Enzymes are becoming an effective tool in industrial processes, from crude applications such as bioremediation to fine processes such as chirally selective biocatalysis. The ligninolytic enzymes have recently received considerable attention for industrial application due to both their broad substrate range and their ability to degrade the most recalcitrant natural polymer, lignin. This group of enzymes was therefore identified as the target group for this study. Improved enzyme properties are constantly being sought to enhance the range of applications for enzymes. Biodiversity provides a wide variety of enzymes. Several researchers have concentrated on extremophiles as their primary source of superior enzymes, consequently neglecting temperate environments in their search for these enzymes. The relatively neglected fungal biodiversity of South Africa provided an opportunity to test the hypothesis that potentially important industrial enzymes with unusual properties could be isolated from mesophilic basidiomycetous fungi. Subsequent screening of Eastern Cape biodiversity for thermostable ligninolytic enzymes from basidiomycetes resulted in the isolation of a novel laccase enzyme from a basidiomycetous species. This fungus was identified as Peniophora sp. UD4 by phylogenetic analysis of rDNA ITS sequences. Initial studies indicated a superior optimum temperature of 70°C and thermostability, indicated by no loss in activity at 60°C over nine hours. Further characterization of the laccase revealed a broader than usual substrate range through its unusual ability to oxidatively couple DMAB and MBTH. The laccase also exhibited a broad pH oxidation range for ABTS (pH 2 – 6.8), and a relatively high affinity (K_m_ = 0.0123 mM) and catalytic efficiency (63 252 mM^(-1)^s^(-1)^) for ABTS as a substrate. The laccase activity from Peniophora sp. UD4 was shown to be comprised of three isozymes with a molecular weight of 62 kDa and pI’s of 6.33, 6.45 and 6.50. Investigation of the nutrient and physical factors affecting ligninolytic enzyme production and growth of Peniophora sp. UD4 indicated that the wild-type organism was unsuitable for large scale production of the thermostable laccase due to the low levels of laccase production. The thermostable laccase was applied to defouling of ultrafiltration membranes, bioremediation of industrial waste streams, biocatalysis, and biosensor technology as potential applications. Application of the Peniophora sp. UD4 laccase to defouling of membranes used for ultrafiltration of brown water showed large flux recoveries of 31, 21 and 21% after the first three defouling recycles respectively, compared to 3% for the control without immobilized enzyme. The novel laccase showed potential for the bioremediation of industrial waste streams, the most successful being that of bleach plant effluent, where a reduction of 66% of the phenolic load was achieved. Application of the novel laccase to biocatalytic oxidation of ferulic acid and (±)-α-pinene showed higher product yield as compared to oxidation of these compounds by Trametes versicolor laccase in mediated and non-mediated systems. The major products of (±)-α-pinene oxidation were identified as verbenol and trans-sorberol. The Peniophora sp. UD4 laccase was successfully applied to biosensor technology, which benchmarked significantly better than Trametes versicolor laccase for the detection of 4-chlorophenol. The biosensor developed with laccase from UD4 by covalent binding to a glassy carbon electrode exhibited the best combination of sensitivity and stability. This thesis shows that a laccase with superior properties was obtained from a mesophilic South African basidiomycete. The catalytic properties displayed by the novel laccase from Peniophora sp. UD4 all contribute to the increased industrial applicability of laccases, and may be the most industrially feasible enzyme of its class isolated to date.

