高产L-乳酸米根霉的原生质体制备与再生条件研究

为建立原生质体融合法选育高产L-乳酸米根霉(Rhizopus oryzae)菌株,对米根霉菌株原生质体制备及再生条件进行研究。考察酶种类、酶质量浓度、时间、温度、pH值、渗透压、预处理等因素对原生质体制得量及再生率的影响,获得原生质体制备及其再生的优良条件：不添加甘氨酸,渗透压稳定剂为0.6mol/L的NaCl缓冲液,按1.5mg/mL组合酶质量浓度,蜗牛酶、纤维素酶和溶壁酶质量浓度配比为1:1:1进行混合,35℃、pH6、酶解2h,可获得原生质体制得量最大为2.74×106个/mL,再生率最高为6.8%。     

1 株产酸性α-淀粉酶黑曲霉原生质体制备和再生条件优化

通过单因素试验、Plackett-Burman（PB）试验和响应面优化获得产酸性α-淀粉酶黑曲霉孢子原生质体制备和再生的最佳工艺参数：黑曲霉种龄72 h、孢子浓度7.5×106CFU/mL、渗透压稳定剂甘露醇浓度0.85 mol/L,以10 g/L蜗牛酶＋ 10 g/L 纤维素酶＋ 10 g/L 溶菌酶为破壁酶、pH 6.8、酶解温度37 ℃、酶解2 h。在此条件下,原生质体制备率可达86.92%,再生率可达42.67%。     

红曲霉M9x的原生质体形成与再生条件的研究

对红曲霉M9x用混合酶制取原生质体及原生质体再生条件进行了探索。其原生质形成和再生最佳条件为:以豆芽汁培养液为菌丝培养基,采用60h菌龄的菌丝体,以0.6mol/LMgSO4作为渗稳剂,用1.5%混合酶(蜗牛酶:纤维素酶=7:3)在30℃处理2h,原生质体形成数可以达到1.61×107个/ml,再生率为8.49%。     

啤酒酵母原生质体制备与再生条件的筛选

试验正交设计方法,对啤酒酵母原生质体制备与再生进行了研究。试验表明:以静置培养12h的细胞,0.6mol/L KCl作为渗透压稳定剂,经EDTA-Na2+预处理,在2%蜗牛酶作用下,32℃酶解90min为条件获得最佳制备率,制备率为99%以上;以静置培养14h的细胞,0.6mol/L蔗糖作为渗透压稳定剂,不经过预处理,在1.5%蜗牛酶作用下,30℃酶解120min,涂布于山梨醇为渗稳剂的再生培养基中为条件获得最佳再生率,再生率为65%。     

Cell fusion of Saccharomyces cerevisiae fragile mutants

Fragile mutants of Saccharomyces cerevisiae are defective in the structure of the cell wall and plasma membrane. The mutant cells lyse in hypotonic solutions but grow exponentially when osmotic stabilizer is included in the medium. These mutants display a general increase in the permeability of the plasma membrane. We show here that fragile yeast cells of the same mating type can fuse without protoplast formation. The frequency of cell x cell fusion is lower than that observed for protoplast x protoplast fusion and can be significantly increased if the cells of one partner are converted to protoplasts. Microscopic observations and genetic analysis demonstrate that the hybrids obtained are fusion products. The fusion between fragile cells is explained in terms of the existence of local defects on their surface where the cell wall is thinner (or even missing), thus allowing a direct contact of cells by means of their plasma membranes.     

阿魏菇原生质体制备及再生条件的建立

以新疆阿魏菇为材料,研究菌龄、酶解时间、酶解液对阿魏菇菌丝原生质体产量的影响,同时还研究了不同渗透压稳定剂对原生质体再生的影响。结果显示,在0.6 mol/L KCl稳渗剂条件下,菌龄为8 d阿魏菇菌丝体(0.1 g)在0.8 mL混合酶解液中(1.5%离析酶+0.5%纤维素酶+2%溶菌酶)制备原生质体,35℃酶解3 h获得原生质体达5×106个/mL。原生质体再生结果显示,在0.8 mol/L蔗糖稳渗剂下,原生质体再生时间为16 d,其再生率可达0.6%。该文通过对阿魏菇原生质体分离和再生条件的研究,旨在从细胞水平上探索一条食用菌育种新途径,为发展和完善食用菌新菌株选育提供理论依据和技术借鉴。     

