氨基肽酶的测定方法及氨基肽酶性质的研究

酱油是深受广大人民欢迎的大众化调味品,在酱油生产中,麦、豆是不可缺少的原料。大豆蛋白质被水解为肽和氨基酸,是酱油鲜味的主要来源。在生产过程中大豆蛋白质首先被蛋白酶作用,长链蛋白质无规则断裂成分子量较小的多肽碎片。蛋白酶是内肽酶,它能从蛋白质内部切断肽键形成肽,但却不能再把短肽的肽键切断,释放出自由氨基酸,因而在酱油酿造过程中蛋白质的溶解与蛋白酶活力有密切关系。高活力的蛋白酶有助于提高酿造酱油的蛋白质利用率,却不会使氨基酸生成率增加,只有肽酶才能将小分子多肽从自由氨基末端或自由羧基末端将氨基酸逐一切下,释放自由氨基酸。高活力的肽酶使酿造酱油中的氨基酸量增加,从而提高了酱油的氨基酸生成率,增加酱油的鲜     

自然发酵黄豆酱酱曲培养过程中蛋白酶的形成及蛋白质的分解

豆酱是中国传统发酵的豆制品,酱曲是发酵的前期阶段,该过程中蛋白质被水解酶分解为氨基酸和肽类。水分含量及pH值变化可以影响酱曲培养,试验结果表明水分含量呈下降趋势,pH值第1～20天下降,第20～45天升高。中性蛋白酶活力最高,碱性蛋白酶活力最低,在发酵第30天时酸性、中性蛋白酶活力达到高峰而后降低,碱性蛋白酶活力则持续升高,蛋白质分解产物NSI、多肽氮含量持续上升,最终平均NSI可达53.98%(干基),多肽氮均值为28.35%(干基),氨态氮均值为0.45%(干基)。     

不同生化因子对米曲霉蛋白酶系活力的影响

研究不同生化因子（金属离子、乙二胺四乙酸（EDTA）和十二烷基磺酸钠（SDS））对米曲霉蛋白酶系（包括酸性蛋白酶、中性蛋白酶、碱性蛋白酶、氨肽酶和羧肽酶等5种）活力的影响,结果显示：生化因子都对羧肽酶活力有促进作用,影响最强的是Ca2+,其相对酶活达到310%;对酸性蛋白酶活力促进作用最强的是的Mn2+,相对酶活达到317%;而SDS完全抑制其活性。生化因子对中性蛋白酶、碱性蛋白酶和氨肽酶活力主要起抑制作用,抑制能力最强的分别是Ge2+,Fe3+和SDS。Ag+对氨肽酶活力促进显著,相对酶活力达到483%。     

曲霉的蛋白水解酶类对大豆蛋白的水解作用

一、蛋白酶在酱油酿造中具有不同的作用酱油酿造的整个过程主要是培养微生物,使之产生酶,分解原料中的各种成分,构成酱油的色、香、味、体。所用的微生物以米曲霉为主,原料以大豆蛋白为主,味以鲜为主.因而米曲霉蛋白酶活力的高低一直是广大酿造工作者研究的目标.从基本原理上理解,蛋白酶分解蛋白质成(月示)、胨、肽、氨基酸,应该说蛋白酶活力越高,蛋白质被分解得就越彻底,原料的蛋白质利用率就越高.国内许多单位在选育高蛋白酶菌株、促进蛋白质利用率     

米曲霉固体发酵核桃粕条件优化

核桃粕中的蛋白质是一种优质植物蛋白,其含量高达51.36%,对其进行加工利用,将有力地促进核桃产业的发展。该文利用米曲霉固态发酵核桃粕,以蛋白酶活力为评价指标,通过试验分别探讨发酵温度、发酵时间、米曲霉添加量对核桃粕中蛋白酶活力的影响。在单因素试验的基础上进行L9（34）正交试验,对产酶条件进行优化。结果显示,发酵温度、米曲霉添加量、发酵时间对核桃粕中蛋白酶活力的大小均有极显著影响。获得最佳产酶条件为发酵温度31℃、米曲霉添加量0.015%、发酵时间50 h,此时蛋白酶活力为1 187.20 U/g。并在此条件下,对米曲霉在核桃粕和豆粕中的蛋白酶活力进行比较,发现米曲霉更适宜在核桃粕中发酵产蛋白酶。     

