Efficient acetoin production from pyruvate by engineered Halomonas bluephagenesis whole-cell biocatalysis

Acetoin is an important platform chemical,which has a wide range of applications in many industries.Halomonas bluephagenesis,a chassis for next generation of industrial biotechnology,has advantages of fast growth and high tolerance to organic acid salts and alkaline environment.Here,α-acetolactate synthase andα-acetolactate decarboxylase from Bacillus subtilis 168 were co-expressed in H.bluephagenesis to produce acetoin from pyruvate.After reaction condition optimization and further increase ofα-acetolactate decarboxylase expression,acetoin production and yield were significantly enhanced to 223.4 mmol·L^(-1) and 0.491 mol·mol^(-1) from 125.4 mmol·L^(-1) and 0.333 mol·mol^(-1),respectively.Finally,the highest titer of 974.3 mmol·L^(-1)(85.84 g·L^(-1))of acetoin was accumulated from 2143.4 mmol·L^(-1)(188.6 g·L^(-1))of pyruvic acid within 8 h in fed-batch bioconversion under optimal reaction conditions.Moreover,the reusability of the cell catalysis was also tested,and the result illustrated that the whole-cell catalysis obtained 433.3,440.2,379.0,442.8 and 339.4 mmol·L^(-1)(38.2,38.8,33.4,39.0 and 29.9 g·L^(-1))acetoin in five repeated cycles under the same conditions.This work therefore provided an efficient H.bluephagenesis whole-cell catalysis with a broad development prospect in biosynthesis of acetoin.

Tandem Biocatalysis by CotA-TJ102@UIO-66-NH2 and Novozym 435 for Highly Selective Transformation of HMF into FDCA

2,5-Furandicarboxylic acid (FDCA) is a potential biorenewable chemical for applications including plastics, polyamides, drugs, etc. The selective biosynthesis of FDCA from 5-hydroxymethylfurfural (HMF) by a speci c enzyme poses a great challenge. In this study, we reported an e cient strategy to produce FDCA from HMF by the tandem biocatalysis of laccase (CotA-TJ102@UIO-66-NH 2 ) and Novozym 435. For the rst step, a nanoparticle metal organic framework was synthesized as a carrier to immobilize CotA-TJ102@UIO-66-NH 2 , which was assigned for the production of 5-formyl-2-furancarboxylic acid (FFCA) and featured an enzyme loading of 255.54 mg/g, speci c activity of 135.90 U/mg, and solid loading ratio of 99.65%. Under optimal conditions, an ideal FFCA yield of 98.5% was achieved, and the CotA-TJ102@UIO-66-NH2 pre- sented a high recycling capacity after 10 cycles. For the second step, Novozym 435 was applied for the further conversion of FFCA into FDCA, presenting a high FDCA yield of 95.5% under the optimized conditions. Novozym 435 also exhibited a high recyclability after eight cycles. As a result, the tandem biocatalysis strategy provided a 94.2% FDCA yield from HMF, indicating its excellence as a method for FDCA production.

Recent Progress on Biocatalysis and Biotransformations in Ionic Liquids

Ionic liquids have negligibly low vapor pressure, high stability and polarity. They are regarded as green solvents. Enzymes, especially lipases, as well as whole-cell of microbe, are catalytically active in ionic liquids or aqueous-ionic liquid biphasic systems. Up to date, there have been many reports on enzyme-exhibited features and enzyme-mediated reactions in ionic liquids. In many cases, remarkable results with respect to yield, catalytic activity, stability and (enantio-, regio-) selectivity were obtained in ionic liquids in comparison with those observed in conventional media. Accordingly, ionic liquids provide new possibilities for the application of new type of solvent in biocatalytic reactions.