桦褐孔菌原生质体制备与再生

研究桦褐孔菌原生质体制备与再生条件。采用正交试验法确定复合酶比例,用单因素试验测定不同等渗液、酶解时间、桦褐孔菌菌龄、酶解温度、再生培养基对桦褐孔菌原生质体制备与再生的影响。结果表明：混合酶(纤维素酶5mg/mL、蜗牛酶15mg/mL、溶壁酶15mg/mL、崩溃酶15mg/mL)、菌龄为7d的菌丝,采用0.6mol/L MgSO4为渗透压稳定液,酶解温度35℃,酶解时间5.5h,有利于原生质体的制备。就原生质体再生而言,酶解时间为3.5h制得的原生质体,以MgSO4作为渗透压稳定液,以完全再生培养基(CYM)和麦芽酵母葡萄糖再生培养基(GYM)有利于原生质体再生,再生率最高为0.13%。     

长枝木霉原生质体制备及再生条件研究

研究优化了长枝木霉（Trichoderma longibrachiatum）JK-15菌株菌丝体制备原生质体细胞及再生的条件。通过单因素试验和正交试验对影响原生质体形成和再生的菌龄、破壁酶组成、酶解反应温度及时间、渗透压稳定剂等因素进行了筛选优化。结果表明,长枝木霉菌株JK-15的最适条件为菌丝体菌龄18 h,混合酶组成配比为1.5%蜗牛酶∶1.5%纤维素酶∶0.1%溶壁酶,酶解条件为30 ℃条件下酶解2.5 h,原生质体制备过程中渗透压稳定剂为0.7 mol/L山梨醇,原生质体再生培养基中渗透压稳定剂为0.7 mol/L蔗糖,实验结果为长枝木霉JK-15的育种工作奠定了良好的技术基础。     

钝齿棒杆菌AS1.998原生质体形成与再生条件的研究

研究了影响L-异亮氨酸(L-Isoleucine)产生菌钝齿棒杆菌(Corynebacterium crenatum)AS1.998原生质体形成和再生的各个因素。结果表明:菌体培养至对数生长期,0.8 U/mL青霉素G处理3 h后,1.0mg/mL的蛋清溶菌酶37℃,150 r/min酶解15 h,原生质体形成率可达98.0%以上,在含8.5%蔗糖的再生完全培养基上,再生率可达20.0%～30.0%。     

灵芝原生质体诱变选育高产漆酶突变株的研究

对一株产漆酶活性较高的灵芝(Ganoderma.lucidumKarstNo.1)原生质体的制备、再生及紫外诱变育种进行了研究,获得了1株漆酶活性比对照菌株提高2.83倍的突变株G301。实验所确定的原生质体最佳制备条件为:以蔗糖为稳渗剂,采用4%(W/V)溶壁酶与2%(W/V)蜗牛酶等体积混合酶液,在30℃、pH6.0下,对液体培养48h的灵芝菌丝体酶解1h;同时实验所确定的原生质体诱变的适宜条件为:30W紫外灯距离40cm照射30s。     

冬虫夏草原生质体制备与再生条件的研究

采用正交分析方法,对影响冬虫夏草菌株原生质体制备与再生的主要因素进行研究。结果表明,最佳条件组合为培养3d 的菌丝体,在2.0% 溶壁酶+1.0% 蜗牛酶的酶液中,0.6mol/L 的KCl 作为原生质体制备的渗稳剂,0.6mol/L 的甘露醇作为再生培养基的渗稳剂,32℃酶解2h。在此条件下,原生质体得率可达到2.98 × 108/ml,再生率可达到42.09 × 10-2。     

双亲灭活米曲霉原生质体融合中原生质体制备的研究

对双亲灭活米曲霉原生质体融合育种中原生质体制备的条件进行了研究,将菌丝的预处理与细胞壁的酶解合为一步,并讨论了DTT的加入量。结果表明,原生质体制备的最佳破壁酶为纤维素酶、溶壁酶、蜗牛酶3种酶混合浓度比为5:3:1;菌丝培养15h;酶解时加入3mol/L DTT;酶解2.5h;0.8mol/L NaCl作为渗压稳定剂。所得双亲菌株原生质体的融合率为3.31%。     

Flow cytometric analysis of Saccharomyces cerevisiae autolytic mutants and protoplasts.

