The family 1 cellulose-binding modules (CBM1) form a groupof small, stable carbohydrate-binding proteins. These modulesare essential for fungal cellulosedegradation. This thesisdescribes both functional studies of the CBM1s as well asprotein engineering of the modules for several objectives. The characteristics and specificity of CBM1s from theTrichoderma reeseiCel7A and Cel6A, along with severalother wild type and mutated CBMs, were studied using bindingexperiments and transmission electron microscopy (TEM). Datafrom the binding studies confirmed that the presence of onetryptophan residue on the CBM1 binding face enhances itsbinding to crystalline cellulose. The twoT. reeseiCBM1s as well as the CBM3 from theClostridium thermocellumCipA were investigated by TEMexperiments. All three CBMs were found to bind in lineararrangements along the sides of the fibrils. Further analysesof the bound CBMs indicated that the CBMs bind to the exposedhydrophobic surfaces, the so called (200) crystalline face ofValoniacellulose crystals. The function and specificity of CBM1s as a part of an intactenzyme were studied by replacing the CBM from the exo-actingCel7A by the CBM1 from the endoglucanase Cel7B. Apart fromslightly improved affinity of the hybrid enzyme, the moduleexchange did not significantly influence the function of theCel7A. This indicates that the two CBM1s are analogous in theirbinding properties and function during cellulosehydrolysis. The CBM1 was also used for immobilization studies. Toimprove heterologous expression of a CBM1-lipase fusionprotein, a linker stability study was carried out inPichia pastoris. A proline/threonine rich linker peptidewas found to be stable for protein production in this host. Forwhole bacterial cell immobilization, theT. reeseiCel6A CBM1 was expressed on the surface of thegram-positive bacteria,Staphylococcus carnosus. The engineeredS. carnosuscells were shown to bind cellulosefibers. To exploit the stable CBM1 fold as a starting point forgenerating novel binders, a phage display library wasconstructed. Binding proteins against an amylase as well asagainst a metal ion were selected from the library. Theamylase-binding proteins were found to bind and inhibit thetarget enzyme. The metal binding proteins selected from thelibrary were cloned on the surface of theS. carnosusand clearly enhanced the metal bindingability of the engineered bacteria. <b>Keywords</b>: cellulose-binding, family 1carbohydrate-binding module, phage display, bacterial surfacedisplay, combinatorial protein library, metal binding, proteinengineering,Trichoderma reesei, Staphyloccus carnosus.

<p>The family 1 cellulose-binding modules (CBM1) form a groupof small, stable carbohydrate-binding proteins. These modulesare essential for fungal cellulosedegradation. This thesisdescribes both functional studies of the CBM1s as well asprotein engineering of the modules for several objectives.</p><p>The characteristics and specificity of CBM1s from the<i>Trichoderma reesei</i>Cel7A and Cel6A, along with severalother wild type and mutated CBMs, were studied using bindingexperiments and transmission electron microscopy (TEM). Datafrom the binding studies confirmed that the presence of onetryptophan residue on the CBM1 binding face enhances itsbinding to crystalline cellulose. The two<i>T. reesei</i>CBM1s as well as the CBM3 from the<i>Clostridium thermocellum</i>CipA were investigated by TEMexperiments. All three CBMs were found to bind in lineararrangements along the sides of the fibrils. Further analysesof the bound CBMs indicated that the CBMs bind to the exposedhydrophobic surfaces, the so called (200) crystalline face of<i>Valonia</i>cellulose crystals.</p><p>The function and specificity of CBM1s as a part of an intactenzyme were studied by replacing the CBM from the exo-actingCel7A by the CBM1 from the endoglucanase Cel7B. Apart fromslightly improved affinity of the hybrid enzyme, the moduleexchange did not significantly influence the function of theCel7A. This indicates that the two CBM1s are analogous in theirbinding properties and function during cellulosehydrolysis.</p><p>The CBM1 was also used for immobilization studies. Toimprove heterologous expression of a CBM1-lipase fusionprotein, a linker stability study was carried out in<i>Pichia pastoris</i>. A proline/threonine rich linker peptidewas found to be stable for protein production in this host. Forwhole bacterial cell immobilization, the<i>T. reesei</i>Cel6A CBM1 was expressed on the surface of thegram-positive bacteria,<i>Staphylococcus carnosus</i>. The engineered<i>S. carnosus</i>cells were shown to bind cellulosefibers.</p><p>To exploit the stable CBM1 fold as a starting point forgenerating novel binders, a phage display library wasconstructed. Binding proteins against an amylase as well asagainst a metal ion were selected from the library. Theamylase-binding proteins were found to bind and inhibit thetarget enzyme. The metal binding proteins selected from thelibrary were cloned on the surface of the<i>S. carnosus</i>and clearly enhanced the metal bindingability of the engineered bacteria.</p><p><b>Keywords</b>: cellulose-binding, family 1carbohydrate-binding module, phage display, bacterial surfacedisplay, combinatorial protein library, metal binding, proteinengineering,<i>Trichoderma reesei, Staphyloccus carnosus</i>.</p>

