Chitin, the second most abundant polysaccharide in nature after cellulose, is found in the exoskeleton of insects, fungi, yeast, and algae, and in the internal structures of other vertebrates. Chitinases are enzymes that degrade chitin. Chitinases contribute to the generation of carbon and nitrogen in the ecosystem. Chitin and chitinolytic enzymes are gaining importance for their biotechnological applications, especially the chitinases exploited in agriculture fields to control pathogens. Chitinases have a use in human health care, especially in human diseases like asthma. Chitinases have wide-ranging applications including the preparation of pharmaceutically important chitooligosaccharides and N-acetyl D glucosamine, preparation of single-cell protein, isolation of protoplasts from fungi and yeast, control of pathogenic fungi, treatment of chitinous waste, mosquito control and morphogenesis, etc. In this review, the various types of chitinases and the chitinases found in different organisms such as bacteria, plants, fungi, and mammals are discussed.

Chitinases: An update.

Chitin is the second most abundant organic and renewable source in nature, after cellulose. Chitinases are chitin-degrading enzymes. Chitinases have important biophysiological functions and immense potential applications. In recent years, researches on fungal chitinases have made fast progress, especially in molecular levels. Therefore, the present review will focus on recent advances of fungal chitinases, containing their nomenclature and assays, purification and characterization, molecular cloning and expression, family and structure, regulation, and function and application.

Review of fungal chitinases.

We have compared expression systems based on autonomously replicating vectors in the yeasts Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Hansenula polymorpha and Yarrowia lipolytica in order to identify a more suitable host organism for use in the expression cloning method (Dalboge and Heldt-Hansen, 1994) in which S. cerevisiae has traditionally been used. The capacity of the expression systems to secrete active forms of six fungal genes encoding the enzymes galactanase, lipase, polygalacturonase, xylanase and two cellulases was examined, as well as glycosylation pattern, plasmid stability and transformation frequency. All of the examined alternative hosts were able to secrete more active enzyme than S. cerevisiae but the relative expression capacity of the individual hosts varied significantly in a gene-dependent manner. One of the most attractive of the alternative host organisms, Y. lipolytica, yielded an increase which ranged from 4.5 times to more than two orders of magnitude. As the initially employed Y. lipolytica XPR2 promoter is unfit in the context of expression cloning, two novel promoter sequences for highly expressed genes present in only one copy on the genome were isolated. Based on sequence homology, the genes were identified as TEF, encoding translation elongation factor-1 alpha and RPS7, encoding ribosomal protein S7. Using the heterologous cellulase II (celII) and xylanase I (xylI) as reporter genes, the effect of the new promoters was measured in qualitative and quantitative assays. Based on the present tests of the new promoters. Y. lipolytica appears as a highly attractive alternative to S. cerevisiae as a host organism for expression cloning.

Comparison of expression systems in the yeasts Saccharomyces cerevisiae, Hansenula polymorpha, Klyveromyces lactis, Schizosaccharomyces pombe and Yarrowia lipolytica. Cloning of two novel promoters from Yarrowia lipolytica.

The use of specific mycolytic soil microorganisms to control plant pathogens is an ecological approach to overcome the problems caused by standard chemical methods of plant protection The ability to produce lytic enzymes is a widely distributed property of rhizosphere-competent fungi and bacteria Due to the higher activity of Trichoderma spp lytic enzymes as compared to the same class of enzymes from other microorganisms and plants, effort is being aimed at improving biocontrol agents and plants by introducing Trichoderma genes via genetic manipulations An overview is presented of the data currently available on lytic enzymes from the mycoparasitic fungus Trichoderma

Significance of lytic enzymes from Trichoderma spp. in the biocontrol of fungal plant pathogens