Optimization of Two-species Whole-cell Immobilization System Constructed with Marine-derived Fungi and Its Biological Degradation Ability

Mycelia pellet formed spontaneously in the process of cultivation was exploited as a biological carrier for whole-cell immobilization due to its unique structural characteristic. An innovative two-species whole-cell im- mobilization system was achieved by inoculating the marine-derived fungus Pestalotiopsis sp. J63 spores into cul- ture medium containing another fungus Penicillium janthinellum P1 pre-grown mycelia pellets for 2 days without any pretreatment. In order to evaluate the biological degradation capacity of this novel constructed immobilization system, the immobilized pellets were applied to treat paper mill effluent and decolorize dye Azure B. The use of the constructed immobilization system in the effluent resulted in successful and rapid biodegradation of numerous in- soluble fine fibers. The optimum conditions of immobilized procedure for maximum biodegradation capacity were determined using orthogonal design with biomass of P1 pellets 10 g （wet mass）, concentration of J63 spore 2x109 mlq, and immobilization time 2 d. The results demonstrate that immobilized pellets have more than 99% biodegradation capacity in a ten-hour treatment process. The kinetics of biodegradation fits the Michaelis-Menten equation well. Besides, the decolorization capability of immobilized pellets is more superior than that of P1 mycelia pellets. Overall, the present study offers a simple and reproducible way to construct a two-species whole-cell immobiliza- tion system for sewage treatment.

Whole-cell recordings of calcium and potassium currents in acutely isolated smooth muscle cells

AIM： To record calcium and potassium currents in acutely isolated smooth muscle cells of mesenteric arterial branches in rats. METHODS： Smooth muscle cells were freshly isolated by collagenase digest and mechanical trituration with polished pipettes. Patch clamp technique in whole-cell mode was employed to record calcium and potassium currents. RESULTS： The procedure dissociated smooth muscle cells without impairing the electrophysiological characteristics of the cells. The voltage-gated Ca^2＋ and potassium currents were successfully recorded using whole-cell patch clamp configuration. CONCLUSION： The method dissociates smooth muscle cells from rat mesenteric arterial branches. Voltage-gated channel currents can be recorded in this preparation.

Hybrid enzyme catalysts synthesized by a de novo approach for expanding biocatalysis

The two major challenges in industrial enzymatic catalysis are the limited number of chemical reaction types that are catalyzed by enzymes and the instability of enzymes under harsh conditions in industrial catalysis.Expanding enzyme catalysis to a larger substrate scope and greater variety of chemical reactions and tuning the microenvironment surrounding enzyme molecules to achieve high enzyme performance are urgently needed.In this account,we focus on our efforts using the de novo approach to synthesis hybrid enzyme catalysts that can address these two challenges and the structure-function relationship is discussed to reveal the principles of designing hybrid enzyme catalysts.We hope that this account will promote further efforts toward fundamental research and wide applications of designed enzyme hybrid catalysts for expanding biocatalysis.

Facilitation of cascade biocatalysis by artificial multi-enzyme complexes——A review

Multi-enzyme complexes are the results of natural evolution to facilitate cascade biocatalysis.Through enzyme colocalization within a complex,the transfer efficiency of reaction intermediates between adjacent cascade enzymes can be promoted,resulting in enhanced overall reaction efficiency.Inspired by nature,a variety of approaches have been developed for the assembly of artificial multi-enzyme complexes with different spatial organizations,aiming at improving the catalytic efficiency of enzyme cascade.A recent trend of this research area is the creation of enzyme complexes with a controllable spatial organization which helps with the mechanistic studies and bears the potential to further increase metabolic productivity.In this review,we summarize versatile strategies for the assembly of artificial multi-enzyme complexes,followed by an inspection of the mechanistic studies of artificial multi-enzyme complexes for their enhancement of catalytic efficiency.Furthermore,we provide some highlighted in vivo,ex vivo,and in vitro examples that demonstrate the ability of artificial multi-enzyme complexes for enhancing the overall production efficiency of value-added compounds.Recent research progress has revealed the great biotechnological potential of artificial multi-enzyme complexes as a powerful tool for biomanufacturing.

Advancements in biocatalysis:From computational to metabolic engineering

Through several waves of technological research and un‐matched innovation strategies,bio‐catalysis has been widely used at the industrial level.Because of the value of enzymes,methods for producing value‐added compounds and industrially‐relevant fine chemicals through biological methods have been developed.A broad spectrum of numerous biochemical pathways is catalyzed by enzymes,including enzymes that have not been identified.However,low catalytic efficacy,low stability,inhibition by non‐cognate substrates,and intolerance to the harsh reaction conditions required for some chemical processes are considered as major limitations in applied bio‐catalysis.Thus,the development of green catalysts with multi‐catalytic features along with higher efficacy and induced stability are important for bio‐catalysis.Implementation of computational science with metabolic engineering,synthetic biology,and machine learning routes offers novel alternatives for engineering novel catalysts.Here,we describe the role of synthetic biology and metabolic engineering in catalysis.Machine learning algorithms for catalysis and the choice of an algorithm for predicting protein‐ligand interactions are discussed.The importance of molecular docking in predicting binding and catalytic functions is reviewed.Finally,we describe future challenges and perspectives.

