固态发酵黑曲霉产单宁酶发酵条件研究

经紫外线诱变黑曲霉菌株筛选出1株高产单宁酶的黑曲霉B0201,利用五倍子为诱导物,固态发酵该菌株得到的单宁酶活力有较大提高。单因素优化试验表明,五倍子用量为8%,初始水分含量为50%,初始pH6.0,温度30℃为优化产酶条件。在优化的条件下,培养96h后产单宁酶酶活力达到了58.2 U/g(干基)。因此,黑曲霉固态发酵产单宁酶具有很大的研究意义及广阔的应用前景。     

绿僵菌Ma83固态发酵几丁质酶和部分酶学性质的初步研究

研究了金龟子绿僵菌MetarhiziumanisopliaeMa83菌株产几丁质酶的固态发酵条件和粗酶液的酶学性质。研究结果显示 ,当以麸皮∶蚕蛹粉为 3∶1作为培养基 ,最适氮源为1%NaNO3,起始pH为 6 5 ,发酵温度为 31℃ ,接种量为 2mL液态种子时酶活力最高。此外 ,添加稻壳对Ma83产几丁质酶有促进作用。该菌株在生长 2d时 ,酶活力达 33 9U /g干培养基。酶的最适反应温度为 5 0℃ ,最适反应 pH为 6 0 ;不同温度保温 1h后 ,酶的半失活温度为 42℃。不同 pH值的缓冲溶液中 (2 5℃ )放置 1h后 ,酶在pH 5 0～ 6 0条件下稳定性最高     

毕赤酵母工程菌pk53产纤溶酶发酵条件的优化

对1株产纤溶酶毕赤酵母工程菌菌株pk53,利用单因素试验和正交试验确定该菌株的适宜廉价发酵培养基为:麸皮1.5%,豆粕粉2.0%,KH2PO40.5%,MgSO4.7H2O 0.05%;发酵条件为:接种龄14 h,接种量1%,甲醇添加量1.5%,培养基装液量30 mL(250 mL三角瓶),生长pH为5.5,诱导pH为6.0;在此条件下培养,纤溶酶酶活力达429.06 U/mL,是初始发酵条件下酶活力的8.88倍。     

蛋白质谷氨酰胺酶在毕赤酵母中的异源表达

蛋白质谷氨酰胺酶(protein glutaminase, PG)可水解蛋白质侧链的谷氨酰胺残基,生成氨和蛋白质-L-谷氨酸,从而增加负电荷、降低蛋白等电点,进而改善蛋白质的功能特性。但其生产菌株解朊金黄杆菌的产酶量较低,仅有0.258 U/mL,且基因操作效率低,故近年来多采用异源表达的方法,以提高其产量。该实验成功实现蛋白质谷氨酰胺酶酶原(Pro-PG)在毕赤酵母GS115中异源表达,还进行了培养基优化及重组酶学性质的研究。结果表明：重组毕赤酵母pPIC9K-Pro-PG/GS115在摇瓶水平经1%(体积分数)甲醇诱导120 h,表达出的Pro-PG经胰蛋白酶加工后,PG酶活力达到0.878 U/mL。酶学性质研究发现,重组的PG最适作用温度为60℃,在不超过60℃时孵育1 h其相对酶活力可保持在80%以上;该酶作用的最适pH为6.0,在pH 3.0～8.0条件下孵育1 h其相对酶活力可保持在70%以上。     

重组毕赤酵母产果糖基转移酶发酵条件的优化

果糖基转移酶(EC 2.4.1.9)是利用蔗糖为底物制备低聚果糖的关键酶。利用前期构建的产果糖基转移酶的重组毕赤酵母为出发菌株,对其进行发酵优化。最适发酵产酶条件为:装液量30 m L/250 m L、V(甘油)∶V(甲醇)=1∶20、初始pH为5.5、诱导温度为30℃、初始甲醇浓度为1.0%、后续甲醇浓度为1.5%、硫酸铵浓度10 g/L、诱导时间为120 h。在优化的发酵产酶条件下,重组果糖基转移酶酶活力达218.3 U/m L,较优化前提高了5倍。     

