Ultrafiltration for Recovery and Reuse of Peptidoglutaminase in Protein Deamidation

Ultrafiltration (UF) of B. circulans cell extract using a 100kd hollow-fiber membrane had no effect on peptidoglutaminase (PGase) activity and allowed 34 and 26% of PGases I and II to permeate. UF, with 30kd spiral membrane, slightly increased PGase activity in retentate but no activity was detected in permeate. Retentate was used to sequentially deamidate four batches of a soy protein hydrolysate at 30°C for 2 hr, then PGase was recovered by UF. The 100kd membrane resulted in substantial PGase loss in sequential tests due to permeation and inactivation. A 100% conversion and 97% of PGase were obtained after tests with the 30 kd membrane. Multiple recovery and use of PGase by a suitable membrane is possible.     

微生物原果胶酶高产菌株的筛选及发酵特性的研究

从24种不同来源的样品中筛选得到一株编号为XZ3的原果胶酶高产菌株，其酶活达126．4U／ml。通过对菌落形态及孢子囊形态的观察，初步鉴定是曲霉属菌株。在不同发酵时间内测定发酵液中还原糖、pH、湿菌体重、PPase及PGase活性。结果表明：该菌株发酵过程中，发酵液中的还原糖含量在菌体生长初期升高很快，在对数生长期又迅速下降；发酵液的pH保持恒定：菌体产酶与菌体生长几乎同步且PPase与PGase活性的变化规律一致，比值在一定范围内保持恒定。     

红曲霉中PKS基因的结构分析

对实验室保藏的一株高产Monacolin K红曲霉菌株及其野生株进行了初步的PKS基因结构分析。扩增得到红曲霉野生株W菌及其诱变株M菌的目的基因序列,并将测序产物和cDNA序列进行比对,确定该段基因序列不含内含子,且诱变株M菌的突变位置不在此段基因上。分别用ProfileScan、SWISS-MODEL预测了该段蛋白的结构域以及三维结构。     

Papaya Polygalacturonase and its Role in Thermally Injured Ripening Fruit

The polygalacturonase (E.C. 3.2.1.15) (PGase) activity in papayas increased with fruit ripeness. PGase activity was highest in the placenta with the activity decreasing outwardly from the placenta to the exocarp. Extended hot water treatments (46°C for 65 and 90 min) caused delayed softening of the fruit tissue which was correlated to a decrease in PGase activity. The decrease in PGase activity after the heat treatments was more severe in the riper fruits (quarter and half ripe) than the less ripe fruits (colorbreak stage).     

Membrane Reactor for Enzymic Deamidation of Food Proteins

A soy protein hydrolysate was deamidated with peptidoglutaminase (PGase) retained within a 30kd spiral membrane. Reactor was operated at 30°C and 6.5 L/m²/hr flux in recycle mode to 60% conversion, then in diafiltration mode for 2 hr. Time course, predicted by a Michaelis-Menten equation integrated for mixed zero- and first-order kinetics with the corrections for the ultrafiltration (UF) interactions, matched that measured experimentally. The equation could be used to predict the potential activity and performance of PGase in UF reactors to achieve control of reactions for the optimal enzymic process. At the end of the run, 96% conversion and 99% of PGase were obtained. This method has the potential of being scaled-up for the production of enzyme-free deamidated proteins.     

M-31 mutant (virA::Tn5) of Agrobacterium tumefaciens is capable of transferring its T-DNA into the nucleus of host cell, but incapable of integrating it into the chromosome

