构建大肠杆菌合成生物群落利用混合糖同步发酵生产L-乳酸

目的:实现大肠杆菌混合糖发酵产L-乳酸过程中对葡萄糖-木糖的同步利用。方法:以L-乳酸工程菌大肠杆菌JH16(E.coli B,△frdBC△pflB△ackA△adhE,ldhA::ldhL)为出发菌株,通过RED同源重组技术敲除木糖转运及代谢基因xylFGH、xylE和xylA,获得不能利用木糖的菌株大肠杆菌JH16031。以大肠杆菌JH2705(E.coli JH16,△ptsG△mglB)为出发菌株,敲除葡萄糖转运及代谢基因crr、malX和galP,获得不能利用葡萄糖的菌株大肠杆菌JH27071,以构建大肠杆菌合成生物群落。通过摇瓶和5 L发酵罐试验确定该合成生物群落在混合糖发酵时较优的混合接种比例。结果:以60 g/L葡萄糖和40 g/L木糖为碳源进行发酵,当JH16031与JH27071初始接种比为1∶50,初始OD_(600nm)=0.5,混合糖的利用率最高,在84 h内消耗97%的糖,并在96 h内结束发酵。葡萄糖消耗速率为705 mg/(L·h),木糖消耗速率为435 mg/(L·h),L-乳酸产率为951 mg/(L·h),L-乳酸产量达到92 g/L,糖酸转化率为91%。结论:构建大肠杆菌合成生物群落可实现利用混合糖发酵产L-乳酸过程中葡萄糖和木糖的同步利用。研究结果为工业发酵生产中,使用低成本的木质纤维素为原料,降低发酵成本,提高混合糖利用率提供技术支持。

强化厌氧表达dld基因以提高大肠杆菌工程菌发酵产L-乳酸的光学纯度

为去除廉价发酵原料中的D-乳酸从而提高发酵最终产物L-乳酸的光学纯度,该研究通过构建带有不同厌氧诱导启动子(pflBp6、pnirB、pflBp6-pnirB)的D-乳酸脱氢酶基因(dld)的表达质粒,并将其分别转入大肠杆菌(Escherichia coli)HBUT-L,加强其在发酵产L-乳酸的同时消除外源D-乳酸的能力。通过装液量与转速的单因素试验筛选最优的质粒表达条件及菌株,并在无机盐培养基与玉米浆培养基中进行发酵验证。结果表明,在装液量为200 mL/250 mL、转速为150 r/min的条件下,含有启动子pflBp6-pnirB的表达质粒的工程菌株HBUT-L4发酵18 h时D-乳酸脱氢酶活力最高,为81.1 U/g。在此条件下进行无机盐培养基和玉米浆培养基发酵时,工程菌株HBUT-L4相对于出发菌株HBUT-L,D-乳酸消耗速率分别提高250%、217%,L-乳酸的光学纯度分别从96.32%、98.48%提升至99.95%、99.98%。在大肠杆菌工程菌发酵L-乳酸的过程中,带有厌氧诱导启动子pflBp6-pnirB的表达质粒可提高D-乳酸脱氢酶活力,强化菌株消除外源D-乳酸,进而提高L-乳酸的光学纯度的能力,具有重要的工业化应用价值。

Repeated-Batch and Continuous Production of L-Lactic Acid by Rhizopus oryzae Immobilized in Calcium Alginat Beads:ReactorPerformance and Kinetic Model

Repeated-batch and continuous production of L-lactic acid by immobilized Rhizopusoryzae with calcium alginate entrapment method in a three-phase fluidized-bed bioreactor was stud-ied.The operation conditions were optimized.The productivity based on total reactor volume wasabout 3 times higher than that with free cells in a traditional stirred tank bioreactor.A mathemat-ical model was proposed and the model predictions were in good agreement with the experimentaldat.

Synthesis of the Biomimetic Polymer: Aliphatic Diamine and RGDS Modified Poly(d,l-lactic acid)

A novel poly（d, /-lactic acid） （PDLLA） based biomimetic polymer was synthesized by grafting maleic anhydride, butanediamine and arg-gly-asp-ser （RGDS） peptides onto the backbone of PDLLA, aiming to overcome the acidity and auto-accelerating degradation of PDLLA during degradation and to improve its biospecificity and biocompatibility. The synthetic copolymer was characterized by FTIR, ^13C NMR and amino acid analyzer （AAA）.