木聚糖酶XynG1-1在毕赤酵母中的表达及发酵条件优化

将来源于Paenibacillus campinasensis G1-1的木聚糖酶编码基因成功整合到毕赤酵母GS115基因组上,构建了高产木聚糖酶XynG1-1的毕赤酵母工程菌。采用响应面法对该工程菌的发酵条件进行优化。首先使用Design-Expert软件进行Plackett Burman实验设计筛选出影响产酶量的3个主要因素,即甲醇含量、生物素含量和培养时间。在此基础上使用Design-Expert软件进行Box-Behnken实验设计,通过响应面分析得出优化的发酵培养条件为:甲醇含量2.28%,培养时间37.29 h,生物素4 mg/L,酵母粉20 g/L,蛋白胨20 g/L,YNB 30 g/L,装液量100 m L/L,转速250 r/min、温度28℃、磷酸缓冲液pH 6.0。经实验验证,优化后的培养条件下胞外重组酶活达到707.2 IU/m L,与响应面预测结果一致,较优化前木聚糖酶酶活提高了7.9倍,较原始菌株产酶量提高了19.8倍。经10 L发酵罐扩大培养之后,重组木聚糖酶的酶活达到2 703 IU/m L。因此,该研究有效提高了木聚糖酶XynG1-1的发酵产量,并且,该重组酶保持了良好的酶学性质,可为工业化生产及应用奠定基础。     

雪茄烟叶单宁降解菌的筛选、鉴定及发酵工艺优化

为降低雪茄烟叶中的单宁含量（质量分数）,减少雪茄烟叶苦味,改善烟叶品质,从雪茄烟叶表面筛选得到16株对烟叶中单宁有降解效果的细菌菌株,通过测定单宁酶活力选出产酶能力最强的菌株B-3,经鉴定该菌株为成团泛菌（Pantoea agglomerans）。选择不同种类的碳源、氮源和无机盐并设置不同质量浓度梯度,筛选菌株B-3的最优产酶培养基（蔗糖15.45 g/L、酵母膏17.00 g/L、NaCl 4.94 g/L）。产酶培养基优化后菌株B-3所产酶液单宁酶活力高达55.83 U/mL,相比产酶基础培养基提高121.28%。将该酶液用无菌水稀释成单宁酶活力为5.58 U/mL的稀释酶液,并用稀释酶液对印度尼西亚雪茄烟叶进行发酵处理。当稀释酶液添加量为雪茄烟叶质量的25%且在45℃条件下发酵7 d后,雪茄烟叶的单宁降解率为39.69%,烟叶的苦味和杂气减少。     

杂色云芝漆酶基因在甲醇毕赤酵母中表达条件研究

重组甲醇毕赤酵母（Pichia methanolica）工程菌PMADl6／pMETA-Lccl能分泌表达杂色云芝重组漆酶。本文通过对其发酵条件的研究，找出摇瓶发酵表达漆酶的最适培养条件：在BMMY培养基中添加0．2mmol／L的Cu^2＋．装液量为25mL／250mL三角瓶，接种量体积之比为45：25，0．5％甲醇诱导，20℃条件下摇瓶培养5d，漆酶最高酶活力达1192U／L。     

菌株Serratia marcescens SYBC08产过氧化氢酶液态发酵工艺的优化及酶性质研究

利用单因素筛选和正交试验对菌株Serratia marcescens SYBC08液态发酵产酶的培养基和条件进行了优化,其最优工艺为:柠檬酸25 g/L,玉米浆粉36 g/L,初始pH值为6.75,接种量为体积分数4%,装液量50 mL,转速250 r/min,35℃培养36 h产酶活力可达9 553 U/mL,是优化前的5.49倍。通过对硫酸铵沉淀得到的过氧化氢酶进行酶学性质研究,该酶在碱性条件(pH值为9.0)条件下,60℃下保温150 min酶活力几乎不变,65℃半衰期为150 min,其比商品化的牛肝过氧化氢酶具有更高的热稳定性。该酶最佳催化温度是20℃,在0℃依然展示了78%的活力。这些结果表明该酶具有良好的冷适应和热稳定性,在高温、碱性条件或极低温条件有应用潜力。     