A thesis submitted in fulfillment of the requirement for the Degree Master of Technology: Biotechnology, M.L. Sultan technikon, 2000. / The ever-increasing demand for a wide variety of paper products has led to the pulp and paper industry becoming one of the largest industries in the world. In 1988 the United States alone produced almost 71 million metric tonnes of paper and pulp board (Jeffries, 1992). South Africa has also become one of the major international producers of pulp and paper products. Since 1970, the production of paper and board by the South African industry achieved an average growth rate of 5.2% per annum, and in 1997 South Africa was the twelfth largest producer of pulp and 24th biggest supplier of paper and board in the world (Molony, 1999 / M

Cellulomonas find was grown on minimal media with casamino acids and yeast extract added. Avicel was found to be the best cellulosic carbon source for the production of cellulase enzymes. The Millipore Ultrafiltration System was found to be the most efficient method of concentrating the enzyme preparations. Unpretreated sawdust samples of four different softwood species were degraded between 12 and 16 percent over a 15-day treatment. Increasing the concentration of substrate lead to a lower percent degradation but a higher overall degradation. Chemical pretreatment did not appreciably increase degradability of the samples. Physical pretreatments decrease the degrada-bility of the sawdust samples. The lotech pretreatment, which is a combination of chemical and physical pretreatment, gives a substrate that is degraded by the Cellulomonas fimi enzyme preparation. The pretreatment makes 50% of the sample water soluble. In 3 days, a further 35% is degraded from large insoluble chunks into insoluble small particles which remain in suspension. There is 6% degraded into soluble state. This leaves only 9% of the initial sample left in the pellet. With longer incubation, one would expect the degradation to continue. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Submitted in complete fulfillment for the Degree of Master of Technology: Biotechnology, Durban University of Technology, 2010. / Xanthomonas campestris pv phaseoli produced an extracellular endoinulinase on various carbon sources. The highest inulinase production of 9.24 ± 0.03 IU ml¯¹by X. campestris pv. phaseoli was attained using an optimized medium comprising of 3% sucrose and 2.5% tryptone. Inulinase production in X. campestris pv. phaseoli was further enhanced through ethylmethanesulfonate mutagenesis. The resulting mutant, X. campestris pv. phaseoli KM 24 demonstrated enhanced inulinase production of 22.09 ± 0.03 IU ml¯¹after 24 h, which was 2.4 – fold higher than that of the wild type. Inulinase production by this mutant was scaled up in a 5 L fermenter yielding a final activity of 21.87 ± 0.03 IU ml¯¹with an inulinase/invertase (I/S) ratio of 2.6 after 18 h. Maximum volumetric (21 865 IU 1¯¹ h¯¹) and specific (119 025 IU g¯¹ h¯¹) productivities of inulinase were attained in a fermenter after 18h growth. Inulin hydrolysis by the crude inulinase and subsequent detection of mono- and oligosaccharides indicated the presence of an endoinulinase. The extracellular endoinulinase from the mutant KM 24 was purified to homogeneity by gel filtration chromatography and had a specific activity of 174.74U/mg. the optimum pH and temperature of the purified enzyme were found to be 6.0 and 50°C, respectively. The enzyme was stable up to 60°C, retaining over 60% activity for 30 min, but activity rapidly declined at temperatures above 60°C. The pure inulinase enzyme was also found to be stable between pH 6-9. The Lineweaver-Burk plots showed that the apparent Km and Vmax values of the inulinase for inulin were 1.15 mg/ml and 15µM/min, respectively. The Kcat value was found to be 0.145 min¯¹ with an enzyme catalytic efficiency of 0.126 mg¯¹.ml.min¯¹.This mutant demonstrated good potential for large scale production of inulinase and fructooligosaccharides. / National Research Foundation

Submitted in fulfilment for the requirement of a Degree of Doctor of Technology: Biotechnology, Durban University of Technology, 2010. / The aim of this study was to evaluate T. lanuginosus for the production of hemicellulases, its yield enhancement using mutagenesis and application of a selected xylanase on bagasse pupl to assess the improvement of pulp properties. The objectives were: To determine the localization of hemicellulases in T. lanuginosus strains, To develop high yielding strains of T. lanuginosus through mutagenensis, To investigate the synthesis of xylanase by T. lanuginosus MC134, To optimize the medium components and cultural conitions of T. lanuginosus MC134 strain, To study the influence of agitation and aeration on the production of xylanase by T. lanuginosus MC134 in a fermenter, To evaluate the bleach boosting abilities of T. lanuginosus xylanase on bagasse pulp, To evaluate simultaneous xylanase production and biobleaching potential of T. lanuginosus.