An avirulent mutant (M-31 strain) was produced by the transposon (Tn5) mutagenesis of Agrobacterium tumefaciens (A-208 strain). A binary vector, pIG121-Hm, containing a kanamycin resistance gene (nptII) and beta-glucuronidase (GUS) gene with an intron, was introduced into M-31 and A-208 strains. The resultant Agrobacteria were inoculated onto leaves of Kalanchoe daigremontiana and to tobacco BY-2 cells to assay GUS activity to monitor the T-DNA transfer into the nuclei of host cells. The results indicated that T-DNA was transferred into the nuclei of cells of both host plants inoculated with the M-31 mutant. The M-31 mutant strain had an insertion of Tn5 in the virA gene on its Ti plasmid. The introduction of the virA gene in the M-31 mutant complemented its avirulent phenotype. No kanamycin-resistant cells were observed when the M-31 mutant harboring the pIG121-Hm was inoculated to tobacco BY-2 cells. The M-31 mutant (virA::Tn5) seems to transfer T-DNA into the nucleus of the host cell, but is unable to integrate it to the chromosome.     

干酪乳杆菌yhaI基因敲除菌株的构建及其耐药表型的分析

为了深入研究干酪乳杆菌Zhang在抗生素环境中适应性进化机制,构建yhaI基因敲除体系,并对突变体的耐药表型进行研究。运用PCR技术分别扩增yhaI基因的同源臂序列,构建带有内部缺失yhaI基因的敲除载体。采用Cre/lox基因重组系统,筛选双交换子L.casei Zhang-G-1200-yhaI::lox66-P32-cat-lox71,构建突变株L.casei Zhang-G-1200-Che yhaChe I,通过稀释法研究突变株的耐药表型。通过PCR和PCR产物经测序验证,yhaI基因缺陷的菌株构建成功。耐药表型结果表明,突变株在庆大霉素浓度为8和16 μg/mL时,浊度变为原始菌株的1/2;在庆大霉素浓度为16 μg/mL时,活菌数变为原始菌株的7/10。成功构建干酪乳杆菌Zhang的基因敲除体系,为研究干酪乳杆菌Zhang的基因功能提供了技术平台。     

luxS基因对盐胁迫下植物乳杆菌生长及细菌素合成的影响

以植物乳杆菌KLDS1.0391野生株及其luxS基因缺失突变株为研究对象,探究luxS基因对该菌盐耐受能力的影响,并测定在0%、2%、3%、4%和6%质量分数NaCl胁迫条件下,luxS基因缺失对该菌生长及细菌素合成的影响,采用实时荧光定量聚合酶链式反应（polymerase chain reaction,PCR）技术在转录水平上测定该菌细菌素合成相关基因的表达水平。结果表明：随NaCl质量分数的升高,2 株菌的存活率均逐渐下降,当NaCl质量分数大于3%时,相同条件下野生株对盐的耐受能力显著强于突变株（P＜0.05）;除此之外,NaCl质量分数越高,菌株进入稳定期的时间越向后延迟,luxS基因缺失突变株在各NaCl质量分数下生长及细菌素合成能力均显著弱于野生株（P＜0.05）。实时荧光定量PCR结果显示,当培养至稳定期24 h时,细菌素结构基因plnEF以及双组分调节基因plnD和plNC8HK均因luxS基因的缺失,表达显著下调（P＜0.05）。因此,luxS基因缺失显著降低植物乳杆菌KLDS1.0391盐耐受能力,并且在NaCl胁迫条件下,该菌生长及细菌素合成能力也因luxS基因缺失而显著下降。     

Purification and Characterization of a Novel Protopectin-Solubilizing Enzyme from Aspergillus niger Z-25

A mutant, Aspergillus niger Z-25 derived from A. niger XZ-131 with N⁺ supplementation, produced a protopectin-solubilizing enzyme (protopectinase). The enzyme was purified to homogeneity by a procedure involving ammonium sulfate fractionation and gel chromatographies on DEAE-Sephadex A-50 and Sephadex G-100 columns. The molecular weight of the protopectinase was estimated at 68.4 KD by SDS-polyacrylamide gel electrophoresis. The enzyme was stable from pH 3.0 to 7.0 and up to 60°C. The optimum pH and temperature for enzyme activity was 4.0 and 50°C, respectively. The purified enzyme had 268-U/mL PPase (protopectinase) activity on citrus protopectin, and also showed 100-U/mL PGase (polygalacturonase) activity, which catalyzed the hydrolysis of polygalacturonic acid. The Km value for protopectin was 0.215 mg/mL, while the Km value for polygalacturonic acid was 39.09 mg/ml. The PPase/PGase q-value was 2.68, more than 0.5. Therefore the enzyme was considered a novel pectinase and classified as protopectinase.     