高产阿魏酸酯酶菌株的筛选及其固态发酵的研究

从自然界中采集土壤样本,使用阿魏酸乙酯为唯一碳源的选择性培养基;进行初筛、固态发酵产酶试验、复筛得到1株高产阿魏酸酯酶的菌株,通过形态学观察鉴定为黑曲霉。对该菌株固态发酵产阿魏酸酯酶的培养条件进行了研究,单因素实验结果表明,相同汽爆条件下汽爆稻草的发酵产酶酶活较高;在固液比1∶3、初始pH3、麸皮添加量25%、30℃下发酵3d后酶活最高,可达445.1mU/g。     

1 株产单宁酶嗜热真菌的鉴定、酶学性质分析及碳水化合物活性酶表达

对产单宁酶的1 株嗜热真菌HBHF5进行鉴定,开展单宁酶酶学性质的分析及碳水化合物活性酶（carbohydrate?active enzymes,CAZymes）的转录组学研究,探究嗜热真菌HBHF5在食品酶制剂开发中的潜力。经对菌株的菌落、孢子形态观察及ITS序列比对分析,最终鉴定嗜热真菌HBHF5为烟曲霉（Aspergillus fumigatus）。经分析,菌株HBHF5在固态发酵培养时不产单宁酶,而液态诱导培养时,菌株HBHF5胞内和胞外均检测到单宁酶活性,且以胞外酶为主（94%）,酶活力最高达136?U/mL。单宁酶最适反应温度为60?℃,在60?℃处理30?min,能够维持酶原活力的90%以上。该酶最适反应pH值为6.0,在pH?5.0～9.0范围内,能够维持60%以上的酶活力。不同金属离子对酶活力的影响存在差异,Cu2＋、Fe3＋、Mn2＋和Zn2＋对该单宁酶活性抑制较强。经转录组学分析,该菌以麸皮为唯一碳源时,共有淀粉酶、纤维素酶和果胶酶等239?个CAZymes基因表达,其中糖苷水解酶类最为丰富,约占CAZymes表达总数的70%。A. fumigatus HBHF5是1 株优良产酶菌株,为具有食品酶制剂开发潜力的嗜热真菌。     

Optimization of Tannase Production and Improvement of Nutritional Quality of Two Potential Low-Priced Plant Feedstuffs under Solid State Fermentation by Pichia kudriavzevii Isolated from Fish Gut

An autochthonous tannase yielding yeast strain Pichia kudriavzevii (GU939629), isolated from the gut of an Indian major carp (mrigal), Cirrhinus cirrhosus, has been used for extracellular tannase production and subsequent processing of two plant feedstuffs, Groundnut oil cake (GOC) and Pistia leaves (PL), under solid state fermentation (SSF). Of the two plant materials studied, GOC supported maximum tannase activity (0.82 ± 0.024 U/gds) degrading 94.1% of the initial tannin content, whereas SSF resulted in 0.68 ± 0.02 U/gds tannase activity and 89.1% tannin degradation through the use of PL as substrate. Following SSF for 15 d with optimized culture conditions, analysis of proximate composition revealed that there was significant increase (t-value significant at P < 0.05) in the contents of crude protein, lipid, minerals (Na, K, Ca, Mg, Zn, Fe, Cu, Mn, P), free amino acids and fatty acids; along with reduction in the contents of the other antinutritional factors, for example, crude fiber, phytic acid, and trypsin inhibitor. The results indicate that there is ample scope for further research to appraise potential application of gut microbiota for tannase production, as well as processing of low-cost plant feedstuffs for prospective use as feed ingredients for improved fish protein production.     

