苯丙氨酸脱氨酶发酵工艺及其酶学性质研究

选育出一株具有较高苯丙氨酸脱氨酶活性的菌株巨大芽孢杆菌AS1.127-NJU10。考察了该菌的发酵产酶条件,结果表明:蔗糖为最佳碳源、酵母浸膏和NH4C l组成最佳氮源,其质量浓度分别为:ρ(蔗糖)=20 g/L,ρ(酵母浸膏)=2 g/L和ρ(NH4C l)=10 g/L;发酵培养基最适pH=6.5,培养温度为37℃;诱导物ρ(L-苯丙氨酸)=1 g/L时,酶活最高达1 070 U。同时对苯丙氨酸脱氨酶的性质进行了研究,结果表明:该酶最适pH=5.8,最适温度为40℃,反应液中添加φ(吐温-80)=0.2%和c(K+)=10-5mol/L能明显提高酶活。     

海因酶法制备D-苯丙氨酸的酶催化过程动力学

采用自行筛选的兼有海因酶和N-氨甲酰氨基酸水解酶活性的Burkholderia cepecia 1003菌种,利用海因酶法大规模制备D-苯丙氨酸,对其中涉及的各过程的动力学参数进行了测定.结果表明:L-苄基海因的消旋速率常数为3.975×10^-3min^-1;海因酶的米氏常数为16.7894 mmol•L^-1,最大反应速率为0.6127 mmol•L^-1•min^-1;N-氨甲酰氨基酸水解酶的米氏常数为0.82688 mmol•L^-1,最大反应速率为4.828×10^-4 mmol•L^-1•min^-1.对DL-5-苄基海因的溶解、L-苄基海因的消旋、D-海因的水解开环及其中间产物（N-氨甲酰苯丙氨酸）的水解脱酰氨过程建立了动力学模型,并在此基础上进行了动力学参数显著性分析和优化.结果表明：对于这一级联酶转化反应,D-海因酶的水解反应是快速反应,而N-氨甲酰氨基酸脱氨甲酰的反应速率极小,是该过程的控制步骤.提高氨甲酰水解酶的活力将有助于提高总体的转化速率,而L-海因的消旋速率则是影响外消旋苄基海因转化率的主要因素.     

酶法转化D,L-苄基海因制备N-氨甲酰-D-苯丙氨酸

研究了间歇酶转化和间歇补料酶转化制备N-氨甲酰 -D-苯丙氨酸的过程 ,发现在D ,L-苄基海因 (D ,L-BH)的投入量为 2 0g/L、菌泥与D ,L-BH的质量比为 1∶1的间歇酶转化过程中 ,产物的得率接近 1 0 0 % ,而在初始D ,L-BH的投入量为2 0g/L和菌泥与D ,L-BH之比为 1∶1的间歇补料酶转化过程中 ,则发现在补料的过程中其转化率一直维持在 60 %～ 70 % ,直到底物质量浓度达 50g/L。最终菌泥与D ,L-BH的实际质量比达到了 1∶2 .5 ,提高了酶的利用率。     

巨大芽孢杆菌产青霉素酰化酶培养基的优化

通过正交实验考察了苯乙酸、酶水解酪蛋白和溶氧对产青霉素G酰化酶的影响,实验中苯乙酸作为诱导剂和碳源起作用,其消耗与溶氧具有交互作用,正交实验表采用L9（3^4）。结果表明,苯乙酸含量与溶氧浓度是最主要的因素,最优发酵培养基组成为：酶水解酪蛋白含量为10 g/L,苯乙酸含量为20 g/L,MgCl2·6H2O含量为0.5 g/L,FeCl3·6H2O含量为0.002 g/L,250 mL三角瓶装液量为30 mL。     

海因消旋酶用于制备D-苯丙氨酸的工艺研究

研究酶法制备D-苯丙氨酸的合成工艺,在原有苄基海因水解工艺的基础上增加了海因消旋酶的投入,在转化率相同的情况下,反应温度可以下降25℃。通过优化工艺条件,确定了海因消旋酶的最佳反应温度。通过对比,增加海因消旋酶,可以极大地降低转化温度,有助于海因酶酶活的稳定,也有利于降低生产的能耗。该方法绿色环保,对环境污染小,适合工业化生产。     

化学酶法制备D-丝氨酸

以DL-丝氨酸为原料,先采用氨基酸关环法化学合成酶的作用底物———DL-5-羟甲基海因,收率93.5%。然后,该底物在酶的作用下,起始pH=9.0,培养温度38℃,转化时间42 h,经过一系列的酶解反应即得到D-丝氨酸,收率为81.7%。     