利用同源重组技术调节啤酒中SO2含量研究

二氧化硫在啤酒中具有抗氧化的重要功能,亚硫酸盐还原酶(MET10编码)在啤酒酵母硫代谢过程中起重要作用。利用同源重组技术,采用醋酸锂转化法,将一段目的基因转入到酵母体内,从而获得一株亚硫酸盐还原酶基因突变的工业酿酒酵母。突变株通过驯养,对比发酵栓试验,结果表明在发酵结束时,突变菌株的SO2产量是出发菌株的1.5倍。     

单核细胞性李斯特菌基因dal的敲除及其生物学特性初步分析

在单核细胞性李斯特菌(Listeria monocytogenes)野生株EGDe act A及inl B双基因缺失株(EGDeΔact AΔinl B)的基础上,利用同源重组的方法进一步构建了缺失营养基因dal的菌株(EGDe Δact AΔinl BΔdal),并对该缺失菌株生长状态、毒力基因表达水平、生物被膜的形成量及细胞侵袭等方面作进一步分析。结果显示,37℃摇床培养6 h后,缺失株的菌浓度显著低于EGDe Δact AΔinl B(P0.001),培养基中补充D-丙氨酸的缺失株生长速率与亲本株相比无显著差异;实时荧光定量聚合酶链式反应结果显示,缺失株的sig B基因表达水平变化最明显(P0.01),约下调90%;缺失株生物被膜形成量显著增加(P0.05),培养基补充D-丙氨酸后缺失株生物被膜的生成量与亲本株相比无差异;对Coca-2细胞的侵袭无影响,表明该基因对细菌生长能力及生物被膜形成具有重要的调控作用,并不影响菌株对细胞的侵袭力。此缺失株的构建为进一步研究基因dal的功能提供了理论支持。     

氮离子注入选育高自溶干酪乳杆菌及其肽聚糖水解酶的RT-qPCR分析

通过采用低能N^+注入对干酪乳杆菌116-a进行诱变,以筛选得到高自溶的干酪乳酸菌突变体,并采用实时荧光定量PCR检测N-乙酰胞壁质酶和N-乙酰氨基葡糖胺糖苷酶在野生菌体和突变体中的表达差异。结果显示:经过离子能量30 keV N^+注入诱变,在注入剂量0.5×10^15 ions/cm^2时,筛选得到遗传稳定性良好的最大正突变菌株,其自溶度为56.3%,最大正突变菌株自溶度比野生型菌株的自溶度提高了25.7%。实时荧光定量PCR检测发现,N-乙酰氨基葡糖苷酶和N-乙酰胞壁质酶在突变体中的表达量比野生菌体中的表达量均有所上调,其中N-乙酰氨基葡糖苷酶表达量上调了约55倍,而N-乙酰胞壁质酶的表达量仅上调2.8倍。因此,N-乙酰氨基葡糖苷酶在干酪乳杆菌116-a自溶中起着重要作用,该酶表达量的上调可以有效提高菌体的自溶。     

Effects of high hydrostatic pressure on the growth and beta-carotene production of Rhodotorula glutinis

The effects of high hydrostatic pressure (HHP) on the biomass and beta-carotene biosynthesis of Rhodotorula glutinis R68 were studied. After treatment with five repeated cycles at 300 MPa for 15 min, the barotolerant mutant PR68 was obtained. After 72 h of culture, the biomass of mutant PR68 was 21.6 g/l, decreased by 8.5% compared to the parental strain R68, but its beta-carotene production reached 19.4 mg/l, increased by 52.8% compared to the parental strain R68. The result of restriction fragment length polymorphism (RFLP) analysis suggested that mutant strain PR68 was likely to change in nucleic acid level, and thus enhanced beta-carotene production in this strain as a result of gene mutation induced by HHP treatment.     