炔雌醇月缓释Poloxamer188复合聚L-乳酸电纺纤维的表征

目的考察在聚L-乳酸电纺纤维体系下,Poloxamer188复合量和炔雌醇的载药量对药物包裹和释放行为的影响。方法将炔雌醇、Poloxamer188、聚L-乳酸共同溶解在二氯甲烷中形成均相溶液后静电纺丝,其中Poloxamer188的复合量为聚L-乳酸质量的220%、240%、260%、280%、300%,炔雌醇的载量设为聚L-乳酸质量的5%、10%、15%。扫描电子显微镜观察纤维形态,差示热分析和X-射线衍射考察材料复合状态,高效液相色谱-紫外分光光度法测定释放介质中炔雌醇的含量,绘制释放曲线并拟合。结果所得产品均为微米级直径均匀无珠子结构的纤维。炔雌醇在纤维中复合良好,Poloxamer188在纤维表面有单体存在。炔雌醇在释放全程均为被增溶状态。随着Poloxamer188复合量的增高,药物释放量增加,随着炔雌醇载量的增高,药物释放量减少。Poloxamer188复合量为300%、炔雌醇载量为5%时,药物的百分释放量接近80%,缓释期可达28 d。释放曲线能够被Peppas方程式拟合。结论Poloxamer188复合量为聚L-乳酸的220%~300%时能够得到百分释放量较高的月缓释载炔雌醇电纺纤维。

L-LACTIC ACID FERMENTATION IN A ROTATING-DISC CONTACTOR WITH SIMULTANEOUS PRODUCT SEPARATION BY ION-EXCHANGE

A rotating disk contactor（RDC）was designed to perform L-lactic acid fermentation with afilamentous fungi,Rhizopus oryzae,which was immobilized on the surfaces of the rotating discs.Thebioreactor was operated using repeated-batch method as well as continuous feeding method.Ananionic resin,D354,slightly basic in nature and of high selectivity and capacity was chosen for lacticacid separation.A coupled process of L-lactic acid fermentation and ion-exchange separation wasevaluated experimentally.The results indicated that the pH value of the fermentation broth could bemaintained at about 3-3.5 without any addition of alkali.The conversion ratio of glucose to L-lacticacid was about 0.7 g·g-1 and the fermentation rate reached as high as 62.5 g·h-1·m-2.

Synthesis and Characterization of Chitosan-g-poly- (D, L-lactic acid) Copolymer

Biodegradable chitosan-g-poly (D, L-lactic acid) copolymers were prepared via two methods. (1) The lactide was grafted onto hydroxyl groups of chitosan by using macromolecular initiator sodium of trimethylsilyl-chitosan, (2) poly (D,L-lactic acid)(PLA) with low molecular weight can be linked to the amino group by coupling activated PLA to trimethylsilyl-chitosan. Two graft copolymers had hydrophilic-hydrophobic character and can be applied as carriers for drug delivery.

Optimization of L-lactic Acid Production of Rhizopus Oryzae Mutant RLC41-6 by Ion Beam Implantation at Low-Energy

In order to obtain an industrial strain with a higher L（＋）-lactic acid yield, the strain Rhizopus oryzae RF3608 was mutated by means of nitrogen ion beam implantation and the mutant strain RLC41-6 was isolated. Under optimal conditions the yield of L（＋）-lactic acid produced in a shake-flask reached 133 g/L - 137 g/L after 36 h cultivation, indicating that the conversion rate based on glucose was as high as 88% - 91% and the productivity was 3.75 g/L.h. It was almost a 115% increase in lactic acid production compared with the original strain RF3608.