青霉素酰化酶法制备D-丙氨酸

报道了青霉素酰化酶法制备D-丙氨酸的研究结果,为D-丙氨酸的制备提供了一种方法。考察了pH、温度、DL-丙氨酸与苯乙酰氯的摩尔比、反应时间对制备结果的影响。确定了制备N-苯乙酰-DL-丙氨酸的最佳反应条件:pH=9.5,n(DL-丙氨酸)∶n(苯乙酰氯)=1.1∶1;酶促反应最佳条件为:pH=7.5,温度37℃条件下反应6h,其产物为N-苯乙酰-D-丙氨酸和苯乙酸的混合物。该混合物用c(HC l)=6 mol/L的盐酸高温回流6 h得D-丙氨酸盐酸盐,再经717阴离子树脂处理得D-丙氨酸。光学纯度为95.3%,总收率为72.5%。     

巨大芽孢杆菌(ACCC10011)制备煤矸石肥料的研究

针对全国煤矸石利用率较低,堆积的大量煤矸石污染环境,以贵州煤矸石为例,掺杂磷矿并利用微生物降解法制作肥料,为煤矸石的利用寻找一条可行途径.采用巨大芽孢杆菌(ACCC10011)处理煤矸石与磷矿掺杂物,研究了接菌量、体系pH、两矿比例、培养时间及体系摇动对肥料的影响.研究发现:当接菌量为6 × 1014～ 1.36×1015个/g,pH为6.5,煤矸石与磷矿配比为1∶1,培养3d,目数为120目条件下,制作的肥料中有效磷占全磷的比例由5.65％提高至70.9％,有效硅占全硅的比例由0.039 0％提高至61.5％.     

L-苯丙氨酸甲酯消旋反应改进

对L-苯丙氨酸甲酯消旋方法进行改进。研究了L-苯丙氨酸甲酯在不同催化剂、催化剂用量、反应时间下的消旋制备DL-苯丙氨酸甲酯过程。结果表明:当以水杨醛为催化剂、强酸性D072离子交换树脂为助催化剂、n(水杨醛)∶n(L-苯丙氨酸甲酯)=0.1回流反应时,消旋效果最好。     

酶法转化体系中L-苯丙氨酸的络合萃取

L-苯丙氨酸的分离提取对于促进酶法制备工艺的产业化进程具有重要价值。利用络合萃取分配系数、分离选择性高的特点,对苯丙酮酸酶法制备L-苯丙氨酸转化体系中L-苯丙氨酸的络合萃取进行了研究。以二(2-乙基己基)磷酸为萃取剂,研究了水相平衡pH、萃取剂浓度和稀释剂的种类对分配系数的影响;并利用D2EHPA-正辛醇体系对酶法制备L-苯丙氨酸的转化液进行了络合萃取研究,经4次萃取和1次反萃,L-苯丙氨酸的萃取率达到98 15%;经1次反萃,L-苯丙氨酸的收率达94 33%。     

VC发酵大菌不同组分对小菌生长及产酸的影响

在VC的二步混菌发酵中,作为伴生菌,巨大芽孢杆菌(大菌)对酮古龙酸杆菌(小菌)的生长和产酸有促进作用.通过比较大菌培养液不同分子量的组分对小菌的促进作用,发现分子量小于1 000的组分对小菌的促进作用最明显.     

枯草芽孢杆菌AS1-296产絮凝剂条件的优化

探讨了不同碳源、氮源、培养时间、温度、初始pH和谷氨酸钠添加量对枯草芽孢杆菌AS1-296产絮凝剂条件的研究,并通过数据拟合,创建了相应的数学模型,通过求解得出最佳的培养条件。枯草芽孢杆菌AS1-296产絮凝剂的最佳培养条件为:以蔗糖为碳源,添加量为2.25%;酵母膏为氮源,添加量为0.64%;谷氨酸钠4.50%、初始pH7.1、培养温度33℃。以此条件培养枯草芽孢杆菌AS1-296产絮凝剂的絮凝率为75%以上。     

Hydroliquefaction of Sulcis low-rank coal. 1. Conversion to low-sulphur fuel oil

A low-rank Italian coal was subjected to high-pressure and high-temperature hydroliquefaction with and without a hydrogenation catalyst. A low-sulphur oil was produced, showing properties similar to those of a standard fuel oil. The hydroliquefaction product may be used directly either as a fuel or as raw material for petrochemical processes. Sulcis coal displayed particular reactivity towards hydrogen, as shown also by comparison with a widely used North American coal.     