Whole-cell recording of the robust nucleus of the arcopallium neurons in the adult zebra finch

BACKGROUND： Electrophysiological properties of the song nucleus have been revealed using conventional techniques, such as intracellular and extracellular recording. Research concerning the neuronal activation properties and regulations of the song system at the cellular and ion channel level may help reveal the neural mechanism of song learning. OBJECTIVE： To perform whole-cell recording of robust nucleus of the arcopallium （RA） neurons in brain slices from adult zebra finches （Taeniopygia guttata） and observe the action potential, sodium/potassium current and the spontaneous postsynaptic current of RA neurons. DESIGN, TIME AND SETTING： Self-controlled, neuroelectrophysiological experiment. The study was performed at the Neurophysiology Laboratory of South China Normal University from April to September 2008. MATERIALS： Flaming/Brown puller P-97 was purchased from Sutter Ins, USA; Axopatch 700B amplifier and Digidata 1332A converter were purchased from Axon Instrument, USA; pClamp software was provided by Axon Instrument, USA. METHODS： RA neurons were acutely isolated from 24 healthy male zebra finches. The action potential, voltage-gate sodium/potassium current and spontaneous postsynaptic current were recorded by whole-cell recording technology. Data were analyzed by pClamp software. MAIN OUTCOME MEASURES： The amplitude and frequency of the action potential, and the amplitude of the voltage-dependent and spontaneous postsynaptic currents, were measured. RESULTS： （1） Testing of action potential： Cells exhibited a stable current-voltage relationship following a series of hyperpolarization stepped currents, and an action potential was triggered by the spike threshold. All the recorded cells displayed repetitive firing following depolarizing current injection, with a frequency beyond 100 Hz. （2） Testing of voltage-gate currents： The inward and outward whole-cell currents were observed after a series of depolarizing voltage steps. The inward current disappeared following the application of tetrodotoxin and the outward current was significantly inhibited by application of 4-aminopyfidione and tetraethylammonium chloride. （3） Testing of spontaneous postsynaptic current： The majority of recorded cells exhibited an inward synaptic current when the membrane potential was maintained at -60 mV, with some cells exhibiting a robustly outward current when the membrane potential was maintained at -30 mV. Tetrodotoxin was unable to affect the spontaneous postsynaptic current. Following application of bicuculline [y-aminobutyric acid （A） receptor antagonist] and high concentration kynurenic acid （ionotropic glutamate receptor antagonist）, the inward and outward currents were completely inhibited. CONCLUSION： Under these experimental conditions, the action potential, sodium/potassium current and spontaneous postsynaptic current were recorded successfully in RA neurons. This indicates that the cells preserved relatively intact synaptic connections and normal physiological activity, which is required for investigating ion channels. The inward and outward whole-cell currents were sodium and potassium currents, respectively. The postsynaptic y-aminobutyric acid （A） receptors and ionotropic glutamate receptors contributed to the spontaneous postsynaptic current.

Aureobasidium subglaciale F134 is a bifunctional whole-cell biocatalyst for Baeyer-Villiger oxidation or selective carbonyl reduction controllable by temperature

The microbial production of either ester/lactones or enantio-enriched alcohols through Baeyer-Villiger oxidation or stereoselective reduction of ketones,respectively,is possible by using whole cells of A.subglaciale F134 as a bifunctional biocatalyst.The chemoselective pattern of acetophenone biotransformation catalyzed by these cells can be regulated through reaction temperature,directing the reaction either towards oxidation or reduction products.The Baeyer–Villiger oxidation activity of A.subglaciale F134 whole cells is particularly dependent on reaction temperature.Acetophenone was transformed efficiently to phenol via the primary Baeyer–Villiger product phenyl acetate at 20℃ after 48 h with 100% conversion.In contrast,at 35℃,enantio-enriched(S)-1-phenylethanol was obtained as the sole product with 64% conversion and 89% ee.In addition,A.subglaciale F134 cells also catalyze the selective reduction of various structurally different aldehydes and ketones to alcohols with 40% to 100% yield,indicating broad substrate spectrum and good enantioselectivity in relevant cases.Our study provides a bifunctional biocatalyst systemthat can be used in Baeyer–Villiger oxidation aswell as in asymmetric carbonyl reduction,setting the stage for future work concerning the identification and isolation of the respective enzymes.

