保加利亚乳杆菌诱导产亚油酸异构酶条件研究

亚油酸异构酶可由保加利亚乳杆菌经诱导产生,可以将亚油酸(LA)转化为共轭亚油酸(CLA)。本实验对诱导保加利亚乳杆菌产亚油酸异构酶的条件进行研究,利用紫外和气质联用仪(GC-MS)检测所生成的CLA。结果表明：在培养基中添加1.5‰(V/V) LA 时所产酶的共轭亚油酸转化率最高;温度为36℃,培养36h 为较适的培养条件;单独添加0.1%(m/V)的乳糖或0.1%(m/V)的氯化钠有利于诱导产酶;在培养基中直接添加LA 的效果优于培养3至12h 后再进行添加。诱导所产酶可将LA 转化为CLA,且含有9c,11t-CLA 异构体。     

胆盐水解酶合成条件的优化

以MRS，MRS肉汤，改良MRS为基础培养基，考察了培养基对菌株产胆盐水解酶活力的影响，确定了MRS肉汤为基础培养。对KTx在MRS肉汤中最适碳源、氮源、培养温度、培养基起始pH值、刺激因子作了单因素试验，得出最适碳源为葡萄糖，最适氮源为大豆蛋白胨，最适培养温度37℃，最适培养基起始pH值为5．5～6．0，最适刺激因子吐温80。在此基础上选择葡萄糖、大豆蛋白胨、培养温度、接种量进行L16（4^4）四因素四水平正交试验。结果表明，对胆盐水解酶活力影响因子水平：葡萄糖〉大豆蛋白胨〉培齐温度〉接种量：最佳产胆盐水解酶条件：葡萄糖4％，大豆蛋白胨2％，接种量2％（均为质量分数），培养温度为37℃。优化后菌株的产酶活力可提高到优化前的13．4倍。     

不同发酵条件对Fusarium. solani ZH0101产木聚糖酶的影响

以本实验室筛选到的一株产木聚糖酶但不产纤维素酶的真菌Fusarium solani ZH0101为供试菌株,利用麦秸为诱导物,对产木聚糖酶的液体发酵培养基进行优化。对发酵时间、麦秸添加量、培养基起始pH值、磷酸盐、金属离子、无机氮源和有机氮源对产酶的影响进行研究,优化最佳的培养条件。优化后的液体发酵培养基条件为：麦秸20g/L、KH2PO4 4g/L、CH3COONH4 1g/L、酵母膏2g/L和起始pH值为6.0,发酵时间为14d。优化条件下所产无纤维素酶活力的木聚糖酶酶活力为26.85U/mL,能比未优化前的20.82U/mL提高近30%。     

发酵乳杆菌增殖培养基营养因子优化研究

以MRS 培养基为基础,选择碳源、氮源、生长因子这3 个主要的营养成分进行单因素试验,然后利用L9(34)正交试验优化出发酵乳杆菌增殖培养基营养因子最佳组成为：葡萄糖2.0%,牛肉膏0.5%,胰酶解酪朊0.5%,玉米浆2%,其他MRS 基本成分保持不变。研究结果表明,发酵乳杆菌La-Y1 在优化后的MRS 培养基中,37℃培养16～18h,菌落数高于用原始MRS 培养的发酵液,达到了1010CFU/mL 以上。     

培养基影响大肠杆菌产L-天冬酰胺酶的研究

研究培养基的成分及添加量对大肠杆菌产生抗癌药物-L-天冬酰胺酶的影响.通过对大肠杆菌培养基中碳源、氮源以及微量元素的讨论,得知该菌株以蔗糖为碳源,添加量为0.7%,以0.5%牛肉膏 1.0%蛋白胨为氮源,添加0.01%的硫酸亚铁为微量元素,所产L-天冬酰胺酶的比酶活达到352.54 U/g,相比基本培养基培养提高了31%.     

响应面法分析枯草芽孢杆菌Y-am6产酸性淀粉酶的发酵研究

采用响应面法对产酸性α-淀粉酶的芽孢杆菌Y-am6的发酵培养基进行件优化分析.通过单因素试验筛选得最适碳源、最佳有机氮源及最佳无机氮源分别为麸皮、黄豆面、硫酸铵.然后应用BBD对发酵培养基进行优化设计,试验结果经Design Expert 7.0处理并拟合验证,得发酵培养基关键组分为麸皮8.72％;黄豆面3.19％; (NH4)2SO42.04％.采用优化后发酵培养基,在初始pH值为5.0,37℃,220r/min发酵培养48h条件下,Y-am6的产酶活力提高至434.42U/mL,比初始酶活力提高了4.87倍.     

