米黑毛霉酶凝干酪素生产工艺参数的优化研究

以新鲜牛乳为原料,采用米黑毛霉凝乳酶为凝乳剂,先通过单因素实验研究凝乳酶添加量、Ca Cl2添加量、凝乳p H、凝乳温度对酶凝干酪素得率的影响,然后通过响应面设计进一步优化了米黑毛霉酶凝干酪素生产工艺参数。优化得到的最佳工艺参数为:凝乳酶添加量0.49%、Ca Cl2添加量0.37 mg/m L、凝乳p H6.08、凝乳温度35℃,在此条件下酶凝干酪素的得率可达3.39%。研究结果可为米黑毛霉凝乳酶在干酪素生产中的应用提供参考。     

微小毛霉发酵制取凝乳酶的培养基优化研究

利用响应面分析法优化微小毛霉的发酵培养基,提高微小毛霉凝乳酶的凝乳活力。在单因素实验的基础上,用Design-Expert软件对实验数据进行多元回归分析,建立了3种因素与凝乳酶活力之间的函数关系,得出在基础发酵培养基中加入的氯化钙、乳清粉和葡萄糖的最佳浓度分别为0.58%、0.64%和1.13%,此时,微小毛霉凝乳酶的凝乳活力的理论值为1150.81SU/mL,验证平均值为1109.7SU/mL,与预测值基本一致。     

米黑毛霉产凝乳酶固体发酵培养基优化

对米黑毛霉产凝乳酶固体发酵培养基进行优化,以提高凝乳酶的产量,为米黑毛霉凝乳酶的工业化生产提供技术依据。首先通过单因素实验,得到最佳碳源为葡萄糖,最佳氮源为硝酸铵,最佳产酶诱导物为乳清粉。在此基础上通过正交实验进一步优化,得到培养基外加成分的最优组合为葡萄糖浓度2.5%,硝酸铵浓度1.0%,乳清粉浓度2.0%。培养基优化后米黑毛霉的凝乳活力达到(3649.52±3.62)SU/mL,比优化前提高了54.5%。     

米黑毛霉凝乳酶制备切达干酪的工艺参数优化

为确定米黑毛霉凝乳酶制作切达干酪的最佳工艺条件,以感官评分和出品率为响应值,在单因素试验的基础上,采用响应面法对主要工艺参数进行了优化。试验得到的米黑毛霉凝乳酶制作切达干酪的最佳工艺参数为:酶添加量为2900.38SU/L、凝乳pH为6.2、CaCl_2添加量为0.04%、凝乳温度为34℃、发酵剂添加量为0.14%;在此条件下,干酪感官评分为(95.2±0.34),试验结果与预测值接近,证明模型拟合程度较好。     

微小毛霉凝乳酶基因在毕赤酵母中的诱导表达研究

目的:研究重组毕赤酵母GS115PJ5诱导表达微小毛霉凝乳酶过程中酵母生长、产酶及培养液总蛋白变化情况。方法:测定毕赤酵母生长曲线、凝乳酶凝乳活性、蛋白水解活性、培养基上清液总蛋白含量。结果:毕赤酵母在开始诱导24h后即进入稳定生长期,并保持到240h,然后进入衰亡期。SDS-PAGE显示在分子量约为47000处有目的凝乳酶条带,凝乳酶在培养144h后开始大量积累,酶活性迅速提高,192h时凝乳活性达到最大值300SU/mL,蛋白水解活性为10.75U/mL,凝乳活性与蛋白水解活性的比值(C/P)达到最大值27.9,上清液总蛋白含量为0.189mg/mL。结论:微小毛霉凝乳酶基因在毕赤酵母中得到了有效的表达。     

米黑毛霉凝乳酶的浸提工艺研究

通过单因素试验和正交试验对米黑毛霉凝乳酶的浸提工艺进行优化,确定了米黑毛霉凝乳酶的最优浸提工艺参数为:浸提时间60min,浸提温度30℃,摇床转速180r/min,料液比1∶10,在此提取工艺下,酶液的凝乳酶活力达到1522.87SU/m L。     

微小毛霉凝乳酶蛋白质工程改造研究

微小毛霉凝乳酶(MPC)是微生物凝乳酶的主要来源之一。本研究将微小毛霉凝乳酶基因克隆到表达载体pET30a中并转化宿主BL21(DE3),使微小毛霉凝乳酶蛋白在大肠杆菌中表达。通过易错PCR技术对MPC进行随机突变建库,从中筛选出两株具有优良蛋白特性的突变体,为蛋白质结构与功能研究以及菌种改造奠定基础。     