酱曲蛋白酶活力测定-氨基氮法

氨基氮法测定酱曲蛋白酶活力是在给定的温度，pH及时间下，蛋白酶水解蛋白质转化为氨基酸，通过测定氨基氮的含量表示蛋白酶的活力，方法完善、时间短、操作简单实用。     

酱油曲蛋白酶的分离及催化性质研究

酱油曲提取液,经硫酸铵盐析后,用Sephadex G-75柱层析分离,测定收集液中的蛋白含量并用两种方法测定蛋白酶活力。结果显示,福林法酶活力峰出现在蛋白吸收曲线第二高峰,酶解酪蛋白显示的酶活力峰值出现在蛋白含量第三高峰的左肩。表明酱油曲含有不同分子量大小的蛋白酶,分别作用于蛋白质的不同位点,分子量较大(在20 kDa到14.3 kDa之间)的蛋白酶福林法酶活力最高,蛋白酶水解酪蛋白产生的酪氨酸较多;分子量比较小(小于14.3 kDa)的蛋白酶福林法检测酶活力不高,但分解酪蛋白产生的氨基酸态氮(ANN)含量较高,表明该蛋白酶酶解酪蛋白产生酪氨酸之外的其它氨基酸或肽类较多。     

中国对虾虾壳制备几丁质工艺研究

本实验以中国对虾虾壳为原料制备几丁质,利用柠檬酸脱钙、蛋白酶脱除蛋白质,通过对蛋白酶活力测定,本实验选用Alcalase碱性蛋白酶进行实验。通过单因素实验和Box-behnken响应面设计优化几丁质的最佳制备条件,以灰分含量为脱钙指标,得到柠檬酸脱钙最佳工艺条件为:固液比1∶12,反应时间2.5 h,柠檬酸浓度为8%,灰分含量为0.42%。以蛋白质含量为指标,测得碱性蛋白酶脱蛋白的最佳工艺条件为:加酶量2200 U/g,反应时间5 h,反应温度50℃,脱除的蛋白质浓度可达到31.42%。本实验条件温和、反应迅速、无环境污染,符合我国绿色无公害理念,为工业化生产几丁质提供基础。     

嗜盐古生菌Halobacteriaceae sp.产胞外蛋白酶特性及其酶学性质

对嗜盐古生菌Halobacteriaceae sp.生长过程中的产酶特性做了初步研究,并研究了其胞外蛋白酶的理化特性。试验结果表明:嗜盐古生菌Halobacteriaceae sp.生长周期较长,从对数期开始分泌大量胞外蛋白酶;胞外蛋白酶的最适酶解反应温度是60℃;该蛋白酶具有较好的热稳定性,70℃条件下放置60 min,酶活力基本保持不变;最适反应p H为7.5,在p H 6.5~8.5范围内酶活力保持稳定;此蛋白酶对Na Cl耐受性不强,Na Cl浓度超过2 mol/L时,酶活力被显著抑制(P0.01);金属离子Ca2+对蛋白酶活力具有激活作用,可以增强蛋白酶热稳定性;Mn2+则对蛋白酶活力具有抑制作用,而K+与Mg2+对蛋白酶活力无影响;1.31 mol/L异丙醇以及1mmol/L DTNB对酶活力基本无影响,5 mmol/L的EDTA严重抑制其蛋白酶活力,1 mmol/L的PMSF则使其完全失活,说明此酶可能是一种金属依赖性的丝氨酸蛋白酶。     

天然有机物HFS对酸性蛋白酶酶活的影响

利用酶制剂处理食品原料,进行生产,历史悠久,酶的最大应用对象也是食品工业。在现代食品工业中,酶的应用几乎涉及食品加工的各个领域。随着酶制剂的应用日益广泛,经济效益显著。蛋白酶是水解蛋白质肽链的一类酶的总称,而酸性蛋白酶适宜在酸性条件下水解动植物蛋白质,通过内切和外切作用将蛋白质水解为小肽和氨基酸。本文探讨了天然有机物-HFS对537酸性蛋白酶酶活的影响,测定了该酶的最适反应条件(最适温度为55℃,最适pH值为3.0),考察并探讨了天然有机物-HFS对537酸性蛋白酶最适反应条件的影响。试验结果表明天然有机物-HFS对537酸性蛋白酶具有较好的激活作用,可有效提高酶活约8%,对该酶的最适反应条件无影响,也不改变最适条件曲线的基本变化规律。     

Enhanced stability of crude protease from kiwifruit (Actinidia deliciosa) by adding hydrocolloid for organic processed food uses

Crude protease originating from kiwifruit (Actinidia deliciosa) was extracted for organic processed food uses. The protease included in the kiwifruit can be utilized for organic uses instead of current commercial enzymes from microbial origin, which are not suitable for organic processed food. Crude protease extracted by physical treatment rather than any biochemical purification methods was appropriate for the organic processed food uses. However, crude protease extract has been found to be unstable for processing and storage usage, which has to be modified to be stable by appropriate methods suitable for organic processed food uses. The proteolytic activity of the protease extracted from kiwifruit was measured using casein as a substrate. The decreased inactivation rate constant of crude protease treated with guar gum and locust bean gum within the temperature range of 30–50°C implied the enhanced stability of crude enzymes by treatment with hydrocolloid. The half-times of crude proteases treated with guar gum and locust bean gum were higher than the half-time of native crude protease at 40°C (optimum temperature of the native crude protease), with values of 55.45 min for the guar gum-treated sample, 50.23 min for the locust bean gum-treated sample and 23.26 min for the native sample, demonstrating the quantitative evidence of the enzyme stability. The relatively stable maintenance of the proteolytic activity has helped to realize hydrocolloid-treated enzyme to be used for hydrolytic function in organic processed food applications.     