Wood and wood products are of great economical and environmental importance, both in Sweden and globally. Biotechnology can be used both for achieving raw material of improved quality and for industrial processes such as biobleaching. Despite the enormous amount of carbon that is fixed as wood, the knowledge about the enzymes involved in the biosynthesis, re-organization and degradation of plant cell walls is relatively limited. In order to exploit enzymes more efficiently or to develop new biotechnological processes, it is crucial to gain a better understanding of the function and mechanism of the enzymes. This work has aimed to increase the knowledge about some of the enzymes putatively involved in the wood forming processes in Populus. Xyloglucan endotransglycosylases and a putative xylanase represent transglycosylating and hydrolytic enzymes, respectively. Carbohydrate binding modules represent non-catalytic modules, which bind to the substrate. Among 24 genes encoding for putative xyloglucan endotransglycosylases or xyloglucan endohydrolases that were identified in the Populus EST database, two were chosen for further studies (PttXTH16-34 and PttXTH16-35). The corresponding proteins, PttXET16-34 and PttXET16-35, were expressed in P. pastoris, purified and biochemically characterized. The importance of the N-glycans was investigated by comparing the recombinant wild-type proteins with their deglycosylated counterparts. In order to obtain the large amounts of PttXET16-34 that were needed for crystallization and development of biotechnological applications, the conditions for the large-scale production of PttXET16-34 in a fermenter were optimized. In microorganisms, endo-(1,4)-β-xylanases are important members of the xylan degrading machinery. These enzymes are also present in plants where they might fulfill a similar, but probably more restrictive function. One putative endo-(1,4)-β-xylanase, denoted PttXYN10A, was identified in the hybrid aspen EST library. Sequence analysis shows that this protein contains three putative carbohydrate-binding modules (CBM) from family 22 in addition to the catalytic module from GH10. Heterologous expression and reverse genetics were applied in order to elucidate the function of the catalytic module as well as the binding modules of PttXYN10A. Just as in microorganisms, some of the carbohydrate active enzymes from plants have one or more CBM attached to the catalytic module. So far, a very limited number of plant CBMs has been biochemically characterized. A detailed bio-informatic analysis of the CBM family 43 revealed interesting modularity patterns. In addition, one CBM43 (CBM43PttGH17_84) from a putative Populus b-(1,3)-glucanase was expressed in E. coli and shown to bind to laminarin (β-(1,3)-glucan), mixed-linked β-(1,3)(1,4)-glucans and crystalline cellulose. Due to their high specificity for different carbohydrates, CBMs can be used as probes for the analysis of plant materials. Generally, they are more specific than both staining techniques and carbohydrate-binding antibodies. We have used cellulose- and mannan binding modules from microorganisms as tools for the analysis of intact fibers as well as processed pulps. / QC 20100903

Glykosidhydrolaser (GHs) är enzymer som katalyserar hydrolys av glykosidbindningar i polysackarider och fungerar på endo- eller exo-sätt, beroende på om de riktar sig mot mitten eller änden av en glykan-kedja. De är viktiga i kolcykeln och i olika industrier som använder biomassa som substrat. GHs är fördelaktiga i många industriella processer på grund av deras höga specificitet, omsättningsgrad och biologiska nedbrytbarhet, men de kan vara instabila och är ofta dyra att producera. De varierar i specificitet och har ibland flera katalytiska domäner eller icke-katalytiska tillbehörsdomäner, vilket hjälper till att bryta ner polysackarider och/eller främjar enzymets livslängd. Många GHs kan ha kolhydratbindande moduler (CBMs) som ökar deras termostabilitet och/eller katalytiska aktivitet. CBMs är kopplade till andra domäner i multimodulära domäner av inter-domän