唾液链球菌嗜热亚种Y-2产谷氨酸脱羧酶的影响因子确立

从产酶和细胞生长较好的MRS培养基出发,对Streptococcus salivarius ssp.thermophilus Y-2产谷氨酸脱羧酶(glutamate decarboxylase,GAD)的影响因子进行探讨,结果当培养基组成和培养条件为蛋白胨15g/L、牛肉膏12.5g/L、蔗糖12.5g/L、柠檬酸二铵2.0g/L、乙酸钠5.0g/L、K2HPO42.0g/L、CaCl2 2.0g/L、Tween 80 1.0ml、pH7.0、接种量2%(V/V)、发酵温度37℃、发酵时间12h时,较有利于菌株Y-2产GAD。Plackett-Burman设计法研究表明培养基初始pH值和K2HPO4为影响菌株Y-2产GAD的主要影响因素。经对菌株Y-2产GAD影响因素的筛选,新获得的培养基在组成上与MRS培养基相比已发生显著变化,GAD活力提高了1.3倍。     

有机氮源对三孢布拉霉菌产番茄红素的影响

分别在含基础有机氮源的发酵培养基里添加啤酒废酵母水解液、玉米浆、酵母膏和蛋白胨4种额外有机氮源,发酵培养108 h,测定番茄红素的产量、菌体的生物量及发酵过程中额外有机氮源的添加对三孢布拉霉菌糖的利用情况。结果表明,啤酒废酵母水解液以促进番茄红素合成为主,玉米浆以促进菌体生长为主,酵母膏和蛋白胨的添加对三孢布拉霉菌产番茄红素影响不大。     

发酵生产脂肪酶的菌株筛选及其应用

采用从大庆日月星油厂附近的土壤中筛选得到产脂肪酶的菌株xjA,在固态发酵的条件下生产脂肪酶,并将所产脂肪酶应用于合成共轭亚油酸甘油酯。本实验利用单因素和正交试验方法确定产脂肪酶的最佳固态发酵条件：在种龄36h的情况下,碳源(麸皮)与氮源(豆粕)质量比(m麸皮:m豆粕)为1:4、V培养基:V加水量 1:1、接种量0.3%、发酵温度29℃、发酵时间3d。在此条件下进行固态发酵,固态培养基中脂肪酶的活力最高,可达1450U/g;所得脂肪酶用于合成共轭亚油酸甘油酯,产物中共轭亚油酸(CLA)接入率可达到13.6%。     

1株产胞外葡萄糖氧化酶黑曲霉的液体发酵条件优化

从土壤中筛选得到1株产胞外葡萄糖氧化酶的黑曲霉菌株1504,通过诱变育种选育得到突变菌株UNⅡ021。为进一步提高产酶活力,对其培养基成分和培养条件进行优化,结果最佳培养基组成和培养条件为:以10%的葡萄糖为碳源,0.5%牛肉蛋白胨和0.5%硝酸钠为氮源,碳酸钙添加量30 g/L,培养基初始pH 5.2,接种量0.05(V/V),培养温度29℃,摇床转速240 r/min。在此条件下,培养4 d菌株UNⅡ021产葡萄糖氧化酶活力达到363.04 U/mL,是优化前的2.14倍。     

Effect of Organic Nitrogen Sources on Raw Starch‐Digesting Glucoamylase Production of Rhizopus sp. MKU 40

In a liquid cultivation of Rhizopus sp. MKU 40, supplementation of the medium with 1.5% (w/v) organic nitrogen sources (neopeptone, casein from milk, and meat extract) had a slightly positive effect on glucoamylase (GA) (EC 3.2.1.3) activity compared with the medium lacking organic nitrogen sources. The addition of organic nitrogen sources induced production of protease. Supplementation of the medium with 1.5% (w/v) organic nitrogen sources resulted in an acid and neutral protease activity of 11 — 25 U/mL and 12 — 20 U/mL, respectively. The co‐existence of GA‐I [a highly raw starch‐digesting glucoamylase (RSDG)] and protease in the same medium leads to the production of Ga‐II (an extremely weak RSDG) from GA‐I. As a result the RSDG activity in the medium decreases. Raw starch adsorption rates of a medium without organic nitrogen sources were 100%, because the medium contained only GA‐I. In contrast, the media supplemented with organic nitrogen sources had low starch adsorption rates because the media contained both GA‐I and GA‐II. The results presented in this paper indicate that supplementation of the culture medium of Rhizopus strains with organic nitrogen sources negatively affects GA‐I production.     