一株有机溶剂稳定性蛋白酶产生菌的筛选与鉴定

为了筛选分泌有机溶剂稳定性蛋白酶耐有机溶剂极端微生物,利用添加苯10%(V/V)的MLB培养液从油污染水样中分离获得有机溶剂耐受菌株56株。通过产蛋白酶筛选培养基(SMA 培养基)平板初筛和摇瓶发酵复筛从中筛选获得蛋白酶高产菌株DS1。通过形态学、生理生化及16S rDNA序列(genbank EU578329)构建的系统进化树将菌株DS1鉴定为Bacillus aquaemaris。该菌株能耐受多种有机溶剂,并可以分泌有机溶剂稳定性蛋白酶。该蛋白酶在 25%极性常数(logPOW)大于等于1.8的有机溶剂中稳定性好,于30℃、140 r/min,培养14d仍有70%以上的活力。菌株DS1及其所分泌的蛋白酶均能较好的耐受有机溶剂,可以进行有机相催化,具有潜在的工业应用价值。     

酱香大曲中产四甲基吡嗪细菌的分离鉴定及其功能性研究

采用稀释平板涂布法从酱香型白酒大曲中分离筛选出细菌菌株19株,模拟酱香型白酒生产发酵工艺,获得2株固态发酵产物具有浓郁酱香气味的菌株FBKL1.0199和FBKL1.0201。结合菌株形态学观察、生理生化实验和分子生物学方法,鉴定其为地衣芽孢杆菌（Bacillus licheniformis）。研究发现,菌株FBKL1.0199具有高产蛋白酶的特性,其中性蛋白酶活力达3 925. 80 U/g,酸性蛋白酶活力也相对较高,达139.27 U/g。同时,利用固相微萃取-气质联用技术对菌株FBKL1.0199和FBKL1.0201模拟白酒固态发酵的挥发性香味物质进行分析,发现其中吡嗪类物质相对含量较高,分别为46.01%和48.32%,且均以四甲基吡嗪为主,含量分别为44.11%和47.41%。分离得到的菌株FBKL1.0199和FBKL1.0201可以作为产四甲基吡嗪的功能菌。     

Purification and properties of cysteine protease from rhizomes of Curcuma longa (Linn.)

BACKGROUND: Turmeric rhizome (Curcuma domestica Linn.) contains proteases and has proteolytic activity. Curcumin from turmeric rhizomes has been used for healing manu ailments, including cancer have been used for healing many ailments, including cancer. The purpose of this study was to purify turmeric protease and to research their biochemical characteristics. RESULTS: Cysteine protease from C. domestica has been purified to homogeneity using acetone precipitation followed by preparatory native polyacrylamide gel electrophoresis (PAGE). This protocol resulted in six fold purification with 28% final recovery. The purified turmeric protease showed a prominent single peak and band on high‐performance liquid chromatography and sodium dodecyl sulfate–PAGE, respectively, and an estimated molecular weight of 43 KDa, and exhibited optimal activity between 37 and 60 °C. The protease activity of the turmeric protease was significantly inhibited by iodoacetic acid. The turmeric protease had higher alanine and glutamate content and cleaved synthetic peptides N‐Cbz‐Ile‐Pro and N‐Cbz‐Phe‐Leu in a time‐dependent manner. Peptide mass fingerprint using matrix‐assisted laser desorption/ionization–time of flight mass spectroscopy revealed peptide matches to proteasome subunit alpha type 3 of Oryza sativa ssp. japonica (Rice). The turmeric protease showed antifungal activity at 10 µg mL^?1 towards pathogens Pythium aphanidermatum, Trichoderma viride and Fusarium sp. CONCLUSION: Cysteine addition significantly activated turmeric protease. The protease inhibition test suggested that turmeric protease belonged to the cysteine type. The biochemical characteristics of turmeric protease described in this paper can provide useful information for potential end uses of turmeric protease for pharmaceutical industry applications such as therapeutics. Copyright © 2009 Society of Chemical Industry     