Simple methods, based on the technique of flow cytometry, have been developed for the phenotypic characterization of yeast autolytic mutants and for the analysis of the formation and regeneration of the yeast protoplasts. The expression of lytic mutations determined uptake of the fluorescent dye propidium iodide, which could be carefully monitored by flow cytometry. Mixed populations of lysed and viable cells were precisely quantified and sorted, and the technique was also applied to demonstrate protection from lysis of mutant cells with cell wall defects, in the presence of osmotic stabilizers. Protoplast formation and regeneration was monitored by analysing relative cell size; this was facilitated by the preparation of homogeneous protoplast preparations. The technique of flow cytometry proved superior to other conventional methods for these types of study.     

双歧杆菌原生质体的制备及其转化系统的建立

考察了长双歧杆菌WBL001菌体生长状态、酶解浓度、时长、温度以及不同渗透压稳定剂等因素,获得长双歧杆菌WBL001原生质体制备及再生的优化条件:长双歧杆菌以3%接种量活化至MRS培养基,培养至对数中期(OD600∶1),5μg/mL变溶菌素,37℃孵育30 min,以原生质体稳定液SMM为反应缓冲液,原生质体生成率达到81%,其再生率达到48%;在此基础上,通过聚乙二醇PEG融合,将穿梭表达载体pBAX转入双歧杆菌原生质体,成功建立双歧杆菌转化体系。     

米曲霉3.951菌株与RIB40菌株高产原生质体的制备及再生条件的研究

针对2株米曲霉进行原生质体制备条件的研究,比较了制备材料、菌龄、渗透压稳定剂、复合酶配比、酶解时间以及酶解温度对米曲霉原生质体形成和再生的影响。结果表明,2株米曲霉原生质体制备的最适宜条件稍有差异,其中米曲霉3.951菌株原生质体制备和再生的适宜条件是:以菌丝为制备材料,菌龄为14 h,渗透压稳定剂为0.8 mol/L NaCl,复合酶配比为1.0%纤维素酶、1.0%裂解酶、0.l%蜗牛酶,酶解时间为3 h,酶解温度为35℃;米曲霉RIB40的适宜条件是:以菌丝制备材料,菌龄为12 h,渗透要稳定剂为0.8 mol/L NaCl,复合酶配比是为1.0%纤维素酶、1.0%裂解酶、0.l%蜗牛酶,酶解时间为3 h,酶解温度是35℃。在优化条件下米曲霉3.951菌株释放的原生质体达6.43×107个/g,再生率可达23.5%,米曲霉RIB40释放的原生质体可达4.24×107个/g,再生率达22.41%。     

酱油曲霉孢子原生质体的制备与紫外诱变育种

由1株分离、纯化自曲精的酱油曲霉的分生孢子制备原生质体,并进行紫外诱变,得到7株中性蛋白酶活提高幅度较大的紫外突变株U系,中性蛋白酶活最高的1株达到6 197.4U,为出发株的1.94倍。对原生质体制备条件进行了优化:选取孢子生长旺盛的新鲜斜面培养物(培养5 d左右)制备孢子悬浮液;采用25 mmol/L-巯基乙醇+5 mmol/L Na2EDTA体系预处理20 min;酶解条件是:1%溶菌酶+1%蜗牛酶+1%纤维素酶,山梨醇作为渗透压稳定剂(0.6 mol/L,pH6.88),酶解温度33℃,酶解5 h;再生培养基为0.6 mol/L NaCl高渗透压豆汁培养基,涂布法再生。     

单亲灭活德氏乳杆菌和乳酸乳球菌原生质体融合条件优化

利用单亲灭活原生质体技术对德氏乳杆菌FQ菌株和乳酸乳球菌FL菌株的原生质体进行融合,考察原生质体制备、再生和融合条件的影响因素。结果表明:制备德氏乳杆菌FQ菌株原生质体最适条件为温度37℃,在含有10μg/mL变溶菌素和1mg/mL溶菌酶溶液中超声处理90min。在此条件下,原生质体再生率可达6.36%。乳酸乳球菌FL菌株添加1mg/mL甘氨酸处理后,用10mg/mL溶菌酶37℃恒温酶解90min,原生质体形成率可达99.97%。65℃处理乳酸乳球菌FL菌株原生质体120min,原生质体灭活率可达96.89%。融合实验结果表明,在PEG6000 400g/L(含0.02mol/L MgCl2和0.01mol/L CaCl2)、融合时间5min、融合温度20℃、pH6.5的条件下促融,德氏乳杆菌FQ菌株和乳酸乳球菌FL菌株原生质体的融合率可达2.72×10-6。     