Partial characterization of lipases produced by a newly isolated Penicillium sp. in solid state and submerged fermentation: A comparative study

The aim of this research was the partial characterization of enzymatic extracts produced by a newly isolated Penicillium sp. in submerged (SmF) and solid state fermentation (SSF). The partial characterization of the crude enzymatic extract obtained by SSF and SmF systems showed optimum activity at pH 5.5 and 47 °C, and pH 7.0 and 37 °C, respectively (15.17 U/mL and 11.28 U/mL). The crude enzymatic extracts obtained by SmF and SSF presented the best stability at pH from 4.9 to 8.5 and temperature from 25 °C to 35 °C and pH 7.0 and 25 °C, respectively. These results confirm the interesting potential of SSF, because, besides the higher activities obtained in this system, the half-life time at 25 °C was higher than that observed for the lipase extract obtained in the SmF system.     

茶树单宁酶的原核表达及酶学性质表征

基于大肠杆菌的密码子偏好性，对茶树单宁酶基因CsTanA碱基进行优化，基因优化后GC含量为51.9%，密码子适应指数为0.8，优化前后基因序列的一致性为77.8%，以pET-30a为表达载体对优化基因进行重组表达及酶学性质分析。A600 nm约0.6的重组菌株以1 mmol/L的异丙基-β-D-硫代半乳糖苷在30 ℃诱导12 h，破碎上清液重组单宁酶rCsTanA比活力为0.35 U/mg，镍柱纯化后的rCsTanA比活力为1.53 U/mg，纯化倍数约为4.4。纯化重组单宁酶rCsTanA分子质量为39 kDa，其最适温度和最适pH值分别为40 ℃和7.0。不同金属离子浓度对酶活力的影响存在差异，在低浓度（1 mmol/L）条件下，K＋增强了rCsTanA 37.28%的酶活力，而Ag＋、Cu2＋和Fe3＋使该酶活力均丧失80%以上；而在高浓度（5 mmol/L）条件下，Cu2＋表现出完全抑制rCsTanA活性的特性。表面活性剂（十二烷基硫酸钠、吐温80和十六烷基三甲基溴化铵）和极性溶剂（甲醇、乙醇、丙三醇、异丙醇及丙酮）对rCsTanA活性具有较强抑制作用。本研究不仅实现了茶树单宁酶的表达和酶学性表征，同时也丰富了单宁酶资源，为植物单宁酶的进一步应用研究提供了必要的理论支撑。     

Antioxidant and antihypertensive properties of liquid and solid state fermented lentils

The effect of liquid (LSF) and solid state fermentation (SSF) of lentils for production of water-soluble fractions with antioxidant and antihypertensive properties was studied. LSF was performed either spontaneously (NF) or by Lactobacillus plantarum (LP) while SSF was performed by Bacillus subtilis (BS). Native lactic flora in NF adapted better than L. plantarum to fermentative broth and BS counts increased 4.0 log CFU/g up to 48 h of SSF. LSF water-soluble fractions had higher (P ? 0.05) free amino groups, GABA content, antioxidant and angiotensin I-converting enzyme inhibitory (ACEI) activities than SSF. In addition, GABA and ACEI activity of LSF increased in a time-dependent manner. Proteolysis by BS was limited, with slight changes in free amino groups, while GABA, total phenolic compounds and antioxidant capacity increased throughout fermentation. Higher antihypertensive potential was observed in NF (96 h) characterised by the highest GABA content (10.42 mg/g extract), ACE-inhibitory potency (expressed as IC₅₀) of 0.18 mg protein/ml and antioxidant capacity of 0.26 mmol Trolox equivalents/g extract. Therefore, water-soluble fermented lentil extracts obtained by LSF are particularly promising as functional ingredients in preventing hypertension.     