新疆酸奶中高产蛋白酶与产脂肪酶乳酸菌的筛选

从新疆传统酸奶中,利用透明圈法筛选出一株高产蛋白酶的乳酸菌RA3,其蛋白酶活力为38.87 U/mL;利用铜皂法筛选出产脂肪酶的乳酸菌菌株RC4,其酶活力为8.54 U/mL。经API 50CHL生理生化实验和16S rRNA序列比对鉴定RA3为发酵乳杆菌,RC4为植物乳杆菌,为我国发酵食品提供了新的乳酸菌资源。     

土壤中高产蛋白酶菌株的筛选鉴定及发酵条件优化

从海南土壤中筛选得到一株高产蛋白酶的菌株,并对其进行菌种鉴定、产蛋白酶发酵条件优化及蛋白酶分子质量测定。经筛 选,得到一株高产蛋白酶的菌株,编号为CAUH83,蛋白酶活力达到126 U/mL。 通过形态观察、生理生化试验及16S rDNA序列分析, 鉴定菌株CAUH83为粘质沙雷氏菌（Serratia marcescens）。 通过单因素试验优化,确定该菌株产蛋白酶的最优发酵条件：大豆粉3.0%、 蔗糖3.0%、初始pH 6.0、培养温度30 ℃、培养时间48 h。 在此优化发酵条件下,该菌株产蛋白酶酶活力达到1 932.3 U/mL,较优化前提 高15.3倍。 通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和酶谱分析表明该蛋白酶分子质量约51 kDa。     

青藏高原土壤低温产蛋白酶菌株的筛选及酶学性质研究

从青藏高原土壤样品分离保藏的菌种中筛选到低温条件下产蛋白酶菌株LS20-2,对其进行生理生化试验以及分子生物学鉴定,并对其发酵液进行酶学性质初步研究。结果表明,该菌被鉴定为皮氏类芽孢杆菌（Paenibacillus peoriae）,产蛋白酶的最适反应pH值为7.0,最适温度为40 ℃。在pH5.0～7.0之间、温度0～40 ℃时酶活稳定,金属离子Fe2+、Zn2+及Triten X-100能抑制蛋白酶活性,金属离子Mn2+及表面活性剂Tween-20、Tween-80能提高蛋白酶活性。     

Catalysis and stability of an alkaline protease from a haloalkaliphilic bacterium under non-aqueous conditions as a function of pH, salt and temperature

A haloalkaliphilic bacterium, isolated from Coastal Gujarat (India) was identified as Oceanobacillus sp. (GQ162111) based on 16S rRNA gene sequence. The organism grew and secreted extra cellular protease in presence of various organic solvents. At 30% (v/v) concentration of hexane, heptane, isooctane, dodecane and decane, significant growth and protease production was evident. The alkaline protease was purified in a single step on phenyl sepharose 6 FF with 28% yield. The molecular mass as judged by SDS-PAGE was 30?kDa. The temperature optimum of protease was 50°C and the enzyme retained 70% activity in 10% (v/v) isooctane. Effect of salt and pH was investigated in combination to assess the effect of isooctane. In organic solvents, the enzyme was considerably active at pH 8-11, with optimum activity at pH 10. Salt at 2?M was optimum for activity and enzyme maintained significant stability up to 18?h even at 3?M salt concentration. Patters of growth, protease production, catalysis and stability of the enzyme are presented. The study resumes significance as limited information is available on the interaction of haloalkaliphilic bacteria and their enzymes with organic solvents.     

Partial purification of histone H3 proteolytic activity from the budding yeast Saccharomyces cerevisiae

The proteolytic clipping of histone tails has recently emerged as a novel form of irreversible post‐translational modification (PTM) of histones. Histone clipping has been implicated as a regulatory process leading to the permanent removal of PTMs from histone proteins. However, there is scarcity of literature that describes the identification and characterization of histone‐specific proteases. Here, we employed various biochemical methods to report histone H3‐specific proteolytic activity from budding yeast. Our results demonstrate that H3 proteolytic activity was associated with sepharose bead matrices and activity was not affected by a variety of stress conditions. We have also identified the existence of an unknown protein that acts as a physiological inhibitor of the H3‐clipping activity of yeast H3 protease. Moreover, through protease inhibition assays, we have also characterized yeast H3 protease as a serine protease. Interestingly, unlike glutamate dehydrogenase (GDH), yeast H3 proteolytic activity was not inhibited by Stefin B. Together, our findings suggest the existence of a novel H3 protease in yeast that is different from other reported histone H3 proteases. The presence of histone H3 proteolytic activity, along with the physiological inhibitor in yeast, suggests an interesting molecular mechanism that regulates the activity of histone proteases. Copyright © 2016 John Wiley & Sons, Ltd.