Whole-cell recordings of voltage-gated Calcium,Potassium and Sodium currents in acutely isolated hippocampal pyramidal neurons

Objective：To record Calcium, Potassium and Sodium currents in acutely isolated hippocampal pyramidal neurons. Methods：Hippocampal CA3 neurons were freshly isolated by 1 mg protease/3 ml SES and mechanical trituration with polished pipettes of progressively smaller tip diameters. Patch clamp technique in whole-cell mode was employed to record voltage-gated channel currents. Results：The procedure dissociated hippocampal neurons, preserving apical dendrites and several basal dendrites, without impairing the electrical characteristics of the neurons. Whole-cell patch clamp configuration was successfully used to record voltage-gated Ca^2＋ currents, delayed rectifier K^＋ current and voltage-gated Na^＋ currents. Conclusion：Protease combined with mechanical trituration may be used for the dissociation of neurons from rat hippocampus. Voltage-gated channels currents could be recorded using a patch clamp technique.

Activation of zona incerta gamma-aminobutyric acid-ergic neurons alleviates depression-like and anxiety-like behaviors induced by chronic restraint stress

BACKGROUND Depression is a prevalent affective disorder,but its pathophysiology remains unclear.Dysfunction in the gamma-aminobutyric acid(GABA)-ergic system may contribute to its onset.Recently,antidepressants(e.g.,brexanolone,zuranolone)targeting the GABA-A receptor were introduced.The zona incerta(ZI),an inhibitory subthalamic region mainly composed of GABAergic neurons,has been implicated in emotional regulation.Deep brain stimulation of the ZI in humans affects anxiety and depression symptoms,while activation of ZI neurons in mice can either worsen or alleviate anxiety.Currently,there is no direct evidence linking GABAergic neurons in the ZI to depression-like behaviors in rodents.AIM To explore the relationship between GABAergic neurons in the ZI and depression-like behaviors in mice.METHODS A chronic restraint stress(CRS)model was utilized to induce depression in mice.Whole-cell patch-clamp recordings assessed the excitability changes of GABAergic neurons in the ZI.Additionally,chemogenetic techniques were employed to modulate ZI GABAergic neurons.The performance of the mice in behavioral tests for depression and anxiety was observed.RESULTS The findings indicated that GABAergic neurons in the ZI were closely associated with depression-like behaviors in mice.Twenty-eight days after the CRS model was established,depression-like and anxiety-like behaviors were observed in the mice.The excitability of GABAergic neurons in the ZI was reduced.Chemogenetic activation of these neurons alleviated CRS-induced depression-like and anxiety-like behaviors.Conversely,inhibition of GABAergic neurons in the ZI led to changes in emotion-related behavioral outcomes in mice.CONCLUSION Activity of GABAergic neurons in the ZI was closely associated with depression-like phenotypes in mice,suggesting that these neurons could be a potential therapeutic target for treating depression.

Efficient biosynthesis of creatine by whole-cell catalysis from guanidinoacetic acid in Corynebacterium glutamicum

Creatine is a naturally occurring derivative of an amino acid commonly utilized in functional foods and pharmaceuticals.Nevertheless,the current industrial synthesis of creatine relies on chemical processes,which may hinder its utilization in certain applications.Therefore,a biological approach was devised that employs whole-cell biocatalysis in the bacterium Corynebacterium glutamicum,which is considered safe for use in food production,to produce safe-for-consumption creatine.The objective of this study was to identify a guanidinoacetate N-methyltransferase(GAMT)with superior catalytic activity for creatine production.Through employing whole-cell biocatalysis,a gamt gene from Mus caroli(Mcgamt)was cloned and expressed in C.glutamicum ATCC 13032,resulting in a creatine titer of 3.37 g/L.Additionally,the study employed a promoter screening strategy that utilized nine native strong promoters in C.glutamicum to enhance the expression level of GAMT.The highest titer was achieved using the P1676 promoter,reaching 4.14 g/L.The conditions of whole-cell biocatalysis were further optimized,resulting in a creatine titer of 5.42 g/L.This is the first report of successful secretory creatine expression in C.glutamicum,which provides a safer and eco-friendly approach for the industrial production of creatine.