单增李斯特菌srtA基因敲除菌株的构建及其生物学特性

单核细胞增生性李斯特菌是常见的食源性致病菌,其细胞壁上存在的表面蛋白与其致病性和细菌菌膜的形成密切相关,而srt A基因是介导表面蛋白膜表面定位的关键因子,通过识别特定的蛋白序列将表面蛋白共价结合在细胞壁上,也是单增李斯特菌的重要毒力基因。为深入研究srtA的功能及其对细菌毒力的调控,本研究通过同源重组技术敲除了单增李斯特菌中的srt A基因,并对基因敲除菌株的生物学特性进行了初步研究。通过生长曲线的测定,发现srtA基因敲除株的生长活性低于野生型菌株。进一步利用该菌株对星形胶质细胞系U251的侵袭实验发现,srtA基因敲除菌株的侵袭效率低于野生菌株,提示srtA基因在调控单增李斯特菌的侵袭能力方面发挥重要作用。本研究可为单增李斯特菌毒力基因调控和细菌菌膜的研究提供理论依据。     

碳源对manA基因突变大肠杆菌代谢的影响

ManA基因编码的甘露糖-6-磷酸异构酶在大肠杆菌中催化D-甘露糖和D-果糖的异构化,促进大肠杆菌对碳源的代谢吸收。本文通过研究manA基因突变大肠杆菌对碳源的利用和编码糖代谢基因情况,探讨甘露糖-6-磷酸异构酶对大肠杆菌糖代谢的影响。采用Ⅱ型内含子逆转录突变方法构建manA基因突变大肠杆菌,分析manA基因突变大肠杆菌对不同碳源的利用情况和manA基因突变对大肠杆菌糖代谢相关基因表达的影响,结果显示,大肠杆菌BL21(DE3)ΔmanA以甘露糖、果糖为碳源时,菌株生长受到显著抑制;以淀粉为碳源时,BL21(DE3)ΔmanA菌株的生长显著优于野生型大肠杆菌;以葡萄糖为碳源时,manA基因突变对大肠杆菌的生长无显著影响。通过基因表达分析,发现大肠杆菌BL21(DE3)ΔmanA中甘露糖代谢相关基因的表达显著性降低;果糖代谢途径中6-磷酸果糖激酶Ⅰ亚基的编码基因(pfkA)显著下调表达;水解淀粉的α-淀粉酶编码基因(malS)显著性上调表达。ManA基因突变影响大肠杆菌甘露糖、果糖和淀粉代谢途径中相关基因的表达,从而影响大肠杆菌对碳源的利用。     

大肠杆菌转录调控因子oxyR与抗氧化基因ahpCF、katEG的协同作用

目的探讨大肠杆菌转录调控因子oxyR与抗氧化基因ahpCF、katEG在调控内源过氧化氢(H_2O_2)水平及生长繁殖中的作用。方法荧光法检测大肠杆菌内源H_2O_2,采用以氯化铈做特异染色剂的透射电子显微镜法和AR做染料的荧光分光光度法检测内源H_2O_2的数量变化。结果突变体菌株JI374中ahpCF基因的缺失导致内源H_2O_2积累减少,其生长速度显著提高。与此相反的是,katEG基因缺失的突变体菌株JI372和ahpCF、oxyR基因双突变菌株LC74中,内源H_2O_2水平上升,抑制了细菌的生长速率。结论 oxyR调控下的抗氧化体系相关基因不仅能够调控细菌内源H_2O_2水平,ahpCF、katEG与转录调控因子oxyR之间具有协同作用,共同调控细菌的生长代谢与繁殖。同时,在ahp基因缺失条件下,kat基因起主导作用;在oxyR、ahpCF、katEG基因的协同作用中,oxyR应起决定性的作用。     

