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.

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.

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.

Bovine serum albumin:An efficient and green biocatalyst for the one-pot four-component synthesis of pyrano[2,3-c]pyrazoles

The use of biocatalysts is attracting an increasing amount of attention in chemical catalysis.Here,we have shown that bovine serum albumin（BSA）,a ubiquitous,inexpensive,non-enzymatic transport protein,can serve as an efficient,retrievable catalyst in the one-pot four-component reaction of aryl aldehydes,malononitrile,hydrazine hydrate,and ethyl acetoacetate for the synthesis of pyrano[2,3-c]pyrazoles under mild reaction conditions.The BSA biocatalyst also displayed a high catalytic affinity for acyclic/cyclic ketones to yield the corresponding pyrano[2,3-c]pyrazoles or their spirocyclic variants.The BSA could be used for at least five cycles without serious loss of catalytic activity.This novel,efficient protocol has the merits of high yield,operational simplicity,and a relatively benign environmental impact.Moreover,the method extends the promiscuity of BSA as a biocatalyst.

The Ability of Edible Mushrooms to Act as Biocatalysts: Preparation of Chiral Alcohols Using Basidiomycete Strains

To examine the potential ability of edible mushrooms to act as biocatalysts, 19 basidiomycete strains were screened. Modified media (PG, O, and PGO medium) for liquid cultivation of these basidiomycete strains were designed and tested. Wet cells (>10 g) of 4 basidiomycete strains (Pleurotus salmoneostramineus H7, P. salmoneostramineus H13, Ganoderma lucidum NBRC31863, Flammulina velutipes NBRC31862) were harvested from PGO medium for 7 days. The stereoselective reduction of α-keto esters using the 4 strains was tested. It was found that each of these strains had a reducing activity toward 6 aliphatic α-keto esters. In the presence of L-alanine as an additive, the reduction of ethyl 2-oxobutanoate and ethyl 2-oxopentanoete by P. salmoneostramineus H7 produced the corresponding alcohol with a high conversion ratio and with excellent enantiomeric excess (>99% e.e. (R)). Furthermore, ethyl pyruvate, ethyl 2-oxobutanoate, and ethyl 2-oxopentanoate were predominantly reduced to the corresponding (R)-hydroxy ester (>99% e.e.) by G. lucidum. Thus, we found that these edible mushrooms have great potential to be used as biocatalysts for the stereoselective reduction of carbonyl compounds.

Preparation of Chiral Hydroxy Esters Using Actinobacteria: Biocatalyst Activity of Marine-Derived <i>Micromonospora</i>and <i>Streptomyces</i>Strains

To research the potential ability of marine-derived actinomycetes to act as biocatalysts, 8 Micromonospora strains and 5 Streptomyces strains were screened. Two recommended media (227 and 1076 media) and 2 modified media (1076-25% and P-1076-25% media) for liquid culture of these marine-derived actinomycetes were tested. As a result, 2 Micromonospora strains (Micromonospora sp. NBRC107096 and 107097) cultured with the 1076-25% medium and 2 Streptomyces strains (Streptomyces tateyamensis NBRC105048 and Streptomyces sp. NBRC105896) cultured with P-1076-25% medium showed a good growth. The stereoselective reduction of α-keto esters using these 4 actinomycetes was tested. As a result, it was found that these strains had a reducing activity toward various α-keto esters. The introduction of L-glutamate or sucrose as an additive remarkably increased the conversion ratios in the reduction of substrates by the Micromonospora strain. Furthermore, in the presence of L-alanine, Streptomyces tateyamensis NBRC105048 reduced ethyl pyruvate, ethyl 2-oxobutanoate, ethyl 2-oxopentanoate, ethyl 2-oxohexanoate, and ethyl 3-methyl-2-oxobutyrate to the corresponding α-hydroxy ester with a high conversion ratio and with excellent enantiomeric excess. Thus, we found that these marine-derived actinomycetes have great potential to be used as biocatalysts for stereoselective reduction of carbonyl compounds.