Optimization of L(+)-Lactic Acid Production from Xylose with Rhizopus Oryzae Mutant RLC41-6 Breeding by Low-Energy Ion Implantation

In order to obtain an industrial strain with a higher L（＋）-lactic acid yield, the strain Rhizopus oryzae PW352 was mutated by means of nitrogen ion beam implantation and the mutant strain Rhizopus oryzae RLC41-6 was obtained. An experimental finding was made in surprise that Rhizopus oryzae mutant RLC41-6 is not only an L（＋）-lactic acid producer from corn starch but also an efficient producer of L（＋）-lactic acid from xylose. Under optimal conditions, the production of L（＋）-lactic acid from 100 g/L xylose reached 77.39 g/L after 144 h fed-batch fermentation, A high mutation rate and a wide mutation spectrum of low-energy ion implantation were observed in the experiment.

Improvement of L(+)-Lactic Acid Production of Rhizopus Oryzae by Low-Energy Ions and Analysis of Its Mechanism

The wild type strain Rhizopus oryzae PW352 was mutated by means of nitrogen ion implantation （15 keV, 7.8×10^14 ～ 2.08 ×10^15 ions/cm^2） to find an industrial strain with a higher L（＋）-lactic acid yield, and two mutants RE3303 and RF9052 were isolated. In order to discuss the mechanism primarily, Lactate Dehydrogenase of Rhizopus oryzae was studied. While the two mutants produced L（＋）-lactic acid by 75% more than the wild strain did, their specific activity of Lactate Dehydrogenase was found to be higher than that in the wild strain. The optimum temperature of Lactate Dehydrogenase in Rhizopus oryzae RF9052 was higher. Compared to the wild strain, the Michaelis constant （Km） value of Lactate Dehydrogenase in the mutants was Changed. All these changes show that L（＋）-lactic acid production has a correlation with the specific activity of Lactate Dehydrogenase. The low-energy ions, implanted into the strain, may improve the specific activity of Lactate Dehydrogenase by influencing its gene structure and protein structure.

Enhancement of L(+)-Lactic Acid Production of Immobilized Rhizoous Orvzae Imolanted by Ion Beams

Immobilized Rhizopus oryzae culturing may be a solution to the inhibited production of L（＋）-lactic acid in submerged fermentation, which is caused by aggregated mycelia floc. In the present study, a R. oryzae mutant （RL6041） with a 90% conversion rate of glucose into L-lactic acid was obtained by N＋ implantation under the optimized conditions of a beam energy of 15 keV and a dose of 2.6 ×10^15 ions/cm^2. Using polyurethane foam as the immobilization matrix, the optimal L-lactic acid production conditions were determined as 4 mm polyurethane foam, 150 r/min, 50 g/L ～ 80 g/L of initial glucose, 38℃ and pH 6.0. 15-cycle repeated productions of L-lactic acid by immobilized RL6041 were performed under the optimized culturing conditions and over 80% of the glucose was converted into L-lactic acid in 30 hours on average. The results show that immobilized RL6041 is a promising candidate for continuous L-lactic acid production.

SURFACE OF GELATIN MODIFIED POLY(L-LACTIC ACID)FILM

In this paper, the surface structure of poly(L-lactic acid) (PLLA) film modified with gelatin was investigated. ThePLLA film specimens were treated directly with aqueous alkali solution to provide their surfaces with carboxyl groups, sothat these functional groups could become the reactive sites for gelatin immobilization. The functional groups of the PLLAfilms were identified by ATR-FTIR spectra and XPS spectra, the changes in surface morphology were observed by usingenvironmental scanning electron microscopy (ESEM), and the hydrophilicity of modified PLLA films was examined bywater contact angle measurement. Experimental results showed that the gelatin was immobilized with water-solublecarbodiimide (EDC) onto the PLLA film's surfaces, and the gelatin content on the polymer surface was related to carboxylicgroup formed in the controlled hydrolysis process. Rough surfaces caused by hydrolysis will predominantly favor the adhesion and growth of cell; and the hydrophilicity of these surfaces after the modification procedure is enhanced.