黑曲霉N5-5产单宁酶的酶学性质与固定化

本实验室自主分离的黑曲霉N5-5所产单宁酶已发现对没食子酸丙酯具有良好酶解效果。为了单宁酶的工业化应用,本次研究大批量培养单宁酶,探究其对单宁酸的酶解效果及酶的固定化效果。发酵采用先液体扩培后固体发酵的形式,提酶后利用陶瓷膜过滤技术纯化、浓缩酶液,并对比冷冻干燥和喷雾干燥两种不同干燥方式,还使用树脂载体对酶进行固定化。实验表明,纯化后单宁酶酶活达258.53 U/mL,可水解10%至50%浓度的单宁酸,其中30%以下底物浓度酶解效果较好;冷冻干燥对酶活影响不大而喷雾干燥使酶活降为174.02 U/mL;另外,单宁酶通过树脂载体固定化后,在实验条件下可重复使用至少4次。研究为提高黑曲霉N5-5发酵所产单宁酶的媒介效率、降低酶解反应成本、实现商业化生产建立了理论基础。     

Purification and Biochemical Characterization of Tannase from a Newly Isolated Strain of Paecilomyces Variotii

An extracellular tannase was isolated from Paecilomyces variotii and partially purified using ammonium sulphate precipitation followed by DEAE-Sepharose ion exchange chromatography. Paecilomyces variotii is a newly isolated strain obtained in São Paulo, Brazil, from the screening of 500 fungi evaluated for their production of tannase. The tannase was separated into two peaks. SDS-PAGE analysis indicated that the purified enzyme migrated as a single protein band corresponding to molecular mass of 87.3 kDa (major peak) and 71.5 kDa (minor peak). The peaks eluted very close together between 150 and 250 mM NaCl. DEAE-Sepharose column chromatography led to an overall purification of 19.3 fold. The K_m was found to be 0.61 μmol and the V_max?=?0.55 U.mL^?1. Temperatures from 40 to 65°C and pH values from 4.5 to 6.5 were optimum for tannase activity and stability. This tannase could find potential use in the food-processing industry.     

嗜热真菌单宁酶的克隆表达及在柿子汁中的应用

对嗜热烟曲霉中的单宁酶基因afTanA在毕赤酵母中进行异源表达,分析重组酶的酶学性质及其对柿子汁抗氧化性的影响。研究表明,afTanA基因由1 767 bp碱基组成,编码588个氨基酸和一个终止密码子,存在1个Kex2酶切位点(Lys315-Arg316),其催化活性位点为Ser202、Asp455和His501。重组菌株经48 h诱导,重组AfTanA酶活力达到678U/mL。AfTanA的最适反应温度为40℃,最适pH5.0。不同金属离子浓度对酶活力的影响存在差异,在低浓度(1mmol/L)条件下,Zn2+使AfTanA酶活力提高49.65%,而Cu2+和Fe3+使该酶活力分别降低约78%和98%;在高浓度(5mmol/L)条件下,Zn2+使AfTanA酶活力降低47.12%,Cu2+和Fe3+能够完全抑制AfTanA活性。柿子汁经重组单宁酶AfTanA处理后,其抗氧化性能力显著提高,DPPH、ABTS自由基和·OH清除率分别提高15.79%,12.78%,3.4%。单宁酶AfTanA在毕赤酵母中实现了高效表达,并在果汁加工工业中具有良好的应用潜力。     

Bacillus sphaericus: the highest bacterial tannase producer with potential for gallic acid synthesis

An indigenously isolated strain of Bacillus sphaericus was found to produce 1.21 IU/ml of tannase under unoptimized conditions. Optimizing the process one variable at a time resulted in the production of 7.6 IU/ml of tannase in 48 h in the presence of 1.5% tannic acid. A 9.26-fold increase in tannase production was achieved upon further optimization using response surface methodology (RSM), a statistical approach. This increase led to a production level of 11.2I U/ml in medium containing 2.0% tannic acid, 2.5% galactose, 0.25% ammonium chloride, and 0.1% MgSO(4) pH 6.0 incubated at 37°C and 100 rpm for 48 h with a 2.0% inoculum level. Scaling up tannase production in a 30-l bioreactor resulted in the production of 16.54 IU/ml after 36 h. Thus far, this tannase production is the highest reported in this bacterial strain. Partially purified tannase exhibited an optimum pH of 5.0 with activity in the pH range of 3 to 8; 50°C was the optimal temperature for activity. Efficient conversion of tannic acid to purified gallic acid (90.80%) was achieved through crystallization.