New thermostable endoglucanase from Spirochaeta thermophila and its mutants with altered substrate preferences.

Endoglucanases are key elements in several industrial applications, such as cellulosic biomass hydrolysis, cellulose fiber modification for the production paper and composite materials, and in nanocellulose production. In all of these applications, the desired function of the endoglucanase is to create nicks in the amorphous regions of the cellulose. However, endoglucanase can be diverted from its activity on the fibers by other substrates-soluble oligosaccharides. This issue was addressed in the current study using enzyme engineering and an enzyme evolution approach. To this end, a hypothetical endoglucanase from a thermostable bacterium Spirochaeta thermophila was for the first time cloned and characterized. The wild-type enzyme was used as a starting point for mutagenesis and molecular evolution toward a preference for the higher molecular weight substrates. The best of the evolved enzymes was more active than the wild-type enzyme toward high molecular weight substrate at temperatures below 45 °C (3-fold more active at 30 °C) and showed little or no activity with low molecular weight substrates. These findings can be instrumental in bioeconomy sectors, such as second-generation biofuels and biomaterials from lignocellulosic biomass. KEY POINTS: • A new thermostable endoglucanase was characterized. • The substrate specificity of this endoglucanase was changed by means of genetic engineering. • A mutant with a preference for long molecular weight substrate was obtained and proposed to be beneficial for cellulose fiber modification.

Enzymatic Processes to Unlock the Lignin Value.

Main hurdles of lignin valorization are its diverse chemical composition, recalcitrance, and poor solubility due to high-molecular weight and branched structure. Controlled fragmentation of lignin could lead to its use in higher value products such as binders, coatings, fillers, etc. Oxidative enzymes (i.e., laccases and peroxidases) have long been proposed as a potentially promising tool in lignin depolymerization. However, their application was limited to ambient pH, where lignin is poorly soluble in water. A Finnish biotechnology company, MetGen Oy, that designs and supplies industrial enzymes, has developed and brought to market several lignin oxidizing enzymes, including an extremely alkaline lignin oxidase MetZyme® LIGNO™, a genetically engineered laccase of bacterial origin. This enzyme can function at pH values as high as 10-11 and at elevated temperatures, addressing lignin at its soluble state. In this article, main characteristics of this enzyme as well as its action on bulk lignin coming from an industrial process are demonstrated. Lignin modification by MetZyme® LIGNO™ was characterized by size exclusion chromatography, UV spectroscopy, and dynamic light scattering for monitoring particle size of solubilized lignin. Under highly alkaline conditions, laccase treatment not only decreased molecular weight of lignin but also increased its solubility in water and altered its dispersion properties. Importantly, organic solvent-free soluble lignin fragmentation allowed for robust industrially relevant membrane separation technologies to be applicable for product fractionation. These enzyme-based solutions open new opportunities for biorefinery lignin valorization thus paving the way for economically viable biorefinery business.

Loss of cysteine-rich secretory protein 4 (Crisp4) leads to deficiency in sperm-zona pellucida interaction in mice.

Mammalian sperm gain their ability to fertilize the egg during transit through the epididymis and by interacting with proteins secreted by the epididymal epithelial cells. Certain members of the CRISP (cysteine-rich secretory protein) family form the major protein constituent of the luminal fluid in the mammalian epididymis. CRISP4 is the newest member of the CRISP family expressed predominantly in the epididymis. Its structure and expression pattern suggest a role in sperm maturation and/or sperm-egg interaction. To study the relevance of CRISP4 in reproduction, we have generated a Crisp4 iCre knock-in mouse model through insertion of the iCre recombinase coding cDNA into the Crisp4 locus. This allows using the mouse line both as a Crisp4 deficient model and as an epididymis-specific iCre-expressing mouse line applicable for the generation of conditional, epididymis-specific knockout mice. We show that the loss of CRISP4 leads to a deficiency of the spermatozoa to undergo progesterone-induced acrosome reaction and to a decreased fertilizing ability of the sperm in the in vitro fertilization conditions, although the mice remain fully fertile in normal mating. However, removal of the egg zona pellucida returned the fertilization potential of the CRISP4-deficient spermatozoa, and accordingly we detected a reduced number of Crisp4-deficient spermatozoa bound to oocytes as compared with the wild-type spermatozoa. We also demonstrate that iCre recombinase is expressed in a pattern similar to endogenous Crisp4 and is able to initiate the recombination event with its target sequences in vivo.