不同因素对凝乳酸活力的影响

对犊牛羔羊第四胃及微小毛霉（Ｍｕｃｏｒｐｕｓｉｌｌｕｓ）三种凝乳酶进行了最适温度、ｐＨ稳定范围、热稳定性、不同ｐＨ对效价的影响及各种离子影响的研究．结果表明，牛、羊、微小毛霉凝乳酸的最适温度分别为６０℃、５５℃和６５℃；ｐＨ稳定范围分别是２－７、３－６和３－８；其热稳定性依次为，牛＜羊＜微小毛霉，牛凝乳酸５５℃处理１０ｍｉｎ基本失活，羊凝乳酸６０℃处理１０ｍｉｎ完全失活，微小毛霉凝乳酶６５℃处理１０ｍｉｎ活力全部丧失，三种酶随奶液之ｐＨ值降低而凝乳加快；Ａｌ－－－对上述三种凝乳酸都有较强的促凝乳作用，Ｃａ（＋＋）、Ｆｅ（＋＋）有一定促进作用，而Ｚｎ（－－）、Ｃｕ（－－）对三种酶却有一定抑制凝乳作用，对微小毛霉凝乳酶更为明显，Ｋ＋、Ｌｉ－、Ｍｇ（＋＋）及Ｎａ－均无明显作用。     

豆渣菌糠浅盘发酵制备微小毛霉凝乳酶的研究

为考察将豆渣和金针菇菌糠这两种废弃资源应用于微小毛霉凝乳酶固态浅盘发酵的效果,以医用托盘作为发酵容器,以干燥后的豆渣菌糠1∶1(质量比)混合物为培养基,以固液比1∶1(质量比)加水润湿培养基,121℃灭菌冷却后接种微小毛霉进行发酵.发酵72 h时,加水提取酶,将酶液进行浓缩、盐析、透析以及冷冻干燥后获得凝乳酶粉末.结果显示,以豆渣和菌糠混合物为发酵培养基时,其单位发酵面积最大产酶量可达到1 860 SU/cm2,略高于对照麦麸1 618 SU/cm2的发酵水平,这表明豆渣和菌糠混合物能够替代麦麸用于微小毛霉凝乳酶的固体浅盘发酵.此外,酶提取液经初步分离纯化,可获得凝乳活力为7 497 SU/mg、水解酶活为0.22 Ucas/mg、纯度大于50％的凝乳酶粉末制剂.     

米黑毛霉凝乳酶稳定剂的研究

本文比较了不同种类的醇,金属离子和糖对米黑毛霉凝乳酶保存稳定性的影响。单因素试验结果表明:甘油、Mg~(2+)、可溶性淀粉均可有效提高液体酶的保存稳定性,正交试验结果表明,Mg~(2+)对凝乳酶稳定性影响最大,甘油次之,可溶性淀粉影响最小。试验确定的复合稳定剂的最优配比为:Mg~(2+) 60mmol/L、甘油为25%、可溶性淀粉为4%。在此条件下,酶活的保留率可以达到116.02±1.42%,长效试验结果表明,该复合稳定剂对于保持米黑毛霉凝乳酶稳定性的效果显著。     

Thermal inactivation of chymosin during cheese manufacture

The aspartic proteinase, chymosin (EC 3.4.23.4) is the principal milk clotting enzyme used in cheese production and is one of the principal proteolytic agents involved in cheese ripening. Varietal differences in chymosin activity, due to factors such as cheese cooking temperature, fundamentally influence cheese characteristics. Furthermore, much chymosin is lost in whey, and further processing of this by-product may require efficient inactivation of this enzyme, with minimal effects on whey proteins. In the first part of this study, the thermal inactivation kinetics of Maxiren 15 (a recombinant chymosin preparation) were studied in skim milk ultrafiltration permeate, whole milk whey and skim milk whey. Inactivation of chymosin in these systems (at pH 6.64) followed first order kinetics with a D45.5 value of 100 +/- 21 min and a z-value of 5.9 +/- 0.3 degrees C. D-Values increased linearly with decreasing pH from 6.64 to 6.2, while z-values decreased as pH decreased from 6.64 to 6.4, but were similar at pH 6.4 and 6.2. Subsequent determination of chymosin activity during manufacture of Cheddar and Swiss-type cheese showed good correlations between predicted and experimental values for thermal inactivation of chymosin in whey. However, both types of cheese curd exhibited relatively constant residual chymosin activity throughout manufacture, despite the higher cooking temperature applied in the manufacture of Swiss cheese. Electrophoretic analysis of slurries made from Cheddar and Swiss cheese indicated decreased proteolysis due to chymosin activity during storage of the Swiss cheese slurry, but hydrolysis of sodium caseinate by coagulant extracted from both cheese types indicated similar levels of residual chymosin activity. This may suggest that some form of conformational change other than irreversible thermal denaturation of chymisin takes place in cheese curd during cooking, or that some other physico-chemical difference between Swiss and Cheddar cheese controls the activity of chymosin during ripening.     