用酶组合技术制备水解明胶的研究

本文研究了ASI.398中性蛋白酶和菠萝蛋白酶法制备水解明胶,初步探讨了用酶的组合技术制备小分子量及窄分布水解明胶的可能性。结果表明,酶的不同组合方式是调节产物分子量及分布的极有效手段,菠萝蛋白酶和AS1.398中性蛋白酶组合的先后次序,可以调节产物的分子量,两者以混合方式组合,则可以调节产物的分子量分布。     

Submerged vs. Solid-State Conversion of Soybean Meal into a High Protein Feed Using Aureobasidium pullulans

The goal of this study was to assess the performance of Aureobasidium pullulans in converting soybean meal (SBM) into high protein feed using the submerged and solid-state processes. High solid loading rates (SLR) were evaluated for each process, i.e., 10–25% for submerged and 35–70% for static solid-state fermentation. During the submerged fermentation, 10% SLR was considered the best performer due to the high amount of cell density, low residual carbohydrates, and high protein titers, while 40% SLR resulted in the high protein yields and low residual carbohydrates during the static solid-state fermentation. The solid-state fermentation was conducted in a 14-L paddle-type reactor at 50% SLR, and periodic mixing resulted in a protein titer of ~58% at 72 hours of fermentation. Overall, results showed the feasibility of scaling up these processes in converting SBM to a high protein feed ingredient for animal diet.     

Electrochemical Molecular Conversion of α-Keto Acid to Amino Acid at a Low Overpotential Using a Nanoporous Gold Catalyst

A nanoporous gold (NPG) electrode prepared through a facile anodization technique was employed in the electrochemical reductive amination of biomass-derivable α-keto acids in the presence of a nitrogen source to produce the corresponding amino acids. NPG showed a clear reductive current in the presence of α-keto acid and NH₂OH, and the electrolysis experiments confirmed the production of L-amino acid. A reductive voltammetric signal at the NPG electrode appeared at a more positive potential by 0.18–0.79 V, compared with those at the planar-gold electrode without anodization and other previously reported electrode systems, indicating the high activity of the prepared nanostructure for the electrochemical reaction. Maximum Faradaic efficiencies (FEs) of 74–93% in the reductive molecular conversion to amino acids of Ala, Asp, Glu, Gly, and Leu were obtained under the optimized conditions. The FE values were strongly dependent on the applied potential in the electrolysis, suggesting that the hydrogen evolution reaction at the electrode surface was more significant as the applied potential became more negative. The effect of potential at the NPG was lower than that at the planar-gold electrode. These results indicate that nanostructurization decreases the overpotential for the electrochemical reductive amination, resulting in high FE.     

Olefin as an Intermediate in n-Butane Isomerization on Sulfated Zirconia. An In Situ 13C MAS NMR Study of n-Octene-1 Conversion

By using in situ ¹³C MAS NMR and ex situ GC-MS, the analysis of hydrocarbon products formed from n-octene-1 adsorbed on sulfated zirconia catalyst (SZ) has been performed. It is shown that a mixture of alkanes and stable alkyl substituted cyclopentenyl cations (CPC) is formed as the basic reaction products. Formation of both alkanes and CPC from n-octene-1, a precursor of C₈ ⁺ cation, the key intermediate in n-butane isomerization via a bimolecular pathway, implies that formation of the isomerized alkane occurs by a complex process of conjunct polymerization, rather than isomerization itself. CPC deposited on the SZ surface can be in charge of the catalyst deactivation.     

Unravelling the Specificity of Laminaribiose Phosphorylase from Paenibacillus sp. YM-1 towards Donor Substrates Glucose/Mannose 1-Phosphate by Using X-ray Crystallography and Saturation Transfer Difference NMR Spectroscopy

Glycoside phosphorylases (GPs) carry out a reversible phosphorolysis of carbohydrates into oligosaccharide acceptors and the corresponding sugar 1-phosphates. The reversibility of the reaction enables the use of GPs as biocatalysts for carbohydrate synthesis. Glycosyl hydrolase family 94 (GH94), which only comprises GPs, is one of the most studied GP families that have been used as biocatalysts for carbohydrate synthesis, in academic research and in industrial production. Understanding the mechanism of GH94 enzymes is a crucial step towards enzyme engineering to improve and expand the applications of these enzymes in synthesis. In this work with a GH94 laminaribiose phosphorylase from Paenibacillus sp. YM-1 (PsLBP), we have demonstrated an enzymatic synthesis of disaccharide 1 (β-d -mannopyranosyl-(1→3)-d -glucopyranose) by using a natural acceptor glucose and noncognate donor substrate α-mannose 1-phosphate (Man1P). To investigate how the enzyme recognises different sugar 1-phosphates, the X-ray crystal structures of PsLBP in complex with Glc1P and Man1P have been solved, providing the first molecular detail of the recognition of a noncognate donor substrate by GPs, which revealed the importance of hydrogen bonding between the active site residues and hydroxy groups at C2, C4, and C6 of sugar 1-phosphates. Furthermore, we used saturation transfer difference NMR spectroscopy to support crystallographic studies on the sugar 1-phosphates, as well as to provide further insights into the PsLBP recognition of the acceptors and disaccharide products.