胆固醇氧化酶的发酵条件及酶学性质研究

Brevibacterium sp．可以发酵生产胆固醇氧化酶,实验中对该菌种进行了底物诱导及发酵条件的优化, 确定其最适培养基为(%)：蔗糖0．3,酵母膏0．2,蛋白胨0．3,牛内膏0．3,K_2PO_4 0．1,MgSO_4 0．05,pH6．8。最适培养条件为：接种量5%,24℃培养20h,通气量为50mL 培养基/250mL 三角瓶,200r/min。在最适培养基及最适条件下,胆固醇氧化酶的酶活力可达到24．01U/mg,比未优化前1．71U/mg 提高了14倍。该酶具有较强的酸碱稳定性和热稳定性,最适 pH 为6．5,最适温度为54℃。在最适 pH 和温度条件下测得该酶 km 值为7．1 ×10~(-5)mol/L。     

Production of thermotolerant N-carbamyl-D-amino acid amidohydrolase by recombinant Escherichia coli

A plasmid, pNT4553, was constructed for high level production of N-carbamyl-d-amino acid amidohydrolase (DCase), the thermostability of which has been improved by amino acid substitution. The DCase activity and the stability of the plasmid in the host cells were dependent on the Escherichia coli strains used. E. coli HB101 was the most suitable host strain among the 13 types of E. coli tested. E. coli HB101 exhibited the highest activity, i.e. 6.36 units/ml of culture broth in 2YT medium (1.6% tryptone, 1.0% yeast extract, and 0.5% NaCl, pH 7.0), and the plasmid was stably maintained by cultivation in 5 types of E. coli including HB101. Casamino acids, NZ-amine, peptone, and protein extract (a mixture of hydrolyzates of corn gluten, wheat gluten and soybean), were found to be suitable as natural nitrogen sources for both enzyme activity and growth. When cultivation was carried out in the presence of high concentrations of glycerol (6.5%) as the carbon source, and protein extract (3.0%) as the nitrogen source, in a small volume of the medium (20 ml of medium in a 500-ml shaking flask), in which the aeration level was estimated to be high, growth and activity reached OD550=63.8 (17.1 mg of dry cell weight/ml of culture broth) and 22.9 units/ml of culture broth, respectively. The economical hyperproduction of DCase using only inexpensive constituents for the medium was achieved.     

Utilization of shrimp shellfish waste as a substrate for solid-state cultivation of Aspergillus sp. S1-13: evaluation of a culture based on chitinase formation which is necessary for chitin-assimilation

The utilization of shrimp shellfish waste as a substrate for solid-state cultivation of a filamentous fungus, Aspergillus sp. S1-13, was investigated. The organism was selected from among 220 isolates based on the productivity of its chitinolytic enzyme (chitinase), which might reflect microbial growth. The enzyme was produced only when the organism was grown on medium containing the shellfish waste. The addition of 58-65% water (w/w) to the medium was effective in enhancing production, and a certain amount of enzyme was observed in media of higher water content (up to about 75%). The initial pH and nitrogen source (ammonium sulfate) of the solid-state medium also affected the amount of enzyme. The amount of enzyme increased 2-fold in an optimum solid-state medium: 5 g of shrimp shellfish waste and 3 ml of basal medium (pH 5) containing 0.1% (NH4)2SO4 was inoculated with 4 ml of spore suspension; static cultivation at room temperature. The amount increased further (1.5-fold) when the cultivation was carried out at 37 degrees C, with 1.85 units of the enzyme formed from 1 g of shrimp shellfish waste. An analysis by ion-exchange column chromatography suggested the presence of at least two colloidal chitin-hydrolyzing enzymes and one p-nitrophenyl beta-D-N-acetylglucosaminide-hydrolyzing enzyme in an extract of the solid-state culture. The elution profile was similar to that obtained with a liquid culture filtrate.     

Production of bacterial endoglucanase from pretreated oil palm empty fruit bunch by bacillus pumilus EB3

In this study, endoglucanase was produced from oil palm empty fruit bunch (OPEFB) by a locally isolated aerobic bacterium, Bacillus pumilus EB3. The effects of the fermentation parameters such as initial pH, temperature, and nitrogen source on the endoglucanase production were studied using carboxymethyl cellulose (CMC) as the carbon source. Endoglucanase from B. pumilus EB3 was maximally secreted at 37 degrees C, initial pH 7.0 with 10 g/l of CMC as carbon source, and 2 g/l of yeast extract as organic nitrogen source. The activity recorded during the fermentation was 0.076 U/ml. The productivity of the enzyme increased twofold when 2 g/l of yeast extract was used as the organic nitrogen supplement as compared to the non-supplemented medium. An interesting finding from this study is that pretreated OPEFB medium showed comparable results to CMC medium in terms of enzyme production with an activity of 0.063 U/ml. As OPEFB is an abundant solid waste at palm oil mills, it has the potential of acting as a substrate in cellulase production.     