酶催化固碳过程及其强化技术研究进展

全球范围迅猛发展的工业生产导致温室气体CO_(2)的排放,引发人们对全球气候变化的普遍关切。在发展清洁能源、工业流程再造等减少碳排放的同时,开发高效、经济的CO_(2)捕集、利用与储存(CCUS)技术显得尤为迫切。本文基于CO_(2)资源化利用的目的,对生物体外酶催化固碳过程及其强化技术的研究进展进行综述。首先,介绍了CO_(2)转化过程中涉及的关键催化酶及其优化,阐述了CO_(2)资源化利用的具体策略,涵盖了将CO_(2)催化转化为甲酸、甲醇、甲烷、淀粉以及L-乳酸、丙酮酸等特定产物分子。进而,重点阐述了辅因子的原位再生、酶的固定化、反应系统的优化设计、反应条件优化(pH、温度、底物浓度)以及产物原位分离等技术对CO_(2)生物酶催化反应过程的强化,实现CO_(2)的高效固定与资源化利用。旨在通过多方面交叉论述,为生物酶催化过程及路线设计包括固定化酶催化剂的制备、反应器选择设计与开发、酶催化过程调控、高值化产品定向合成等提供有益的启发和思考。最后,总结了酶催化固碳过程存在的问题和挑战,并对其未来值得研究的方向进行了展望。

ω-转氨酶的筛选和异源表达用于制备S-甲氧基异丙胺

根据转氨酶家族进化分类和底物特异性,构建一个包含36个ω-转氨酶的酶库;将酶库中的基因克隆进大肠杆菌E.coli BL21 (DE3)中进行异源重组表达,考察酶蛋白表达水平并测定相应的酶活以及对映体选择性。通过筛选,获得最佳的ω-转氨酶为来源于巨大芽孢杆菌Bacillus megaterium的ω-转氨酶BmeTA,其重组表达粗酶活为2.0 U/mL,纯酶比活为9.5 U/mg蛋白;酶学性质表征发现其最适温度为35℃,最适pH为8.0。在此基础上进一步优化其催化反应工艺条件,在20 g/L加酶量、0.5 mmol/L辅酶添加量以及1.4的氨基供体/氨基受体比例条件下,反应18 h获得450 mmol/L的S-甲氧基异丙胺,原料转化率达到90%,为实现生物催化制备S-甲氧基异丙胺的工业化奠定基础。

纳米生物催化技术全球专利态势与我国布局对策研究

本文以PatSnap平台的全球纳米生物催化技术专利数据作为研究对象,利用专利计量分析方法,对纳米生物催化技术全球专利申请趋势、申请区域、创新主体、技术构成、法律状态、技术路线等进行分析,揭示纳米生物催化技术最新的发展趋势以及研究热点,并提出该领域技术发展以及专利布局的建议,为我国在纳米生物催化技术研发和应用方向提供参考。

基于创新实践能力培养的“高等生物催化工程”教学案例库建设

“高等生物催化工程”是一门多学科交叉的理论与实践密切结合的课程,在制药工程专业学位的课程体系中处于桥梁和纽带地位。该文以“价值引领、知识传递和能力提升”的人才培养理念为导向,进行了“高等生物催化工程”课程教学案例库建设,构建了以技术案例为核心,课程思政案例为辅的多元化多维度的教学案例库。通过案例教学的实施,有效激发了学生的学习兴趣,引导学生探究式学习,培养创新思维,提高学生分析和解决实际问题的能力。构建课程思政案例将专业知识传授与价值引领相结合,实现立德树人。该案例库建设将有助于研究生工程实践能力和创新能力的培养,对进一步提高制药工程专业学位研究生的教育质量意义重大。