原生质体紫外诱变选育L-半胱氨酸高活力转化菌株

对Pseudomonas sp.HJ-14的原生质体制备和再生条件进行了研究,并对其原生质体紫外辐照诱变选育L-半胱氨酸高活力转化菌株。在37℃、2 mg/mL溶菌酶作用下酶解60 min,其原生质体的形成率和再生率分别为78.6%和28.6%。经过紫外辐照诱变Pseudomonas sp.HJ-14原生质体,在含DL-2-氨基-△~2-噻唑啉- 4-羧酸(DL-ATC)再生平板上筛选抗性菌株,获得1株酶活较高的正突变株B-3,该菌株具有良好的遗传稳定性和酶活稳定性。采用5L罐进行产酶培养,并在pH8.0、DL-ATC·3H_2O浓度为10 g/L、42℃酶促反应9 h,L-半胱氨酸最高产量达4.63 g/L,摩尔转化率为76.5%,较HJ-14提高了117.7%。     

Mass transfer kinetics during osmotic dehydration of pomegranate arils

The mass transfer kinetics during osmotic dehydration of pomegranate arils in osmotic solution of sucrose was studied to increase palatability and shelf life of arils. The freezing of the whole pomegranate at -18 °C was carried out prior to osmotic dehydration to increase the permeability of the outer cellular layer of the arils. The osmotic solution concentrations used were 40, 50, 60°Bx, osmotic solution temperatures were 35, 45, 55 °C. The fruit to solution ratio was kept 1:4 (w/w) during all the experiments and the process duration varied from 0 to 240 min. Azuara model and Peleg model were the best fitted as compared to other models for water loss and solute gain of pomegranate arils, respectively. Generalized Exponential Model had an excellent fit for water loss ratio and solute gain ratio of pomegranate arils. Effective moisture diffusivity of water as well as solute was estimated using the analytical solution of Fick's law of diffusion. For above conditions of osmotic dehydration, average effective diffusivity of water loss and solute gain varied from 2.718 × 10(-10) to 5.124 × 10(-10) m(2)/s and 1.471 × 10(-10) to 5.147 × 10(-10) m(2)/s, respectively. The final product was successfully utilized in some nutritional formulations such as ice cream and bakery products.     

Power generation using different cation, anion, and ultrafiltration membranes in microbial fuel cells

Proton exchange membranes (PEMs) are often used in microbial fuel cells (MFCs) to separate the liquid in the anode and cathode chambers while allowing protons to pass between the chambers. However, negatively or positively charged species present at high concentrations in the medium can also be used to maintain charge balance during power generation. An anion exchange membrane (AEM) produced the largest power density (up to 610 mW/m2) and Coulombic efficiency (72%) in MFCs relative to values achieved with a commonly used PEM (Nafion), a cation exchange membrane (CEM), or three different ultrafiltration (UF) membranes with molecular weight cut offs of 0.5K, 1K, and 3K Daltons in different types of MFCs. The increased performance of the AEM was due to proton charge-transfer facilitated by phosphate anions and low internal resistance. The type of membrane affected maximum power densities in two-chamber, air-cathode cube MFCs (C-MFCs) with low internal resistance (84-91 omega for all membranes except UF-0.5K) but not in two-chamber aqueous-cathode bottle MFCs (B-MFCs) due to their higher internal resistances (1230-1272 omega except UF-0.5K). The UF-0.5K membrane produced very high internal resistances (6009 omega, B-MFC; 1814omega, C-MFC) and was the least permeable to both oxygen (mass transfer coefficient of k(O) = 0.19 x 10(-4) cm/s) and acetate (k(A) = 0.89 x 10(-8) cm/s). Nafion was the most permeable membrane to oxygen (k(O) = 1.3 x 10(-4) cm/s), and the UF-3K membrane was the most permeable to acetate (k(A) = 7.2 x 10(-8) cm/s). Only a small percent of substrate was unaccounted for based on measured Coulombic efficiencies and estimates of biomass production and substrate losses using Nafion, CEM, and AEM membranes (4-8%), while a substantial portion of substrate was lost to unidentified processes for the UF membranes (40-89%). These results show that many types of membranes can be used in two-chambered MFCs, even membranes that transfer negatively charged species.