Dissertation (PhD)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: Cellulose and hemicellulose represents the two most abundant groups of renewable polysaccharides known to man. Apart from their presence in plant material, they also contribute to a significant portion of inexpensive readily available material, such as wastes and bypro ducts from forestry / agricultural origin. The chemical composition of plant material varies, but the biomass content consists of approximately 75% carbohydrate polymers (cellulose and hemicellulose) and 25% lignin. The enzymes required for the degradation of cellulose and hemicellulose are collectively called cellulases and hemicellulases. These enzymes have a broad spectrum of industrial applications including the production of fuel ethanol through fermentations, reducing the amount of chlorine required for bleaching in the pulp and paper industry, increasing dough volume in the baking industry, improving digestion and nutritional value of animal feed, increasing clarification and enhancing the filterability of wine, beer and fruit juice, etc. Therefore, a large potential market exists for cellulases and hemicellulases provided their production is economical and the product, authentic. Aspergilli occur in a wide variety of habitats including soil, stored food and feed products and decaying vegetation. The advantages for using A. niger as host for heterologous enzyme production include good protein secretion, industrial fermentation technology dating as far back as 1919, being a non-pathogenic fungus with GRAS status, no special substrate or cultivation requirements, FDA approval of numerous enzymes (homologous and heterologous) produced, etc. In this study an Aspergillus expression vector was constructed using the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (gpdp) of A. niger and the glucoamylase terminator (glaAT) of Aspergillus awamori. The cDNA copies of the eg! and xyn2 genes of Trichoderma reesei, cbhl-4 of Phanerochaete chrysosporium, man! of Aspergillus aculeatus and xyn3 of Aspergillus kawachii were introduced into the expression vector, respectively. All the plasmids were co-transformed with plasmid p3SR2 to A. niger and transformants selected for stable plasmid integration into the genome of the host. The recombinant enzymes EgI, Xyn2, Cbhl-4, Man! and XynC were successfully expressed and secreted at activity levels of 2300, 8000, 500, 6000 and 900 nkatlml, respectively. The enzymes were produced as functional entities and were subsequently characterized. The EgI, Xyn2 and ManI were evaluated as feed additives for the possible use in the animal feed industry. Improved biomass gain was observed with in vivo studies on poultry. With the possible mass production of heterologous enzymes in mind, a simple medium had to be devised for their inexpensive production. Molasses medium (available from the South African sugar industry) was therefore evaluated and the cultivation conditions optimized for it's possible use as cultivation substrate for A. niger. The evaluation was done on the grounds of EgI and Xyn2 activity produced which was monitored over time. This study highlighted the possible use of A. niger for the heterologous production of enzymes, the use of industrial substrate for cultivation and paved the way for the high level expression of industrially important genes at low cost and a positive environmental impact. / AFRIKAANSE OPSOMMING: Sellulose en hemisellulose verteenwoordig die twee vollopste herwinbare polisakkariede bekend. Behalwe vir hul teenwoordigheid in plantmateriaal, dra hulle ook by tot 'n beduidende fraksie van goedkoop, maklik bekombare materiaal soos afval- en byprodukte van bosbou I landbou oorsprong. Soos te verwagte, varieër die chemiese samestelling van die plantmateriaal, maar die biomassa-inhoud bestaan uit naastenby 25% lignien en 75% koolhidraatpolimere (sellulose and hemicellulose). Die ensieme benodig vir die afbraak van sellulose en hemisellulose staan gesamentlik as sellulases en hemisellulases bekend. Hierdie ensieme het 'n breë spektrum van industriële toepassings insluitende die produksie van brandstofalkohol d.m.v. fermentasies, vermindering in die hoeveelheid chloor benodig vir die bleikproses in die pulp-en-papier industrie, toename in deegvolume in die bakkersindustrie, verbetering van verteerbaarheid en verhoging van voedingswaarde van dierevoer, toename in verheldering en verbeterde filtreerbaarheid van wyn, bier en vrugtesap, ens. Dus bestaan daar 'n groot potensiële