复合酶@ZIF-8的制备及其黑米花青素提取性能

利用体相原位生长法制备了具有优良催化活性和稳定性的α-淀粉酶&纤维素酶@ZIF-8(A&C@ZIF-8)纳米颗粒,结合溶剂萃取过程,可实现黑米中花青素的有效提取。该纳米颗粒中的α-淀粉酶和纤维素酶能分别通过水解多糖链上的α-1,4糖苷键和β-1,4糖苷键来解离植物细胞壁。同时,由于ZIF-8框架良好的空间限域保护作用,这两种酶展现出良好的高温环境耐受性和有机溶剂耐受性。该纳米颗粒的平均粒径为405nm,呈现多角球形颗粒状,其对两种酶的总负载量为18%(质量分数)。A&C@ZIF-8纳米颗粒中的酶可耐受72℃的高温环境,此时α-淀粉酶和纤维素酶的活性分别比其游离酶高出33.2%和247.3%。此外,当利用A&C@ZIF-8纳米颗粒结合溶剂萃取过程进行花青素协同提取时,其每100g提取量达到了200.39mg/g,比仅利用溶剂萃取时的提取效果提升了26.3%。该工作为创新设计和构建用于植物细胞中活性物质提取的固定化酶复合功能材料提供了新策略。

杂化纳米生物催化剂CALB@Fe_(3)O_(4)@ZIF-8的构建及性能

以脂肪酶(lipase,CALB)为模型酶,利用功能性纳米材料四氧化三铁纳米粒子与金属有机骨架(metal-organic framework,MOF)材料ZIF-8相杂化的同时,将CALB包埋在杂化材料中,制备杂化纳米生物催化剂CALB@Fe_(3)O_(4)@ZIF-8,研究了温度、机械搅拌转速、光照条件以及Fe_(3)O_(4)添加量对CALB@Fe_(3)O_(4)@ZIF-8活性的影响,获得制备CALB@Fe_(3)O_(4)@ZIF-8的最佳条件为:温度20℃、转速400 r/min、光照波长380 nm、Fe_(3)O_(4)质量5 mg。在此条件下CALB@Fe_(3)O_(4)@ZIF-8的最大酶活回收率82%,比优化前提高了16%。随着具有光热效应的Fe_(3)O_(4)纳米粒子的加入,CALB@Fe_(3)O_(4)@ZIF-8表现出较强的光热效应,光照(光强和光照波长)显著提高酶与底物的亲和力和反应速率,使CALB@Fe_(3)O_(4)@ZIF-8表现出更高的酶活力。该研究为开发新型功能性纳米杂化MOF固定化酶生物催化剂提供参考。

设计改造羧酸还原酶合成医药中间体(S)-2-氨基丁醇

(S)-2-氨基丁醇同时具有羟基及氨基官能团,是多种重要药物分子的关键手性中间体,生物合成(S)-2-氨基丁醇尚缺少有效的酶元件。以塞格尼氏菌(Segniliparus rugosus)来源的羧酸还原酶SrCAR为研究对象,通过对实验室已有SrCAR突变文库进行筛选测试,结合活性位点共进化分析,同时利用组合活性中心饱和突变策略(Combinatorial active-site saturation test,CAST)构建新的突变体文库,经测试最终获得优势突变体XH7(G430V/E533F/A627N)。该突变体催化底物N-Boc-(S)-2-氨基丁酸到醛产物的活性(kcat/Km)较野生型SrCAR提高2.1倍,热熔值(Tm)提升2.3℃。进一步通过引入荧光假单胞菌(Pseudomonas fluorescens)来源的醇脱氢酶PfADH,可还原N-Boc-(S)-2-氨基丁醛到醇产物。XH7和PfADH的双酶共表达体系反应5 h即可将20 mmol/L底物实现几乎完全转化,转化率达到99%,并经脱Boc保护与分离纯化获得终产物(S)-2-氨基丁醇,得率为60%。通过分子动力学模拟解析最优突变体活性及热稳定性提高的分子机制,为SrCAR酶设计改造提供新的研究思路,拓展酶法合成(S)-2氨基丁醇的生物酶工具箱,可为类似高附加值医药中间体的生物合成提供理论和实践指导。