活力的变化分析,探讨了不同原料配比、水分添加量、制曲温度和蒸料时间等因素对耐盐米曲霉制曲效果的影响,为米曲霉在工业生产中的研究和应用提供理论依据.研究表明,耐盐米曲霉制曲产生的中性蛋白酶和碱性蛋白酶的酶活较高,酸性蛋白酶和淀粉酶酶活较低.复合酶分泌的最佳工艺条件为:原料配比为豆粕:麸皮=2:3,加水量为120％,蒸料时间为30min,30℃制曲42h,此条件下酸性蛋白酶和淀粉酶活力有明显的提高.     

Immobilization of malto‐oligosaccharide forming α‐amylase from Bacillus subtilis KCC103: properties and application in starch hydrolysis

BACKGROUND: A malto‐oligosaccharide forming α‐amylase from Bacillus subtilis KCC103 immobilized in calcium alginate beads was repeatedly used in batch processes of starch hydrolysis. The degree of starch degradation and operational stability of the immobilized system were optimized by varying the physical characteristics and composition of the beads. The products formed from hydrolysis of various starches by α‐amylase immobilized in different supports were analyzed. RESULTS: Immobilized beads prepared from 3% (w/v) alginate and 4% (w/v) CaCl₂ were suitable for up to 10 repeated uses, losing only 25% of their efficiency. On addition of 1% silica gel to alginate prior to gelation, the operational stability of the immobilized enzyme was enhanced to 20 cycles of operation, retaining > 90% of the initial efficiency. Distribution of malto‐oligosaccharides in the starch hydrolyzate depended on the type of starch, reaction time and mode of immobilization. Soluble starch and potato starch formed a wide range of malto‐oligosaccharides (G1–G5). Starches from wheat, rice and corn formed a narrow range of smaller oligosaccharides (G1–G3) as the major products. CONCLUSION: The immobilized beads of α‐amylase from KCC103 prepared from alginate plus silica gel showed high efficiency and operational stability for hydrolysis of starch. This immobilized system is useful for production of malto‐oligosaccharides applied in the food and pharmaceutical industries. Since this KCC103 amylase can be produced at low cost utilizing agro‐residues in a short time and immobilized enzyme can be recycled, the overall cost of malto‐oligosaccharide production would be economical for industrial application. Copyright © 2008 Society of Chemical Industry     

Optimization of medium and process parameters for a constitutive α‐amylase production from a catabolite derepressed Bacillus subtilis KCC103

BACKGROUND: In Bacillus subtilis KCC103 α‐amylase is hyper‐produced and not catabolite repressed by glucose. Various sugars, raw starches and nitrogen sources were tested for their repression effect on α‐amylase synthesis. Enhancement of α‐amylase production by supplementing micronutrients and surfactants was studied. Using optimized medium, process parameters were optimized for improved α‐amylase production. RESULTS: α‐Amylase was produced from KCC103 utilizing simple sugars indicating the absence of catabolite repression. Raw potato and yeast extract were best carbon and nitrogen sources for α‐amylase production. α‐Amylase synthesis was enhanced by micronutrients cysteine, thiamine, Mg²⁺ and SDS. Maximum α‐amylase (394 IU mL^?1) was produced in the optimized medium consisting of (in g L^?1) raw potato (30.0), yeast extract (20.0), cysteine (0.3), thiamine (0.2), SDS (0.2) and MgSO₄ (0.5 mmol L^?1) at 36–48 h under optimal conditions (pH 7.0, 37 °C, 200 rpm). The α‐amylase production was further enhanced to 537.7 IU mL^?1 with shorter time (15–18 h) in a bioreactor with optimized agitation rate of 700 rpm at 30% dissolved oxygen. CONCLUSION: Since there was no carbon catabolite repression of α‐amylase synthesis, sugar mixture from various agro‐residues hydrolysates could be utilized for α‐amylase production. The study showed the feasibility of utilization of raw potato for α‐amylase production from the KCC103, which would lead to a significant reduction in process cost. Copyright © 2008 Society of Chemical Industry