mark vir sellulases en hemisellulases, mits hul produksie ekonomies en die produk outentiek is. Aspergilli kom in 'n wye verskeidenheid van omgewings voor, insluitende grond, gestoorde voedsel- en voerprodukte asook ontbindende plante materiaal. Die voordele vir die gebruik van A. niger as gasheer vir heteroloë ensiemproduksie sluit in 'n goeie proteïen produseerder, industriële fermentasietegnologie dateer sover terug as 1919, 'n nie-patogeniese fungus met GRAS-status, benodig geen spesiale substrate of kwekingskondisies nie, FDA goedkeuring vir 'n groot aantal ensieme (homoloog sowel as heteroloog) wat reeds geproduseer word, ens. In hierdie studie is 'n Aspergillus uitdrukkingsvektor gekonstrueer deur van die konstitutiewe gliseraldehied-3-fosfaat dehidrogenase promoter (gpdp) van A. niger en die glukoamilase termineerder (glaAT) van Aspergillus awamori gebruik te maak. Die cDNA kopiee van die die eg! en xyn2 van Trichoderma reesei, cbhl-4 van Phanerochaete chrysosporium, man! van Aspergillus aculeatus en die xynC van Aspergillus kawachii was onderskeidelik na die uitdrukkingsplasmied oorgedra. Alle plasmiede is gesamentlik met die p3 SR2 plasmied na A. niger getransformeer en vir stabiele integrasie in die gasheergenoom geselekteer. Die rekombinante ensieme Egl, Xyn2, Cbhl-4, Manl en Xyn3 is suksesvol uitgedruk en teen aktiviteitsvlakke van 2300, 8000, 500, 6000 en 900 nkat/ml, onderskeidelik uitgeskei. Die ensieme is as funksionele entiteite geproduseer en vervolgens gekaraktiriseer. Die Egl, Xyn2 en Manl is as voertoevoegings vir die moontlike gebruik in die dierevoerindustrie geëvalueer. Verbeterde biomassa toename is in die in vivo studie op pluimvee waargeneem. Met die moontlikheid van grootskaalse heteroloë ensiemproduksie in gedagte, moes 'n eenvoudige substraat vir hul goedkoop produksie gevind word. Molasse medium (verkrygbaar vanaf die Suid Afrikaanse suiker industrie) was derhalwe geëvalueer en die kwekingskondisies geoptimiseer vir die moontlike gebruik as kwekingssubstraat vir A. niger. Vir die evaluasie is die Egl en Xyn2 aktiwiteite onder verskillende toestande geproduseer en oor tyd gemonitor. Hierdie studie beklemtoon die moontlike gebruik van A. niger vir heteroloë produksie van ensieme, die gebruik van industriële substrate as kwekingsmedium en baan die weg vir ekonomiese, hoëvlakuitdrukking van industrieelbelangrike ensieme met 'n positiewe implikasie op die omgewing.

Submitted in fulfillment of the requirements of the degree of Master of Technology (Biotechnology), Durban University of Technology, Durban, South Africa, 2014. / Microbial enzymes are gaining worldwide attention due to their potential industrial applications. Microorganisms producing thermostable -xylanase and their associated hemicellulases have significant application in the paper and pulp, food, animal feed, and textile industries. The potential of partially purified xylanase from Thermomyces lanuginosus MC 134, Luminase PB 100, Luminase PB 200 (a commercial xylanase) and T. lanuginosus DSM 5826 (Sigma Aldrich) was evaluated in bleaching of bagasse pulp. The temperature and pH optima for all the enzymes were 60°C and pH 6, respectively. The temperature (50- 80°C) and pH (5-8) stability of the enzymes were also assessed. All the enzymes were relatively stable at 60°C and pH 6 for 180 min. T. lanuginosus MC 134 retained 80% of its activity at 60°C and pH 6 for 180 min and PB 200 retained 75% of its activity at 80°C for 180 min. T. lanuginosus MC 134 also exhibited good alkaline stability at pH 8. The commercial xylanases Luminase PB 100, Luminase PB 200, T. lanuginosus DSM 5826 (Sigma Aldrich) were purified to homogeneity using a gel filtration column packed with sephadex G-100 and characterized for Km and Vmax. However extracellular crude xylanases from T. lanuginosus MC 134 was purified to homogeneity using (N )2S04 precipitation and gel filtration column, packed with sephadex G-100. The purified xylanases exhibited a molecular mass of- 26 to 24 kDa, given range as determined by SDS page. The Km and Vmax values of Luminase PB 100, Luminase PB 200, T. lanuginosus MC 134, and T. lanuginosus DSM 5826, xylanases were determined by the Michaelis-Menten equation using birchwood xylan as the substrate. The Km value for Luminase PB 100, Luminase PB 200, T. lanuginosus DSM 5826 and T. lanuginosus MC 134 were, 8.1 mg/mL, 11.7 mg/mL and 14.3 mg/mL respectively. The Vmax for Luminase PB 100, Luminase PB 200, T lanuginosus DSM 5826 and T lanuginosus MC 134 were 232.6, 454.6 and 74.6 !Jl11ol/min/mg. Biobleaching conditions of the xylanases were also optimised and the release of reducing sugars and lignin derived compounds showed that an enzyme dosage of 50U/g of pulp was ideal for biobleaching at pH 6 and 60°C for 180 min. This brightness for T lanuginosus MC 134, Luminase PB 200, Luminase PB 100 was 45.5 ± 0.11%, 44.1 ± 0.007% and 42.7 ± 0.03% respectively at pH 6, compared to untreated samples. Reducing sugars and UV-absorbing lignin-derived compound values were considerably higher in xylanase-treated samples. All the enzymes analysed exhibited similar trends in the release of lignin derived compounds and reducing sugars which indicated their potential in the pulp and paper industry. / PDF Full-text unavailable. Please refer to hard copy for Full-text / M

Made available in DSpace on 2014-08-13T14:50:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-02-21Bitstream added on 2014-08-13T17:59:58Z : No. of bitstreams: 1 000768347_20160221.pdf: 161112 bytes, checksum: 81652a09b62d246f4c78fc239ef06ae2 (MD5) Bitstreams deleted on 2016-02-22T11:12:47Z: 000768347_20160221.pdf,. Added 1 bitstream(s) on 2016-02-22T11:13:41Z : No. of bitstreams: 1 000768347.pdf: 885551 bytes, checksum: 3352268d651e4caf0f5c3131e9467935 (MD5) / As lipases, também chamadas de glicerol éster hidrolases, são enzimas que fazem parte do grupo das serina hidrolases, tendo como substrato, triglicerídeos. O modo de ação das lipases assemelha-se ao das esterases, realizando a hidrólise das ligações ésteres-carboxílicas de acilgliceróis, formando ácidos graxos e glicerol. Processos de bioconversão enzimática têm sido bastante utilizados na produção, transformação e valorização de matérias-primas. Avanços na tecnologia enzimática, como a imobilização de enzimas, possibilitaram a modificação das propriedades cinéticas e da estabilidade destas moléculas contribuindo com o aumento no potencial de aplicações das mesmas. O presente trabalho teve por objetivo estudar diferentes métodos de imobilização de lipases em suportes de sílica, bem como os efeitos deste procedimento, visando melhorar a funcionalidade das enzimas e o maior rendimento econômico nos processos industriais. Os métodos de imobilização escolhidos para os estudos foram: adsorção física, ligação covalente e encapsulação. O processo de imobilização de lipase em Celite (adsorção física) foi otimizado levando em conta o pH, porcentagem da concentração enzima:suporte e temperatura ótimos de atividade enzimática. Também se utilizou Celite como suporte para a imobilização de lipase por ligação covalente, onde se obteve os melhores resultados com atividade enzimática 20% a 40 ºC e eficiência de imobilização de 50%. A celite foi ativada com 3-aminopropiltrietoxisilano e glutaraldeído. Por último, foi avaliada a possibilidade de encapsulação da lipase utilizando o precursor tetraetilortossilicato (TEOS). Os resultados obtidos nesta última metodologia não se mostraram satisfatórios. Logo, com os dados obtidos, podemos dizer que uma boa manutenção da atividade catalítica depende do tipo de retenção (química ou física) e da força de interação ... / Lipases, also known as glycerol ester hydrolases , are enzymes that belong to the group of serine hydrolases. The mode of action of lipases is very similar to esterase group, performing the hydrolysis of carboxylic esters of glycerides - forming fatty acids and glycerol. The enzymatic bioconversion processes have been widely used in manufacturing, processing and recovery of raw materials. Advances methodology for immobilization of enzyme have allowed the modification of the kinetic properties and stability of these molecules contributing to the increase in the potential applications of the same. The present work is aimed to study different methods of immobilization of lipases in silica supports, and the effects of this procedure to improve the functionality of enzymes. The immobilization methods chosen for the studies were: physical adsorption, covalent bonding and encapsulation process. The process of immobilization of lipase on Celite (by physical adsorption) was optimized taking into account several parameters such as: pH, the enzyme concentration:support and temperature for enzyme activity. Celite was also used as a support for the immobilization of lipase by covalent bond, where best results were obtained with 20% enzymatic activity at 40 ° C and immobilization efficiency of 50%. The celite was activated with 3-aminopropyltriethoxysilane and glutaraldehyde. Finally, we have studied the possibility of encapsulation of lipase using the precusor tetraethylorthosilicate (TEOS). The results of this last methodology were not satisfactory. These results show that to maintain a good catalytic activity depends on the type of immobilization chose (chemical or physical) and the strength of the interaction between the enzyme and support, which can cause structural distortions in the protein, leading maintenance or a decrease in catalytic activity

