Fermentation and oxygen transfer characteristics in serine alkaline protease production by recombinant Bacillus subtilis in molasses‐based complex medium

Serine alkaline protease (SAP) production in a complex medium based on physically pretreated molasses by recombinant Bacillus subtilis carrying pHV1431::subC gene is described. The effects of oxygen transfer were investigated in 3.5 dm³ bioreactor systems with controls for agitation rate, dissolved oxygen, pH, temperature, and foam formation under two different agitation rates, ie N = 500 and 750 min^?1, and four different air flow rates, ie Q/V_R = 0.2, 0.5, 0.7, and 1.0 vvm, at a molasses concentration equivalent to initial sucrose concentration (C_So) of 20 kg m^?3. The yield values (Y_X/S, Y_X/O, Y_S/O) and maintenance coefficient of oxygen (m_O), were calculated. m_O decreased with the increase in the air‐inlet rate. Increase in oxygen transfer rate increased the rate of growth and SAP activity, and affected the cultivation time to achieve maximum expression of SAP activity. At Q/V_R = 0.5 vvm and N = 750 min^?1, SAP activity reached 2250 U cm^?3 at t = 36 h. The oxygen transfer coefficient (K_La) and oxygen uptake rate (?r_O) were measured throughout the fermentations and their variation with the oxygen transfer conditions determined. New correlations for the calculation of K_La and ?r_O are proposed. Copyright © 2004 Society of Chemical Industry     

Protease and xylanase activities and thermophilic populations as potential process monitoring tools during thermophilic aerobic digestion

Thermophilic aerobic digestion (TAD) of potato process waste slurry was carried out in a batch process in a continuously stirred tank reactor at 55 °C for a total of 156 h. The pH of the slurry was either unregulated, or regulated at 6.0, 7.0, 8.0, 9.0 and 9.5, and aeration rate was 0.5 vvm. The effect of aeration rate on the digestion process was studied at 0.1, 0.25, 0.5 and 1.0 vvm at pH 7.0 and 55 °C. The development of thermophiles (55 and 65 °C populations) and hydrolytic enzyme activities (protease and xylanase) in the process were monitored. Thermophiles developed rapidly to reach peak populations in 24h or earlier, and remained stable at all fixed pH reactions. The thermophile population was only minimally affected by the aeration rate, and they were present mostly as spores after 96h of digestion. Xylanase enzyme appeared rapidly, reached a peak in approximately 60h, and declined rapidly thereafter. Highest activities were produced in neutral reactions and higher aeration rates. Aeration rates affected protease activity profoundly. The profile of both enzymes closely reflected the development of microbial activity and the overall progress of TAD, in a medium where their substrates were not predominant and not the preferred carbon sources. The relatively simple process of measuring the activities of these enzymes is potentially a more direct measure of the progress of TAD than enumeration of the microbial populations and thus, has greater potential in process monitoring. Copyright © 2003 Society of Chemical Industry     

Enantioselective separation of basic amino acids on talc

Certain amino acid derivatives (ε-basic, anilide group) can be readily adsorbed onto various types of talc (steopac, SS20, C300, C400). For instance, talc is capable of adsorbing the amino acid esters but not the equivalent free amino acids. The types of talc which have high hydrophobicity (00, 15M00) were poor adsorbents. Two applications of these findings are presented: enhancement of the sensitivity of enzymatic tests in the presence of chromogenic substrates and enantioselective separation of ε-basic amino acids (arginine, lysine, ornithine).     

Kinetics of aspartame precursor synthesis catalysed by Pseudomonas aeruginosa elastase

Elastase isolated from Pseudomonas aeruginosa IFO 3455 was found to be an efficient protease to catalyse the synthesis of N-benzyloxycarbonyl-aspartyl-phenylalanine methyl ester, the precursor of the dipeptide sweetener, aspartame. The influence of methanol as a cosolvent in this synthetic reaction was investigated. It was found that the synthesis of the dipeptide precursor was most efficient in 25% (v/v) methanol, pH 7·0 at about 25°C for a reaction time of about 3 h. However, the activity of the enzyme was greatly reduced in 90% methanol. The values of K and k₂ for N-benzyloxycarbonyl-aspartic acid were 0·17 mol dm^?3 and 11·9 mol dm^?3 s^?1 respectively.     