Selection and characterisation of lactic acid bacteria isolated from different origins for ensiling Robinia pseudoacacia and Morus alba L.leaves

The objective of this study was to isolate lactic acid bacteria（LAB） strains from different origins and to select the best strains for ensiling Robinia pseudoacacia（RB） and Morus alba L.（MB）leaves. The LAB strains were inoculated into the extracted liquid obtained from RB and MB leaves to evaluate the fermentation products. 11 LAB strains were selected for further experiments based on the highest products of lactic or acetic acid, including 1 strain of Weissella confusa, 2 of Lactobacillus reuteri and 8 of Lactobacillus plantarum.The API 50 CH fermentation experiment indicated that all of the selected 11 LAB strains utilised most of the carbohydrates. All the strains grew at temperatures between 10 and 45℃ and at a p H of 3.5 to 4.5; however, L. reuteri F7 and F8 tolerated a p H as low as 3.0. All 11 LAB strains showed antibacterial activity against Listeria monocytogens, Escherichia coil, Salmonella sp. and Acetobacter pasteurianus; however, after excluding the effect of organic acids, only F7 and F8 still exhibited antibacterial activity. The present study indicated that the selected 11 LAB strains could be used to prepare silages of RB and MB leaves, especially L. reuteri F7 and F8.

利用竹基纤维素为碳源生物转化L-乳酸的潜力研究

以竹材制浆厂备料工段产生的废弃竹屑为原料,采用蒸汽爆破预处理且提取半纤维素后的竹基纤维素为碳源,同时对预处理前后竹屑采用扫描电子显微镜&能谱仪和激光粒度分析仪进行表征;用分步水解发酵工艺,结合高效液相色谱对发酵L-乳酸工艺关键参数进行了分析,探究了该碳源生物转化L-乳酸的潜力。结果表明,蒸汽爆破预处理提高了原料酶解还原糖释放量,在总固体(total solids,TS)质量浓度140 g/L、酶添加量60 FPU/g、酶解54 h条件下糖化,总糖质量浓度可达31.19 g/L。将糖化液用于发酵试验,在起始糖质量浓度19.43 g/L,发酵初始pH 6.5、菌株接种量5%、发酵17 h条件下,L-乳酸质量浓度可达6.28 g/L,其糖酸转化率高达80.87%。综上,该实验为竹基纤维素的利用提供了高值化途径参考。

抗菌性聚(L-乳酸)/单宁酸接枝薄膜的制备

分别合成聚(衣康酸-co-丁二醇)(PBI)和低分子量聚(L-乳酸)(OLLA),将两者混合后进行缩聚反应,得到聚(L-乳酸-co-衣康酸-co-丁二醇)(PLBI)。然后,采用单宁酸(TA)与甲基丙烯酸缩水甘油酯(GMA)进行开环反应,制备了带有双键的光敏单宁酸(pTA)。最后,采用紫外光照表面接枝法制备了一系列抗菌性PLBI-pTA薄膜,对其化学结构、热学性能、力学性能、亲水性、疏水性及抗菌性能进行测试。结果表明,接枝pTA后,薄膜玻璃化转变温度及结晶度均降低,薄膜的断裂伸长率升高,最高可达328.1%,使PLBI-pTA薄膜具有较好的延展性。薄膜的水接触角由72.20°减小至30.47°,薄膜表面的亲水性得到提高。另外,随着pTA含量的增加,薄膜对大肠杆菌的杀菌率逐渐提升,最高可达86%。

Chitosan——L-Lactic Acid Scaffold for the Regeneration of Peripheral Nerve and Its NGF Release Properties

Chitosan—L-lactic acid composite scaffold for the regeneration of peripheral nerve is obtained by grafting L-lactic acid onto the amino groups in chitosan with combined vacuum freezer drier. The composite scaffold was characterized by ATR-FTIR and SEM. The scaffold has a better graft efficiency and has a dense inner layer and a loose outer layer with porous structure, and the pore size is about 100 μm.The NGF release properties of the scaffold were investigated. The experimental results showed that, at the 1st day, 15.2 ng of NGF on average was released from the scaffold. From day 2 to day 10, the release rate obviously slowed down and 1.64 ng of NGF was released on average every day. After 10 days, the release rate was slower and 10.3 ng of NGF was released on average every day. After 60 days, NGF could also maintained a certain concentration. These properties show that the scaffold is a better carrier for NGF which can be more advantageous to the regeneration of the damaged peripheral nerve. As a result, this composite scaffold would be an ideal candidate for the regeneration of damaged peripheral nerve.