Genome-wide analysis of chitinase genes in the Hypocrea jecorina (anamorph: Trichoderma reesei) genome database revealed the presence of 18 ORFs encoding putative chitinases, all of them belonging to glycoside hydrolase family 18. Eleven of these encode yet undescribed chitinases. A systematic nomenclature for the H. jecorina chitinases is proposed, which designates the chitinases corresponding to their glycoside hydrolase family and numbers the isoenzymes according to their pI from Chi18-1 to Chi18-18. Phylogenetic analysis of H. jecorina chitinases, and those from other filamentous fungi, including hypothetical proteins of annotated fungal genome databases, showed that the fungal chitinases can be divided into three groups: groups A and B (corresponding to class V and III chitinases, respectively) also contained the so Trichoderma chitinases identified to date, whereas a novel group C comprises high molecular weight chitinases that have a domain structure similar to Kluyveromyces lactis killer toxins. Five chitinase genes, representing members of groups A-C, were cloned from the mycoparasitic species H. atroviridis (anamorph: T. atroviride). Transcription of chi18-10 (belonging to group C) and chi18-13 (belonging to a novel clade in group B) was triggered upon growth on Rhizoctonia solani cell walls, and during plate confrontation tests with the plant pathogen R. solani. Therefore, group C and the novel clade in group B may contain chitinases of potential relevance for the biocontrol properties of Trichoderma.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1742-4658.2005.04994.x

A complete survey of Trichoderma chitinases reveals three distinct subgroups of family 18 chitinases.

The synthetic exochitinase substrate 4-methylumbelliferyl N-acetylglucosamine was used to identify seven full-length exochitinase-encoding cDNAs from a Trichoderma harzianum cDNA library by expression in yeast. The cDNA clones represented transcripts of two exochitinase genes, designated as exc1 and exc2, which cross-hybridized under moderate stringency conditions in genomic Southern blots. The exc1 cDNA encodes a 578 amino acid polypeptide showing 72% similarity to the exc2-encoded 602-residue polypeptide. The deduced exochitinase amino acid sequences were found to be homologous with mammalian and fungal hexosaminidases as well as a bacterial chitobiosidase. The substrate specificity of the recombinant enzymes expressed in S. cerevisiae indicates that the enzymes are N-acetylglucosaminidases releasing single N-acetylglucosamine residues from the non-reducing end of the chitin substrate.

Molecular cloning and expression in S. cerevisiae of two exochitinases from Trichoderma harzianum.

A novel endochitinase agar-plate assay has been developed and used to identify 11 full-length cDNAs encoding endochitinase I (ENC I) from aTrichoderma harzianum cDNA library by expression in yeast. The 1473-bpchil cDNA encodes a 424-residue precursor protein including both a signal sequence and a propeptide. The deduced ENC I amino-acid sequence is homologous to other fungal and bacterial chitinases, and the enzyme cross-reacts with a polyclonal antiserum raised against chitinase A1 fromBacillus circulans. TheT. harzianum endochitinase I was secreted into the culture medium by the yeastSaccharomyces cerevisiae in a functionally active form. The purified recombinant enzyme had a molecular mass of 44 kDa, an isoelectric point of 6.3, a pH optimum of 7.0 and a temperature optimum of 20 °C.

Secretion of an enzymatically active Trichoderma harzianum endochitinase by Saccharomyces cerevisiae.

The invention relates to a DNA construct comprising a DNA sequence encoding an enzyme exhibiting exochitinase activity, which DNA sequence comprises the DNA sequence shown in SEQ ID No. 1 or SEQ ID No. 3, or analogues of the DNA sequences encoding polypeptides which are derived from Saccharomyces cerevisiae DSM No. 9944 or Saccharomyces cerevisiae DSM No. 9945. Further, the invention relates to a method of producing said enzyme, an enzyme preparation containing said enzyme, and the use of said exochitinase or said enzyme preparation for a number of purposes. Also claimed is the use of the DNA construct of the invention for producing transgenic plants. Finally, an isolated substantially pure culture of the deposited microorganisms is claimed.

Henriette Draborg

Papers

Molecular cloning and expression in S. cerevisiae of two exochitinases from Trichoderma harzianum.

Secretion of an enzymatically active Trichoderma harzianum endochitinase by Saccharomyces cerevisiae.

An enzyme with exochitinase activity