länkar (IDLs), vilket är polypeptidkedjor som ger strukturell flexibilitet och låter CBMs nå önskade mål på ett substrat, men den fulla funktionen av IDLs i enzymstabilisering har inte dokumenterats. Kitinaser är en grupp av GHs som riktar sig mot det motsträviga polysackaridet kitin, vilket finns i både marina och markbundna miljöer. De finns i organismer såsom insekter med kitinhaltiga exoskelett och i svampar eller andra mikrober med kitininnehållande cellväggar, men de finns även i organismer som inte syntetiserar eller ens metaboliserar kitin, på grund av deras andra relevanta funktioner inom patogenicitet, immunförsvar, etc. Kitin och dess oligosackarid-derivat har flera funktioner i biomass-industrier och kan användas för medicinska ändamål. Många GHs innehåller icke-katalytiska CBMs, varav många är kitinbindande, och spelar därför en roll i att främja kitinbindning och hydrolys av deras enzympartners. Detta projekt fokuserar på ett modulärt GH18-kitinas kodat av genen Cpin_2580. Kitinasdomänen är flankerad av två CBMs. Tidigare forskning har visat att dessa inte är kitinbindande men föreslog att de påverkar enzymets termostabilitet. Däremot undersöktes inte IDL:ernas påverkan i den tidigare studien. För att bestämma rollen av IDLs designades primers för att klona nya genvarianter av Cpin_2580 för att producera nya proteiner med varierande längder av länkar för att bestämma vad för effekt längden har på enzymets termostabilitet. Dessa primers användes till PCR för att skapa gensekvenser med den befintliga Cpin_2580-18s-plasmiden som mall, följt av kloning, proteinproduktion, rening och analys med hjälp av fluoroforbindningsanalys. Nya proteinvarianter kunde genereras och produceras i liten skala, men produktionen upplevde problem, vilket ledde till att IDLs roll inte kunde fastställas fullt ut. / Glycoside hydrolases (GHs) are enzymes that catalyse the hydrolysis of glycosidic bonds in polysaccharides, functioning in endo- or exo-manners, depending on whether they target the middle or the end of a glycan chain. They are crucial in the carbon cycle and various industries that utilise biomass as substrate. GHs are advantageous in many industrial processes due to their high specificity, turnover rates, and biodegradability, but they can be unstable and are often costly to produce. They vary in specificity and sometimes carry multiple catalytic domains or non-catalytic accessory domains, aiding in polysaccharide breakdown and/or promoting the longevity of the enzyme. Many GHs can have carbohydrate binding modules (CBMs) attached that can be considered accessory domains, which increases their thermostability and/or catalytic activity in many cases. CBMs are attached to other domains in multi-modular enzymes by inter-domain linkers (IDLs), which are polypeptide chains that give structural flexibility and allow the CBMs to reach desired targets on a substrate, but the full function of IDLs in enzyme stabilisation has not been documented. Chitinases are a group of GHs that targets the recalcitrant polysaccharide chitin, which exists in both marine and terrestrial environments. They exist in organisms such as insects that have chitinous exoskeletons and in fungi or other microbes with chitin-containing cell walls, but they are also found in organisms that do not synthesise or even metabolise chitin, due to their other functions of relevance in pathogenicity, immune defence, etc. Chitin and its oligosaccharide derivatives have multiple functions in biomass industries, and can be used for medical purposes. Many chitinases contain non-catalytic CBMs, many of which are often chitin-binding, and therefore have a role in promoting chitin attachment and hydrolysis by their enzyme partners. This project focuses on a modular GH18 chitinase encoded by the gene Cpin_2580. The chitinase domain is flanked by two CBMs. Previous research has shown that these are not chitin-binding but suggested they do influence the thermostability of the enzyme. However, the impact of the IDLs was not explored in that previous study. To determine the role of the IDLs, primers were designed with the purpose of cloning new gene variants of the gene Cpin_2580 to produce novel proteins with varying lengths of linkers to determine the effect the length has on the thermostability of the enzyme. These primers were used for PCR to create novel gene sequences using the pre-existing Cpin_2580-18s plasmid as a template, followed by cloning, protein production, purification, and analysis using fluorophore binding assay. Novel protein variants could be generated and produced at small scale, but scaled-up protein production experienced problems, which led to the role of IDLs not being fully determined.