Enantioselective Resolution of(R,S)-Carvedilol to(S)-(2)-Carvedilol by Biocatalysts

Among the microorganisms employed in the study,Aspergillus niger(GUFCC5443),Escherichia coli(ATCC9637),Streptomyces halstedii(CKM-2),Pseudomonas putida(NCIB9494),Cunninghamella elegans(NCIM689)and Sphingomonas paucimobilis(NCTC11030)were capable for the enantioselective conversion of racemic Carvedilol.Immobilization technique enhanced the enantioselectivity of microorganisms and thus increased the enantiomeric purity of the drug.Excellent enantiomeric ratios(E)were found in reactions catalyzed by immobilized A.niger and E.coli with values 174.44 and 104.26,respectively.Triacylglycerol lipase from Aspergillus niger was also employed in this study as a biocatalyst which resulted in the product with 83.35%enantiomeric excess(ee)and E of 11.34 while the enzyme on immobilization has yielded 99.08%ee and 216.39 E.The conversion yield(C%)of the drug by free-enzyme was 57.42%,which was enhanced by immobilization to 90.51%.Hence,our results suggest that immobilized triacylglycerol lipase from A.niger(Lipase AP6)could be an efficient biocatalyst for the enantioselective resolution of racemic Carvedilol to(S)-(-)-Carvedilol with high enantiomeric purity followed by immobilized cultures of A.niger and E.coli.

Construction of the yeast whole-cell Rhizopus oryzae lipase biocatalyst with high activity

Surface display is effectively utilized to construct a whole-cell biocatalyst.Codon optimization has been proven to be effective in maximizing production of heterologous proteins in yeast.Here,the cDNA sequence of Rhizopus oryzae lipase (ROL) was optimized and synthesized according to the codon bias of Saccharomyces cerevisiae,and based on the Saccharomyces cerevisiae cell surface display system with α-agglutinin as an anchor,recombinant yeast displaying fully codon-optimized ROL with high activity was successfully constructed.Compared with the wild-type ROL-displaying yeast,the activity of the codon-optimized ROL yeast whole-cell biocatalyst (25 U/g dried cells) was 12.8-fold higher in a hydrolysis reaction using p-nitrophenyl palmitate (pNPP) as the substrate.To our knowledge,this was the first attempt to combine the techniques of yeast surface display and codon optimization for whole-cell biocatalyst construction.Consequently,the yeast whole-cell ROL biocatalyst was constructed with high activity.The optimum pH and temperature for the yeast whole-cell ROL biocatalyst were pH 7.0 and 40 °C.Furthermore,this whole-cell biocatalyst was applied to the hydrolysis of tributyrin and the resulted conversion of butyric acid reached 96.91% after 144 h.

红花钓钟柳中新型糖基转移酶的筛选与鉴定

苯乙醇苷类化合物因具有抗氧化、抗肿瘤等生理活性在食品、医药等领域备受关注。糖基转移酶作为生物催化剂可用于各种糖苷产物的制备。为丰富苯乙醇苷类化合物的糖苷多样性,在红花钓钟柳转录组中筛选糖基转移酶候选基因并进行功能鉴定。该研究基于不同植物组织中基因差异表达分析筛选到7条候选基因,将其克隆到pET-MBP表达载体上,并导入大肠杆菌Escherichia.coli Transetta(DE3)异源表达后利用体外酶促反应进行其生物催化活性研究。获得一个新型糖基转移酶UGT712A2,可以催化桂叶苷B C4-OH的葡萄糖基化反应生成新的苯乙醇苷类化合物桂叶苷B-4-葡萄糖苷。同时,UGT712A2也可以催化毛蕊花糖苷、芦丁、胡黄连苷Ⅱ、7-去甲基软木花椒素等化合物的糖基化,具有一定的底物宽泛性。因此,UGT712A2可能是一种潜在的酶工具,用于苯乙醇苷类化合物和其他多种新型糖苷产物的制备,从而发现新的糖苷化合物应用于食品、医药领域。

Recent Advances in Photoenzymatic Catalysis

Photoenzymatic catalysis has become an emerging field in organic synthetic chemistry that provides eco-friendly alternatives to traditional methods. This comprehensive review examines the developing field of photoenzymatic catalysis, categorized by reaction types and focusing on its application in organic synthesis. This article highlights recent advances in the use of photoenzymatic reactions in carbon-carbon cross-coupling, ketone and alkene reduction, hydroamination, and hydrosulfonylation, mostly by flavin-dependent “ene”-reductases and nitroreductases. In each case, we exemplified the substrate scope that produces products with high yield and enantioselectivity. Additionally, the emerging trends in developing new enzymatic variants and novel reaction pathways that broaden the scope and enhance yield of these reactions were discussed.

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.