核桃浆对鼠李糖乳杆菌培养特性及其富硒后抗缺氧活性的影响

为考察以核桃浆为原料开发廉价、高效的鼠李糖乳杆菌培养体系的可行性,本文用核桃浆代替MRS培养基中氮源,研究了核桃浆的颗粒度、料液比、用量、脱脂处理以及摇床转速对鼠李糖乳杆菌的生长、发酵液抗菌活性和抗氧化活性的影响,并用所得菌体经富硒处理后饲喂小鼠3 d,监测小鼠在暴露于低压缺氧条件下（海拔5000 m）6 h后的血细胞和血红蛋白含量,评价其抗缺氧潜力。结果发现,采用料液比为1:10 (g/mL),经120目筛网过滤的去脂核桃浆完全替代MRS培养基中氮源（牛肉膏、蛋白胨）,可使培养基成本降低70%,使菌体产量提高2.5倍（从2.6×109至7.6×109 CFU/mL）,但不影响发酵液的抗菌活性和抗氧化活性（P>0.05）。连续口服富硒菌体3 d,可显著提高小鼠在低压缺氧条件下的血红蛋白含量（P<0.05）,优于MRS培养基所得菌体。综上,用核桃浆能够完全代替MRS培养基中氮源用于培养鼠李糖乳杆菌,并能有效提高菌体产量和活性。     

蛹虫草菌静息细胞培养方法及纤溶酶的诱导研究

以纤溶酶活力和生物量为评价指标,建立了蛹虫草菌静息细胞培养方法,并研究了酪蛋白肽对纤溶酶的诱导作用。 结果表明,静息细胞培养基中碳源和氮源确定为葡萄糖0.08%和尿素0.06%;培养条件为培养基初始pH 7.0,接种量10%,摇床转速180 r/min, 23 ℃培养12 h。 在此条件下,纤溶酶在血纤维蛋白平板上形成的溶圈面积为34.97 mm2,蛹虫草菌生物量为2.38 g/L。 酪蛋白肽组分C2 是合成纤溶酶的直接诱导物,当其添加量为0.1 g/L时,纤溶酶活力较对照提高了264%。     

Characteristics of alpha-glucosidase production from recombinant Aspergillus oryzae by membrane-surface liquid culture in comparison with various cultivation methods

alpha-Glucosidase was produced using recombinant Aspergillus oryzae by membrane-surface liquid culture (MSLC), a method previously developed by the authors and the results compared with other methods, including shaking flask culture (SFC), agar-plate culture (APC), culture on urethane sponge supports (USC), and liquid surface culture (LSC) to determine possible reasons for the advantageous features of MSLC. When yeast extract was used as a nitrogen source, the amount of enzyme produced by MSLC was 5 or more times higher than those for SFC and LSC, but similar to that using APC. Enzyme production in USC was slightly lower than in MSLC and APC. Cell growth was similar irrespective of the cultivation method used. When NaNO3, a typical inorganic nitrogen source was used, enzyme production in all the cultures was lower than that using yeast extract. However, even using NaNO3, the amount of the enzyme produced by MSLC was 8 to 20 times higher than those by SFC, APC, USC, and LSC. Although cell growth using NaNO3 was similar to that for yeast extract in MSLC, it was markedly decreased in SFC, APC, and LSC. The reason for the difference in enzyme productivity for various cultivation methods using yeast extract and NaNO3 as a nitrogen source is discussed, on the basis of the experimental findings. The role of the oxygen transfer effect and gene expression levels in enzyme production were also examined.     

Extracellular α-Amylase Production by Bacillus brevis MTCC 7521

Alpha amylases have various applications in food processing industries, for example, baking, brewing and distillery industries. Studies of the Ca²⁺ independent α-amylase production were carried out by a strain of Bacillus brevis MTCC 7521 isolated from a brick kiln soil. The optimum temperature, pH and incubation period for amylase production were 50°C, 6.0 and 36 h, respectively. The enzyme secretion was at par in the presence of any of the carbon sources (soluble starch, cassava starch and cassava flour). B. brevis produced more amylase in presence of beef extract as nitrogen source in comparison to other organic nitrogen sources (peptone, yeast extract and casein) and asparagine, potassium nitrate, ammonium sulphate, ammonium nitrate and urea reduced the enzyme activity. The addition of Ca²⁺ (10–40 mM) or surfactants (Tween 20, Tween 40, Tween 60, Tween 80, and sodium lauryl sulphate at 0.02% concentration) in culture medium did not result in further improvement in the enzyme production. The purified enzyme had a molecular mass of 205 kDa in native SDS-PAGE.     

一株产酯化酶细菌的筛选

从汾酒大曲（后火曲、红心曲、清茬曲）中筛选出1株产酯化酶较高的菌株,从菌落外形及显微镜观察初步确认是葡萄球菌。该菌株在液体发酵培养基中,以37℃恒温摇床培养48 h,发酵液酶活力可达25.00 U/mL;对该菌株的发酵培养基进行碳源和氮源优化。结果表明,以豆饼粉作氮源,玉米粉为碳源,于37℃、180 r/min下摇床培养48 h,酶活力可达到33.33 U/mL。