Proteolytic activity of proteinases on macropeptide isolated from kappa-casein.

Proteolytic activities of chymosin, bovine pepsin, Mucor miehei rennet, Cryphonectria parasitica (formerly Endothia parasitica) rennet, trypsin, and chymotrypsin on kappa-casein macropeptide were measured. Macropeptide solutions (10 mg/ml of .05 M, pH 6.6 phosphate buffer) were incubated with the enzymes at 37 degrees C for various times, and their reactions were stopped by adding .025 ml of pepstatin (1 mg/ml of methanol). Peptides released from kappa-casein macropeptide were then fractionated using reverse-phase HPLC. At the pH of milk (pH 6.6), kappa-casein macropeptide was resistant to enzymic action by chymosin, bovine pepsin, and M. miehei and C. parasitica rennets. Bovine pepsin hydrolyzed kappa-casein macropeptide at pH 3. kappa-Casein macropeptide was readily hydrolyzed at pH 6.6 by trypsin and chymotrypsin. Possible physiological functions of the kappa-casein macropeptide are discussed in light of these findings.     

SECONDARY STRUCTURE OF SOME ASPARTYL PROTEINASES

A structural study of some aspartyl proteinases was undertaken. Secondary structure prediction methods indicate that chymosin, pepsin, penicillopepsin and Mucor miehei proteinase have relatively high proportions of β-sheet with active site aspartic acid residues located in β-turn regions. Secondary structure determination from far-UV CD spectral data support the above finding that the aspartyl proteinases have a high proportion of β-sheet. The proportion of β-sheet generally decreased at pH values greater than 6.3. More extensive unfolding occurred with pepsin and penicillopepsin than chymosin, Mucor miehei proteinase, Mucor pusillus proteinase and Endothia parasitica proteinase in the neutral to alkaline pH range. Results obtained from the near-UV CD spectra of the aspartyl proteinases indicate a change in spectra in the neutral to alkaline pH range which suggests the importance of aromatic groups to tertiary structure stability.     

Recombinant lamb chymosin as an alternative coagulating enzyme in cheese production

Recombinant lamb chymosin (RLC) was prepared and tested for its potential use in cheese production. The milk clotting activity and proteolytic activity of RLC were evaluated in comparison with commercial recombinant calf chymosin (RCC), cow rennet (CR), and microbial coagulant (MC). RLC, RCC, and MC showed similar responses to pH, with a sharp increase of the coagulation time at pH 6.6 to 6.8 and decrease of curd firmness at the pH 6.5 to 6.6. In the case of CR, we observed two clear increases in the coagulation time and decreases in the curd firmness, at pH 6.4 to 6.5 and 6.6 to 6.8. Optimal clotting activity was obtained for RLC at 40 degrees C, for both CR and RCC at 45 degrees C, and for MC at 60 degrees C. The temperature instability of RLC at temperatures above 45 degrees C could constitute a benefit in making hard cheese varieties. The additon of CaCl2 to milk resulted in enhanced clotting activity of all coagulants, most prominently for CR. The proteolytic activity of RLC was significantly lower from that of CR but not significantly different from the activity of RCC. The lower proteolytic activity in the cheese made with RLC did not have negative effect on organoleptic properties. The overall quality of the cheese made with RLC was at least comparable to that of the cheese made with RCC, and both cheeses were better scored than the cheese made with CR.     