Optimization of L(+)-Lactic Acid Fermentation Without Neutralisation of Rhizopus Oryzae Mutant RK02 by Low-Energy Ion Implantation

In order to get an industrial strain which can yield a high concentration of lactic acid for ISPR （in situ product removal）, the original strain Rhizopus oryzae RE3303 was mutated by low-energy ion beam implantation. A mutant RK02 was screened, and the factors such as the substrate concentration, nitrogen source concentration, inoculum size, seed age, aeration and temperature that affect the production of lactic acid were studied in detail. Under optimal con- ditions, the maximum concentration of L（＋）-lactic acid reached 34.85 g/L after 30 h shake-flask cultivation without adding any neutralisation （5% Glucose added）, which was a 146% increase in lactic acid production after ion implantation compared with the original strain. It was also shown that RK02 can be used in ISPR to reduce the number of times of separation.

Preparation of Poly ( L-lactic acid ) / Vermiculites Composites by in situ Copolymerization of Lactic Acid and Hydroxylic Vermiculites

L-lactic acid (L-LA) based copolymer/hydroxylation vermiculites composites (PLLA-co-bis A/HVMTs) were prepared by in situ reaction among L-LA, adipic acid, and hydroxylation lamellar vermiculites (HVMTs) using bisphenol-A epoxy resin as chain extending agent. HVMTs were obtained by sulfuric acid-leaching of lamellar vermiculites (VMTs). The effects of sulfuric acid leaching on the VMTs structure were characterized by X-ray diffraction (XRD), 29Si magic-angle spinning nuclear magnetic resonance(29Si NMR), Fourier transform infrared spectroscopy (FTIR), field emission scanning electron microscopy (FE-SEM), and thermogravimetric analysis (TGA). FTIR, FE-SEM, and TGA were used to characterize the reaction activity of HVMTs. The results indicated that VMTs with increased hydroxyl groups had been successfully obtained and could react with -COOH of the reaction system. The amount of L-LA based copolymer grafted on the surface of HVMTs was more than 22%. The onset decomposition temperature of L-LA based copolymer grafted on the surface of HVMTs was 30℃ higher than that of free L-LA based copolymer.

Multi-porous electroactive poly(L-lactic acid)/ polypyrrole composite micro/nano fibrous scaffolds promote neurite outgrowth in PC12 cells

In this study, poly（L-lactic acid）/ammonium persulfate doped-polypyrrole composite fibrous scaffolds with moderate conductivity were produced by combining electrospinning with in situ polymerization. PC12 cells were cultured on these fibrous scaffolds and their growth following electrical stimulation （0-20.0 μA stimulus intensity, for 1-4 days） was observed using inverted light microscopy, and scanning electron microscopy coupled with the MTT cell viability test. The results demonstrated that the poly（L-lactic acid）/ammonium persulfate doped-polypyrrole fibrous scaffold was a dual multi-porous micro/nano fibrous scaffold. An electrical stimulation with a current intensity 5.0- 10.0 μAfor about 2 days enhanced neuronal growth and neurite outgrowth, while a high current intensity （over 15.0 μA） suppressed them. These results indicate that electrical stimulation with a moderate current intensity for an optimum time frame can promote neuronal growth and neurite outgrowth in an intensity- and time-dependent manner.

Effects of cultivation conditions on the optical purity of L(+)-lactic acid produced from corncob hydrolysate by Rhizopus oryzae

The effect ofcultivation conditionson the opticalpurity ofL(+)-lactic acid produced by Rhizopusoryzae HZS6 from corncob hydrolysate was investigated. The isomeric composition of lactic acid was influenced by the supplementation of L(+)-lactic acid to fermentation medium. L(+)-Isomer increased with the dosage,no(-)-lactic acid was observed when the concentration of supplemented L(+)-lactic acid in matrix was≥1.5g l-1. However,the L(+)-lactic acid yield decreased with the dosage. Under suitable conditions,100g l-1 initial corncob xylose,2g l-1 NH4NO3,1.5 g l-1 supplemented L(+)-lactic acid,R. oryzae HZS6 could produce 100% L(+)-form lactic acid with the yield of 75% and final concentration of 81.2 g l-1,at pH 6.0 and temperature 34℃.