酸性蛋白酶在羊毛染色前处理中的应用

羊毛纤维及羊毛织物通过酸性蛋白酶处理，可获得减量率为1％－2％，且纤维强度并未显著下降的最佳工艺条件。从扫描电镜照片发现，经处理的羊毛纤维表面磷片明显受到破坏，棱角变得圆滑。处理后羊毛织物的上染率和上染速率都有所提高。     

Cloning,nucleotide sequence and functions of XPR6, which codes for a dibasic processing endoprotease from the yeast Yarrowia lipolytica

Yarrowia lipolytica DO613, carrying the xpr6-13 mutation, secretes an inactive precursor of alkaline extracellular protease that has not been cleaved after the Lys-Arg at the end of the pro-region. Compared to wild type, DO613 membrane preparations had significantly reduced ability to cleave after Lys-Arg of an artificial substrate. The XPR6 gene was cloned by complementation by screening for restoration of production of alkaline protease activity. Sequencing of a 3735 base pair SalI-SphI XPR6 fragment revealed a large open reading frame with a coding capacity of 976 amino acids (molecular weight, 110 016). The deduced amino acid sequence had significant homology to Saccharomyces cerevisiae Kex2p, a processing endoprotease that cleaves after pairs of basic amino acids. Disruption of the XPR6 gene was not lethal, but it resulted in several phenotypic changes. First, essentially no mature alkaline extracellular protease was produced indicating that the low levels produced by strains carrying previously isolated xpr6 alleles were due to leaky mutations. Second, mating type B strains carrying the disrupted XPR6 gene did not mate, but mating type A strains did. Third, the XPR6 disruption strains grew poorly on rich media at pH 5·5 and above. Cells remained physically attached after budding and continued to bud forming large dog balloon-like structures. In addition, these structures aggregated forming visible clumps in liquid culture. These growth aberrations were largely eliminated by growing cells in medium at pH 4. Fourth, no mycelial forms were observed regardless of the pH.     

Assay Systems and Characterization of Pacific Whiting (Merluccius productus) Protease

Commonly used protease assays and substrates were compared for sensitivity and simplicity in analyzing proteolytic activity in Pacific whiting causing gel weakening of surimi during heat-setting. Assay based on detection of trichloroacetic acid (TCA)-soluble products, using azocasein as substrate, showed highest sensitivity. By that assay, optimal pH of the protease was 5.5, and optimal temperature, 55°. The validity of the assay for measuring activity was confirmed by pH profiles of residual proteolytic and autolytic activities of uncooked surimi. These analyses showed pH profiles similar to those of fish juice with a pH optimum of 5.5.     

马铃薯蛋白酶抑制剂酶解产物超滤组分的抗氧化活性与结构特征

本实验以马铃薯蛋白酶抑制剂为原料,通过超声联合蛋白酶K酶解处理后,对其酶解产物(PPIHs)的理化性质、抗氧化稳定性及其超滤后各组分的抗氧化活性及结构特性进行分析。结果表明,超声联合酶解处理后,PPIHs的溶解度增加,乳化性和乳化稳定性增强;进一步研究发现,其在80 °C以下的抗氧化稳定性较好,超过80 °C时,有下降趋势;在酸性或中性环境下,除了对ABTS自由基的抗氧化稳定性较差,PPIHs对其他自由基的抗氧化稳定性较好。PPIHs经过超滤得到PPIHs-I(> 10 kDa)、PPIHs-II(3-10 kDa)和PPIHs-III(< 3 kDa)的三种超滤产物,PPIHs-I对ABTS自由基的清除能力最强,Zeta电位的绝对值显著增大;PPIHs-II的油脂氧化抑制能力以及对-OH和DPPH自由基的清除能力较强,粒径均显著减小。通过紫外吸收光谱、傅里叶变换红外光谱和荧光光谱中可以看出,酶解产物的结构发生明显改变,如无规卷曲和β-转角相对含量提高,β-折叠含量和 α-螺旋相对减少,疏水性增强等,进而改善其抗氧化活性。     

中性蛋白酶限制性水解对高温菜籽粕蛋白功能性质的影响

研究了中性蛋白酶限制性水解菜籽蛋白的功能性质。结果表明,中性蛋白酶水解能显著改善高温菜籽粕蛋白的各种功能性质,限制性水解菜籽蛋白的溶解性随水解度的增加而增加,DH=10的限制性水解菜籽蛋白在pH7.0时溶解度达57.11%;DH=2的限制性水解菜籽蛋白乳化性最好,其在测定的pH范围内乳化性均较菜籽蛋白提高40%以上;DH=4的限制性水解菜籽蛋白起泡性最佳,可达162.5%;DH=2的限制性水解菜籽蛋白吸油性与吸水性均最好,且分别较原菜籽粕蛋白提高了2.6倍与1.9倍。     

羊毛蛋白酶防毡缩加工综述

综述了羊毛化学法防毡缩加工中存在的问题,列举了现有不同羊毛蛋白酶防毡缩方法。根据羊毛鳞片层的结构特点,探讨了酯酶(脂肪酶、角质酶)、角蛋白酶、改性蛋白酶和谷酰胺转氨酶组合应用于羊毛生物法防毡缩加工的可行性。详细介绍了酶法预处理与蛋白酶相结合的新型羊毛防毡缩方法。