微小毛霉凝乳酶的酶学性质研究

目的：研究微小毛霉(HL-1)凝乳酶的酶学性质。方法：研究了酶的最适温度,酶的pH值稳定性和热稳定性,探讨了金属离子、化学物质和钙离子对酶活力的影响,加酶量对酶反应的影响,测定了酶的蛋白水解活力、相对分子量和酶的Km与Vm值。结果：酶的最适温度为60℃; 最适pH值为5.5;酶在65℃保温5min活力损失95%;钙离子是酶的激活剂,其含量与酶活力正相关,而钾离子则对酶有抑制作用;氯化钠对酶活力的影响不大,但是SDS可导致酶严重失活;加酶量与反应时间成反比;酶的蛋白水解活力为485.3U/g;相对分子质量是42000,Km为0.02380mol/L,Vm为1.1227mg/min。结论：得出了微小毛霉(HL-1)凝乳酶的酶学性质。     

枯草芽孢杆菌克隆与表达微小毛霉凝乳酶基因

从自制的酒酿利用酪蛋白培养基分离纯化到了一株产凝乳酶的微小毛霉菌株（ZZMZ-19）,从ZZM-19菌株的cDNA文库筛选到了两个凝乳酶基因chl和ch2并实现了凝乳酶基因ch1和ch2在枯草芽孢杆菌菌株（ZZMZ-01）中的的克隆与表达。chl和曲2阳性克隆菌株在酪蛋白培养基r1]发酵30h左右的时间凝乳酶酶活达到最人,分别为48．27SU／mL和41．02SU／mL,相比出发菌株发酵时间缩短了15h。用交联葡聚糖凝胶柱G100分离纯化其发酵卜清液后,用十二烷基硫酸钠聚丙烯酰胺凝胶电泳方法测得CHII和cHIII分子草分别为32ku和30ku。     

Effect of Heat-Stable Proteases on the Kinetic Parameters of Milk Clotting by Chymosin

The milk clotting per unit casein hydrolytic activities of proteases from 14 psychrotrophic pseudomonad isolates of raw milk ranged from 0.77 to 9.97 at 30°C. The milk clotting activities of chymodn and T16 protease were not completely additive, especially at high chymosin concentration when clotting time was relatively fast. The T16 protease was not effective in catalyzing the enzymic step of milk clbtting at O°C in the time expected on the basis of its milk clotting at 30°C. Milk incubated with the T25 motease for 8 days at 4°C and then clotted with chymosin at 30°C exhibited weak curd consistency.     

微小毛霉凝乳酶基因的克隆与表达

微小毛霉(Mucor pusillus)凝乳酶是微生物凝乳酶的主要来源之一,但与传统的牛凝乳酶比较具有一定的缺陷。为将其采用基因工程的方法进行改造获得理想的凝乳酶,本研究克隆到微小毛霉凝乳酶基因,将其插入原核表达载体pTWlN1中,使之与几丁质结合域(CBD)一内含肽(intein)融合,获得原核表达质粒pTWIN1/M。转化大肠杆菌BL21(DE3),后经IPTG诱导后进行SDS-PAGE电泳分析,获得了重组蛋白。     

Purification and characterisation of milk‐clotting and caseinolytic activities of pepsin isolated from adult sheep abomasa

Ovine pepsin was isolated and assessed for its milk‐clotting properties and caseinolytic activity in comparison with commercial chymosin. Ovine pepsin showed similar responses to variations in pH, temperature and CaCl₂ concentration of milk compared with chymosin, although its pH sensitivity was higher. SDS‐PAGE electrophoretic analysis of the casein fractions treated with ovine pepsin showed that alpha‐casein was more susceptible to proteolysis than beta‐casein, in contrast to chymosin. Curd‐firming properties of skim milk gels obtained with ovine pepsin and chymosin were evaluated by Gelograph under the same conditions. Curd produced using ovine pepsin was less firm than that made with chymosin.     

Extracellular protease from Mucor pusillus: purification and characterization

Extracellular protease from Mucor pusillus was purified 18-fold with 7.56% recovery by ion-exchange chromatography and gel filtration. The enzyme was found to be monomeric in nature, having a molecular mass of 49 kDa. The enzyme acted optimally at 50°C and was stable in the temperature range 30–50°C. It was completely inactivated by heating for 30 min at 65°C. The optimum of activity for the purified extract was observed at milk CaCl₂ concentration of 0.02  m and at milk pH of 5. These properties, except for temperature, were similar to those of rennet.