Mutagenizing brewing yeast strain for improving fermentation property of beer

A brewing yeast mutant with perfect sugar fermentation capacity was isolated by mutagenizing the Saccharomyces pastorianus transformant, which carries an integrated glucoamylase gene and has one copy of non-functional alpha-acetolactate synthase gene. The mutant was able to utilize maltotriose efficiently, and the maltotriose fermentability in YNB-2% maltotriose medium increased from 32.4% to 72.0% after 5 d in shaking culture. The wort fermentation test confirmed that the sugar fermentation property of the mutant was greatly improved, while its brewing performances were analogous to that of the wild-type strain and the characteristic trait of shortened beer maturation period was retained. Therefore, we believe that the brewing yeast mutant would benefit the beer industry and would be useful for low caloric beer production.     

脱酰胺对热诱导小麦面筋蛋白构象及凝胶性质的影响

利用蛋白质谷氨酰胺酶(PGase)对小麦面筋蛋白进行处理,研究PGase脱酰胺及热诱导对小麦面筋蛋白构象及其凝胶性质的影响。结果表明,PGase脱酰胺能够在一定程度上抑制小麦面筋蛋白的热诱导聚集行为,提高小麦面筋蛋白热诱导凝胶的强度和保水性。当PGase添加量为3 U/g时,其凝胶强度和保水性均达到最大值,分别为903. 27 N和78. 70%。研究还表明,PGase处理后小麦面筋蛋白与水相互作用增加,从而提高了脱酰胺小麦面筋蛋白热诱导凝胶中非可冻结水含量。扫描电镜结果显示PGase脱酰胺结合热诱导处理的小麦面筋蛋白形成了更加有序的凝胶网络结构。     

米曲霉RIB40高效同源重组和尿苷/尿嘧啶营养缺陷型菌株的构建

目的:以米曲霉RIB40为出发菌株，构建具有高同源重组效率的营养缺陷型菌株。方法:首先通过同源重组技术和5-氟乳清酸（5-Fluoroorotic Acid，5-FOA）对尿苷/尿嘧啶营养缺陷的筛选作用，获得AopyrG基因缺失的米曲霉RIB40。然后利用烟曲霉AfpyrG基因替换Aoku70得到?Aoku70敲除菌株，再通过5-FOA的筛选作用使得?Aoku70菌株基因组发生自身环化，丢失AfpyrG基因。最后为了检验?Aoku70?AopyrG菌株的可用性，将编码红色荧光蛋白的mCherry基因敲入至双突变菌株的组蛋白H2B基因上，并通过激光共聚焦显微镜观察红色荧光蛋白质的亚细胞定位。结果:通过AopyrG和Aoku70的双基因敲除，获得了高同源重组效率的营养缺陷型菌株?Aoku70?AopyrG。用激光共聚焦观察到红色荧光蛋白定位于细胞核，表明?Aoku70?AopyrG菌株可用于米曲霉的基因改造。结论:?Aoku70?AopyrG菌株可作为一个高同源重组效率的营养缺陷型出发菌株用于今后米曲霉RIB40的遗传改良。     

ATF1过表达和BAT2敲除酿酒酵母发酵性能的研究

以啤酒酵母工业菌株S5为对照,对过表达醇乙酰基转移酶编码基因ATF1同时敲除氨基酸转氨酶编码基因BAT2酿酒酵母工程菌株S5-1进行发酵性能的研究。结果表明,与出发菌株相比,突变株生长状况、发酵速度、酒精度等基本发酵性能没有明显变化,而突变株发酵后的乙酸酯总量有较大程度的提升,为85.44mg/L,提高了6.96倍,高级醇总量有较大程度的下降,为79.25mg/L,降低了27.28%。研究结果为啤酒风味的改善奠定了良好的基础。