Efficient stereoselective hydroxylation of deoxycholic acid by the robust whole-cell cytochrome P450 CYP107D1 biocatalyst

Deoxycholic acid(DCA)has been authorized by the Federal Drug Agency for cosmetic reduction of redundant submental fat.The hydroxylated product(6β-OH DCA)was developed to improve the solubility and pharmaceutic properties of DCA for further applications.Herein,a combinatorial catalytic strategy was applied to construct a powerful Cytochrome P450 biocatalyst(CYP107D1,OleP)to convert DCA to 6β-OH DCA.Firstly,the weak expression of OleP was significantly improved using pRSFDuet-1 plasmid in the E.coli C41(DE3)strain.Next,the supply of heme was enhanced by the moderate overexpression of crucial genes in the heme biosynthetic pathway.In addition,a new biosensor was developed to select the appropriate redox partner.Furthermore,a cost-effective whole-cell catalytic system was constructed,resulting in the highest reported conversion rate of 6β-OH DCA(from 4.8%to 99.1%).The combinatorial catalytic strategies applied in this study provide an efficient method to synthesize high-value-added hydroxylated compounds by P450s.

微生物酶在有机化学工艺上的应用

微生物酶凭借其特有的催化活性、特异性以及环境友好性,可以在常温且温和的条件下催化多种有机反应。尽管微生物酶在有机化学中的应用具有许多优势,但仍面临酶的稳定性、催化活性、大规模生产的成本效益等挑战。基于此,文章将详细探讨微生物酶在有机化学工艺中的应用以及在应用中面临的挑战和可采取的解决策略,以期促进微生物酶技术在有机合成领域中的广泛应用。

生物触媒在AAO工艺中的工程应用研究

生物触媒是一种新型的高效环保水处理药剂,能够提高AAO工艺的去污能力并减少系统剩余污泥量的产生。本次研究根据小试试验的研究结论,将生物触媒药剂投加到生化反应池中,进一步验证该药剂在AAO工艺中的实际应用效果。结果表明,生物触媒药剂加入到系统后能够显著提升AAO工艺的去污能力,对污水中COD、氨氮、总氮、总磷的去除率分别为90.55%、99.06%、74.68%、64.98%;显著降低了剩余污泥量的排放以及除磷剂、PAM阳离子和电量的使用,节省运营成本费约36万元,具有十分良好的环保与经济效益。

南极假丝酵母脂肪酶B的酿酒酵母表面展示及其催化己酸乙酯的合成

从南极假丝酵母(Candida antarctica)基因组克隆得到南极假丝酵母脂肪酶B(Candida antarctica Lipase B,CALB)全基因片段,利用连接肽celA Linker将CALB与酿酒酵母细胞表面展示蛋白α-凝集素的C端连接融合,构建表面展示载体pICAS-celAL-CALB,转化酵母后获得重组酵母菌Saccharomyces cerevisiae pICAS-celAL-CALB。该重组酵母菌经葡萄糖诱导表达及分析,表明CALB已在酿酒酵母细胞表面成功展示,水解活力达26.26 u/(g·dry cell)。重组酵母菌经冻干能有效地实现在非水相中全细胞催化己酸和乙醇酯化合成己酸乙酯。反应物己酸与乙醇的摩尔比为1:1.25,己酸乙酯的产率为98.0%,具有较好的操作稳定性。

酶法合成生物柴油的研究进展

生物柴油是一种清洁可再生的生物能源 ,是石油燃料的理想替代物。酶法合成生物柴油具有提取简单、反应条件温和、醇用量小、甘油易回收和无废物产生等优点。综述了脂肪酶与固定化脂肪酶、全细胞生物催化剂在生物柴油生产中应用的新进展 ,并对我国生物柴油产业的发展提出了建议。

酿酒酵母表面展示南极假丝酵母脂肪酶B的酶学性质研究

本文对酿酒酵母表面展示南极假丝酵母脂肪酶B(Candida antarctica lipase B,CALB)的一般酶学性质进行研究,得出如下结论:酵母表面展示CALB在碱性条件下具有很好的稳定性和水解活性。其水解对硝基苯酚酯的最适反应pH值范围是8.0～8.5,与游离的CALB商品酶相比,向碱性条件偏移。最适反应温度范围是40～45℃。最适反应底物为对硝基苯酚丁酸酯,表现出极为明显的底物特异性。Ca2+、Mg2+对其有明显的激活作用。K+对其有微弱的激活作用。而Co2+、Cu2+对其有明显的抑制作用。本研究为酵母表面展示脂肪酶的应用研究奠定理论基础。

脂肪酶催化合成生物柴油的研究进展

环保型燃料生物柴油有望解决能源短缺的问题,脂肪酶催化动植物油脂合成生物柴油的方法具有反应条件温和、产物易分离和不污染环境等优点。综述了酶催化法在提高脂肪酸酯产率和减少生产成本等方面的研究进展。