Orientador: Valter de Albuquerque Pedrosa / Coorientador: Luciana Francisco Fleuri / Banca: Maria José Queiroz de Freitas Alves / Banca: Haroldo Yukio Kawaguti / Resumo: As lipases, também chamadas de glicerol éster hidrolases, são enzimas que fazem parte do grupo das serina hidrolases, tendo como substrato, triglicerídeos. O modo de ação das lipases assemelha-se ao das esterases, realizando a hidrólise das ligações ésteres-carboxílicas de acilgliceróis, formando ácidos graxos e glicerol. Processos de bioconversão enzimática têm sido bastante utilizados na produção, transformação e valorização de matérias-primas. Avanços na tecnologia enzimática, como a imobilização de enzimas, possibilitaram a modificação das propriedades cinéticas e da estabilidade destas moléculas contribuindo com o aumento no potencial de aplicações das mesmas. O presente trabalho teve por objetivo estudar diferentes métodos de imobilização de lipases em suportes de sílica, bem como os efeitos deste procedimento, visando melhorar a funcionalidade das enzimas e o maior rendimento econômico nos processos industriais. Os métodos de imobilização escolhidos para os estudos foram: adsorção física, ligação covalente e encapsulação. O processo de imobilização de lipase em Celite (adsorção física) foi otimizado levando em conta o pH, porcentagem da concentração enzima:suporte e temperatura ótimos de atividade enzimática. Também se utilizou Celite como suporte para a imobilização de lipase por ligação covalente, onde se obteve os melhores resultados com atividade enzimática 20% a 40 ºC e eficiência de imobilização de 50%. A celite foi ativada com 3-aminopropiltrietoxisilano e glutaraldeído. Por último, foi avaliada a possibilidade de encapsulação da lipase utilizando o precursor tetraetilortossilicato (TEOS). Os resultados obtidos nesta última metodologia não se mostraram satisfatórios. Logo, com os dados obtidos, podemos dizer que uma boa manutenção da atividade catalítica depende do tipo de retenção (química ou física) e da força de interação ... / Abstract: Lipases, also known as glycerol ester hydrolases , are enzymes that belong to the group of serine hydrolases. The mode of action of lipases is very similar to esterase group, performing the hydrolysis of carboxylic esters of glycerides - forming fatty acids and glycerol. The enzymatic bioconversion processes have been widely used in manufacturing, processing and recovery of raw materials. Advances methodology for immobilization of enzyme have allowed the modification of the kinetic properties and stability of these molecules contributing to the increase in the potential applications of the same. The present work is aimed to study different methods of immobilization of lipases in silica supports, and the effects of this procedure to improve the functionality of enzymes. The immobilization methods chosen for the studies were: physical adsorption, covalent bonding and encapsulation process. The process of immobilization of lipase on Celite (by physical adsorption) was optimized taking into account several parameters such as: pH, the enzyme concentration:support and temperature for enzyme activity. Celite was also used as a support for the immobilization of lipase by covalent bond, where best results were obtained with 20% enzymatic activity at 40 ° C and immobilization efficiency of 50%. The celite was activated with 3-aminopropyltriethoxysilane and glutaraldehyde. Finally, we have studied the possibility of encapsulation of lipase using the precusor tetraethylorthosilicate (TEOS). The results of this last methodology were not satisfactory. These results show that to maintain a good catalytic activity depends on the type of immobilization chose (chemical or physical) and the strength of the interaction between the enzyme and support, which can cause structural distortions in the protein, leading maintenance or a decrease in catalytic activity / Mestre

Submitted in partial fulfillment of the requirements for the Degree of Master of Technology: Biotechnology, Durban Institute of Technology, 2000 / M